Enhanced early production of inflammatory chemokines improves viral control in the lung.

Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung.

The mechanisms by which a primary tumor affects a selected distant organ before tumor cell arrival remain to be elucidated. This report shows that Gr-1+CD11b+ cells are significantly increased in lungs of mice bearing mammary adenocarcinomas before tumor cell arrival. In the premetastatic lungs, these immature myeloid cells significantly decrease IFN-gamma production and increase proinflammatory cytokines. In addition, they produce large quantities of matrix metalloproteinase 9 (MMP9) and promote vascular remodeling. Deletion of MMP9 normalizes aberrant vasculature in the premetastatic lung and diminishes lung metastasis. The production and activity of MMP9 is selectively restricted to lungs and organs with a large number of Gr-1+CD11b+ cells. Our work reveals a novel protumor mechanism for Gr-1+CD11b+ cells that changes the premetastatic lung into an inflammatory and proliferative environment, diminishes immune protection, and promotes metastasis through aberrant vasculature formation. Thus, inhibition of Gr-1+CD11b+ cells could normalize the premetastatic lung environment, improve host immunosurveillance, and inhibit tumor metastasis.

Memory CD4+ T cells induce innate responses independently of pathogen

Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4+ T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4+ T cell–induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (TH1) or TH17 polarized but are independent of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4+ T cells induce an early innate response that enhances immune protection against pathogens.

Tissue exit: a novel control point in the accumulation of antigen-specific CD8 T cells in the influenza a virus-infected lung.

Memory/effector T cells efficiently migrate into extralymphoid tissues and sites of infection, providing immunosurveillance and a first line of defense against invading pathogens. Even though it is a potential means to regulate the size, quality, and duration of a tissue infiltrate, T cell egress from infected tissues is poorly understood. Using a mouse model of influenza A virus infection, we found that CD8 effector T cells egressed from the infected lung in a CCR7-dependent manner. In contrast, following antigen recognition, effector CD8 T cell egress decreased and CCR7 function was reduced in vivo and in vitro, indicating that the exit of CD8 T cells from infected tissues is tightly regulated. Our data suggest that the regulation of T cell egress is a mechanism to retain antigen-specific effectors at the site of infection to promote viral clearance, while decreasing the numbers of bystander T cells and preventing overt inflammation.

Resolution of acute inflammation in the lung.

Acute inflammation in the lung is essential to health. So too is its resolution. In response to invading microbes, noxious stimuli, or tissue injury, an acute inflammatory response is mounted to protect the host. To limit inflammation and prevent collateral injury of healthy, uninvolved tissue, the lung orchestrates the formation of specialized proresolving mediators, specifically lipoxins, resolvins, protectins, and maresins. These immunoresolvents are agonists for resolution that interact with specific receptors on leukocytes and structural cells to blunt further inflammation and promote catabasis. This process appears to be defective in several common lung diseases that are characterized by excess or chronic inflammation. Here, we review the molecular and cellular effectors of resolution of acute inflammation in the lung.

Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality

Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of naïve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.

[Neutrophils and macrophages related to the pathogenesis and disease development of chronic obstructive pulmonary disease by the inflammatory reaction].

Chronic obstructive pulmonary disease (COPD) is a chronic airway disorder characterized by obstructive airflow limitation which is not completely reversible with treatment. Inflammatory changes in the peripheral airways, especially those with the diameter less than 2mm (so-called small airway disease) have been speculated to be initial steps of COPD. And so it must be quite clear that neutrophils and macrophages play an essential role in the pathogenesis of these lesions. Studies with bronchoalveolar lavage demonstrated an increase in neutrophil numbers and the neutrophil chemoattractant interleukin-8. Recent studies demonstrated that neutrophils and macrophages are increased and contain a variety of proteases, which are involved in cell infiltration and activation. Studies with gene-engineered animals and anti-cytokine treatment will facilitate better understanding the role of neutrophils and macrophages, and eventual novel therapy.

Dual role of interleukin-10 in the regulation of respiratory syncitial virus (RSV)-induced lung inflammation.

RSV lower respiratory tract infections (LRTI) are among the most common diseases necessitating hospital admission in children. In addition to causing acute respiratory failure, RSV infections are associated with sequelae such as secondary bacterial infections and reactive airway disease. One characteristic host response observed in severe RSV-induced LRTI and/or subsequent development of asthma is increased expression of interleukin (IL)-10. However, contradictory results have been reported regarding whether IL-10 inhibits asthmatic responses or intensifies the disease. We aimed to reconcile these discordant observations by elucidating the role of IL-10 in regulating the host response to RSV LRTI. In this study, we used a lung-specific, inducible IL-10 over-expression (OE) transgenic mouse model to address this question. Our results showed that the presence of IL-10 at the time of RSV infection not only attenuated acute inflammatory process (i.e. 24 h post-infection), but also late inflammatory changes [characterized by T helper type 2 (Th2) cytokine and chemokine expression]. While this result appears contradictory to some clinical observations where elevated IL-10 levels are observed in asthmatic patients, we also found that delaying IL-10 OE until the late immune response to RSV infection, additive effects rather than inhibitory effects were observed. Importantly, in non-infected, IL-10 OE mice, IL-10 OE alone induced up-regulation of Th2 cytokine (IL-13 and IL-5) and Th2-related chemokine [monocyte chemoattractant protein 1 (MCP-1), chemokine (C-C motif) ligand 3 (CCL3) and regulated upon activation normal T cell expressed and secreted (RANTES)] expression. We identified a subset of CD11b(+)CD11c(+)CD49b(+)F4/80(-)Gr-1(-) myeloid cells as a prinicipal source of IL-10-induced IL-13 production. Therefore, the augmented pathological responses observed in our 'delayed' IL-10 over-expression model could be attributed to IL-10 OE alone. Taken together, our study indicated dual roles of IL-10 on RSV-induced lung inflammation which appear to depend upon the timing of when elevated IL-10 is expressed in the lung.

Cancer-related inflammation

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumours. In some types of cancer, inflammatory conditions are present before a malignant change occurs. Conversely, in other types of cancer, an oncogenic change induces an inflammatory microenvironment that promotes the development of tumours. Regardless of its origin, 'smouldering' inflammation in the tumour microenvironment has many tumour-promoting effects. It aids in the proliferation and survival of malignant cells, promotes angiogenesis and metastasis, subverts adaptive immune responses, and alters responses to hormones and chemotherapeutic agents. The molecular pathways of this cancer-related inflammation are now being unravelled, resulting in the identification of new target molecules that could lead to improved diagnosis and treatment.

Nanotoxicology: An Emerging Discipline Evolving from Studies of Ultrafine Particles

Although humans have been exposed to airborne nanosized particles (NSPs; < 100 nm) throughout their evolutionary stages, such exposure has increased dramatically over the last century due to anthropogenic sources. The rapidly developing field of nanotechnology is likely to become yet another source through inhalation, ingestion, skin uptake, and injection of engineered nanomaterials. Information about safety and potential hazards is urgently needed. Results of older biokinetic studies with NSPs and newer epidemiologic and toxicologic studies with airborne ultrafine particles can be viewed as the basis for the expanding field of nanotoxicology, which can be defined as safety evaluation of engineered nanostructures and nanodevices. Collectively, some emerging concepts of nanotoxicology can be identified from the results of these studies. When inhaled, specific sizes of NSPs are efficiently deposited by diffusional mechanisms in all regions of the respiratory tract. The small size facilitates uptake into cells and transcytosis across epithelial and endothelial cells into the blood and lymph circulation to reach potentially sensitive target sites such as bone marrow, lymph nodes, spleen, and heart. Access to the central nervous system and ganglia via translocation along axons and dendrites of neurons has also been observed. NSPs penetrating the skin distribute via uptake into lymphatic channels. Endocytosis and biokinetics are largely dependent on NSP surface chemistry (coating) and in vivo surface modifications. The greater surface area per mass compared with larger-sized particles of the same chemistry renders NSPs more active biologically. This activity includes a potential for inflammatory and pro-oxidant, but also antioxidant, activity, which can explain early findings showing mixed results in terms of toxicity of NSPs to environmentally relevant species. Evidence of mitochondrial distribution and oxidative stress response after NSP endocytosis points to a need for basic research on their interactions with subcellular structures. Additional considerations for assessing safety of engineered NSPs include careful selections of appropriate and relevant doses/concentrations, the likelihood of increased effects in a compromised organism, and also the benefits of possible desirable effects. An interdisciplinary team approach (e.g., toxicology, materials science, medicine, molecular biology, and bioinformatics, to name a few) is mandatory for nanotoxicology research to arrive at an appropriate risk assessment.

Epigenetic Therapy Ties MYC Depletion to Reversing Immune Evasion and Treating Lung Cancer

Combining DNA-demethylating agents (DNA methyltransferase inhibitors [DNMTis]) with histone deacetylase inhibitors (HDACis) holds promise for enhancing cancer immune therapy. Herein, pharmacologic and isoform specificity of HDACis are investigated to guide their addition to a DNMTi, thus devising a new, low-dose, sequential regimen that imparts a robust anti-tumor effect for non-small-cell lung cancer (NSCLC). Using in-vitro-treated NSCLC cell lines, we elucidate an interferon α/β-based transcriptional program with accompanying upregulation of antigen presentation machinery, mediated in part through double-stranded RNA (dsRNA) induction. This is accompanied by suppression of MYC signaling and an increase in the T cell chemoattractant CCL5. Use of this combination treatment schema in mouse models of NSCLC reverses tumor immune evasion and modulates T cell exhaustion state towards memory and effector T cell phenotypes. Key correlative science metrics emerge for an upcoming clinical trial, testing enhancement of immune checkpoint therapy for NSCLC.

Local neuroinflammation and the progression of Alzheimer’s disease

Postmortem immunohistochemical studies have revealed a state of chronic inflammation limited to lesioned areas of brain in Alzheimer’s disease. Some key actors in this inflammation are activated microglia (brain macrophages), proteins of the classical complement cascade, the pentraxins, cytokines, and chemokines. The inflammation does not involve the adaptive immune system or peripheral organs, but is rather due to the phylogenetically much older innate immune system, which appears to operate in most tissues of the body. Chronic inflammation can damage host tissue and the brain may be particularly vulnerable because of the postmitotic nature of neurons. Many of the inflammatory mediators have been shown to be locally produced and selectively elevated in affected regions of Alzheimer’s brain. Moreover, studies of tissue in such degenerative processes as atherosclerosis and infarcted heart suggest a similar local innate immune reaction may be important in such conditions. Much epidemiological and limited clinical evidence suggests that nonsteroidal anti-inflammatory drugs may impede the onset and slow the progression of Alzheimer’s disease. But these drugs strike at the periphery of the inflammatory reaction. Much better results might be obtained if drugs were found that could inhibit the activation of microglia or the complement system in brain, and combinations of drugs aimed at different inflammatory targets might be much more effective than single agents.

Targeted nanoparticles containing the proresolving peptide Ac2-26 protect against advanced atherosclerosis in hypercholesterolemic mice

Chronic, nonresolving inflammation is a critical factor in the clinical progression of advanced atherosclerotic lesions. In the normal inflammatory response, resolution is mediated by several agonists, among which is the glucocorticoid-regulated protein called annexin A1. The proresolving actions of annexin A1, which are mediated through its receptor N-formyl peptide receptor 2 (FPR2/ALX), can be mimicked by an amino-terminal peptide encompassing amino acids 2–26 (Ac2-26). Collagen IV (Col IV)–targeted nanoparticles (NPs) containing Ac2-26 were evaluated for their therapeutic effect on chronic, advanced atherosclerosis in fat-fed Ldlr−/− mice. When administered to mice with preexisting lesions, Col IV–Ac2-26 NPs were targeted to lesions and led to a marked improvement in key advanced plaque properties, including an increase in the protective collagen layer overlying lesions (which was associated with a decrease in lesional collagenase activity), suppression of oxidative stress, and a decrease in plaque necrosis. In mice lacking FPR2/ALX in myeloid cells, these improvements were not seen. Thus, administration of a resolution-mediating peptide in a targeted NP activates its receptor on myeloid cells to stabilize advanced atherosclerotic lesions. These findings support the concept that defective inflammation resolution plays a role in advanced atherosclerosis, and suggest a new form of therapy.

Cancer cell–autonomous contribution of type I interferon signaling to the efficacy of chemotherapy

Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell–mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-β receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN–related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway

Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited. Eosinophils have been described mainly in allergy settings but are increasingly appreciated as being involved in other aspects of immunity. Albert and colleagues use a clinically approved inhibitor of the dipeptidyl peptidase DPP4 to facilitate the recruitment of eosinophils to mouse tumors, where they are essential in tumor destruction.

Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A.

Neutrophils are the main effector cells during inflammation, but they can also control excessive inflammatory responses by secreting anti-inflammatory cytokines. However, the mechanisms that modulate their plasticity remain unclear. We now show that systemic serum amyloid A 1 (SAA-1) controls the plasticity of neutrophil differentiation. SAA-1 not only induced anti-inflammatory interleukin 10 (IL-10)-secreting neutrophils but also promoted the interaction of invariant natural killer T cells (iNKT cells) with those neutrophils, a process that limited their suppressive activity by diminishing the production of IL-10 and enhancing the production of IL-12. Because SAA-1-producing melanomas promoted differentiation of IL-10-secreting neutrophils, harnessing iNKT cells could be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses.

Differential role of CbpA and PspA in modulation of in vitro CXC chemokine responses of respiratory epithelial cells to infection with Streptococcus pneumoniae.

Respiratory epithelial cells play an active part in the host response to respiratory pathogens, such as Streptococcus pneumoniae, by releasing chemokines responsible for neutrophil recruitment. In order to investigate the role of specific pneumococcal virulence factors in eliciting CXC chemokine responses, type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562) were infected with S. pneumoniae D39 or mutants lacking choline-binding protein A (CbpA), pneumococcal surface protein A (PspA), or specific domains thereof. In response to wild-type D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA and interleukin-8 protein. This response was increased twofold when a cbpA deletion mutant (DeltaCbpA) was used, suggesting that CbpA inhibits CXC chemokine induction. All three N-terminal domains of CbpA are required for this effect, as in-frame deletion of the respective region of cbpA had the same effect on the CXC chemokine response as deletion of cbpA altogether. Infection with a pspA deletion mutant (DeltaPspA) led to a twofold decrease in the CXC chemokine response of A549 but not Detroit-562 cells, compared to infection with D39 at 2 h. Thus, PspA appears to have the ability to stimulate early CXC chemokine release from A549 cells. Deletion of the region of pspA encoding the first N-terminal alpha-helical domain reduced the ability of S. pneumoniae to elicit a chemokine response to the same degree as deletion of pspA altogether. Thus, the N termini of CbpA and PspA exert differential effects on CXC chemokine induction in epithelial cells infected with S. pneumoniae.

Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.

PSGL-1 function in immunity and steady state homeostasis.

The substantial importance of P-selectin glycoprotein ligand 1 (PSGL-1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL-1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin-PSGL-1 interaction requires branched O-glycan extensions on specific PSGL-1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O-glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL-1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O-glycans on PSGL-1 and provide the molecular interactions responsible for the velcro-like catch bonds that support leukocyte rolling. Such binding of PSGL-1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL-1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T-cell homing to secondary lymphoid organs. PSGL-1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O-glycan modifications of PSGL-1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL-1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T-cell traffic. The multi-level influence of PSGL-1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O-glycans. However, central as specific O-glycosylation is to PSGL-1 function, in vivo regulation of PSGL-1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL-1 glycosylation and to update understanding of PSGL-1 functional scope.

A link between inflammation and metastasis: serum amyloid A1 and A3 induce metastasis, and are targets of metastasis-inducing S100A4

S100A4 is implicated in metastasis and chronic inflammation, but its function remains uncertain. Here we establish an S100A4-dependent link between inflammation and metastatic tumor progression. We found that the acute-phase response proteins serum amyloid A (SAA) 1 and SAA3 are transcriptional targets of S100A4 via Toll-like receptor 4 (TLR4)/nuclear factor-κB signaling. SAA proteins stimulated the transcription of RANTES (regulated upon activation normal T-cell expressed and presumably secreted), G-CSF (granulocyte-colony-stimulating factor) and MMP2 (matrix metalloproteinase 2), MMP3, MMP9 and MMP13. We have also shown for the first time that SAA stimulate their own transcription as well as that of proinflammatory S100A8 and S100A9 proteins. Moreover, they strongly enhanced tumor cell adhesion to fibronectin, and stimulated migration and invasion of human and mouse tumor cells. Intravenously injected S100A4 protein induced expression of SAA proteins and cytokines in an organ-specific manner. In a breast cancer animal model, ectopic expression of SAA1 or SAA3 in tumor cells potently promoted widespread metastasis formation accompanied by a massive infiltration of immune cells. Furthermore, coordinate expression of S100A4 and SAA in tumor samples from colorectal carcinoma patients significantly correlated with reduced overall survival. These data show that SAA proteins are effectors for the metastasis-promoting functions of S100A4, and serve as a link between inflammation and tumor progression.

In situ regulation of DC subsets and T cells mediates tumor regression in mice.

Vaccines are largely ineffective for patients with established cancer, as advanced disease requires potent and sustained activation of CD8(+) cytotoxic T lymphocytes (CTLs) to kill tumor cells and clear the disease. Recent studies have found that subsets of dendritic cells (DCs) specialize in antigen cross-presentation and in the production of cytokines, which regulate both CTLs and T regulatory (Treg) cells that shut down effector T cell responses. Here, we addressed the hypothesis that coordinated regulation of a DC network, and plasmacytoid DCs (pDCs) and CD8(+) DCs in particular, could enhance host immunity in mice. We used functionalized biomaterials incorporating various combinations of an inflammatory cytokine, immune danger signal, and tumor lysates to control the activation and localization of host DC populations in situ. The numbers of pDCs and CD8(+) DCs, and the endogenous production of interleukin-12, all correlated strongly with the magnitude of protective antitumor immunity and the generation of potent CD8(+) CTLs. Vaccination by this method maintained local and systemic CTL responses for extended periods while inhibiting FoxP3 Treg activity during antigen clearance, resulting in complete regression of distant and established melanoma tumors. The efficacy of this vaccine as a monotherapy against large invasive tumors may be a result of the local activity of pDCs and CD8(+) DCs induced by persistent danger and antigen signaling at the vaccine site. These results indicate that a critical pattern of DC subsets correlates with the evolution of therapeutic antitumor responses and provide a template for future vaccine design.

Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β–dependent adaptive immunity against tumors

The therapeutic efficacy of anticancer chemotherapies may depend on dendritic cells (DCs), which present antigens from dying cancer cells to prime tumor-specific interferon-γ (IFN-γ)–producing T lymphocytes. Here we show that dying tumor cells release ATP, which then acts on P2X7 purinergic receptors from DCs and triggers the NOD-like receptor family, pyrin domain containing-3 protein (NLRP3)-dependent caspase-1 activation complex ('inflammasome'), allowing for the secretion of interleukin-1β (IL-1β). The priming of IFN-γ–producing CD8+ T cells by dying tumor cells fails in the absence of a functional IL-1 receptor 1 and in Nlpr3-deficient (Nlrp3−/−) or caspase-1–deficient (Casp-1−/−) mice unless exogenous IL-1β is provided. Accordingly, anticancer chemotherapy turned out to be inefficient against tumors established in purinergic receptor P2rx7−/− or Nlrp3−/− or Casp1−/− hosts. Anthracycline-treated individuals with breast cancer carrying a loss-of-function allele of P2RX7 developed metastatic disease more rapidly than individuals bearing the normal allele. These results indicate that the NLRP3 inflammasome links the innate and adaptive immune responses against dying tumor cells.

Morin Attenuates Ovalbumin-Induced Airway Inflammation by Modulating Oxidative Stress-Responsive MAPK Signaling.

Asthma is one of the most common inflammatory diseases characterized by airway hyperresponsiveness, inflammation, and remodeling. Morin, an active ingredient obtained from Moraceae plants, has been demonstrated to have promising anti-inflammatory activities in a range of disorders. However, its impacts on pulmonary diseases, particularly on asthma, have not been clarified. This study was designed to investigate whether morin alleviates airway inflammation in chronic asthma with an emphasis on oxidative stress modulation. In vivo, ovalbumin- (OVA-) sensitized mice were administered with morin or dexamethasone before challenge. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained to perform cell counts, histological analysis, and enzyme-linked immunosorbent assay. In vitro, human bronchial epithelial cells (BECs) were challenged by tumor necrosis factor alpha (TNF-α). The supernatant was collected for the detection of the proinflammatory proteins, and the cells were collected for reactive oxygen species (ROS)/mitogen-activated protein kinase (MAPK) evaluations. Severe inflammatory responses and remodeling were observed in the airways of the OVA-sensitized mice. Treatment with morin dramatically attenuated the extensive trafficking of inflammatory cells into the BALF and inhibited their infiltration around the respiratory tracts and vessels. Morin administration also significantly suppressed goblet cell hyperplasia and collagen deposition/fibrosis and dose-dependently inhibited the OVA-induced increases in IgE, TNF-α, interleukin- (IL-) 4, IL-13, matrix metalloproteinase-9, and malondialdehyde. In human BECs challenged by TNF-α, the levels of proteins such as eotaxin-1, monocyte chemoattractant protein-1, IL-8 and intercellular adhesion molecule-1, were consistently significantly decreased by morin. Western blotting and the 2',7'-dichlorofluorescein assay revealed that the increases in intracellular ROS and MAPK phosphorylation were abolished by morin, implying that ROS/MAPK signaling contributes to the relief of airway inflammation. Our findings indicate for the first time that morin alleviates airway inflammation in chronic asthma, which probably occurs via the oxidative stress-responsive MAPK pathway, highlighting a novel profile of morin as a potent agent for asthma management.

Inducing Tumor Immunity through the Selective Engagement of Activating Fcγ Receptors on Dendritic Cells

Induction of tumor-specific immunity requires that dendritic cells (DCs) efficiently capture and present tumor antigens to result in the expansion and activation of tumor-specific cytotoxic T cells. The transition from antigen capture to T cell stimulation requires a maturation signal; in its absence tolerance, rather than immunity may develop. While immune complexes (ICs) are able to enhance antigen capture, they can be poor at inducing DC maturation, naive T cell activation and protective immunity. We now demonstrate that interfering with the inhibitory signal delivered by FcγRIIB on DCs converts ICs to potent maturation agents and results in T cell activation. Applying this approach to immunization with DCs pulsed ex-vivo with ICs, we have generated antigen-specific CD8+ T cells in vivo and achieved efficient protective immunity in a murine melanoma model. These data imply that ICs may normally function to maintain tolerance through the binding to inhibitory FcγRs on DCs, but they can be converted to potent immunogenic stimuli by selective engagement of activating FcγRs. This mechanism suggests a novel approach to the development of tumor vaccines.

Neutrophil elastase-mediated lung disease.

Elastases of both the neutrophil and macrophage have been implicated in lung disease initiation and progression. Although it is unlikely that these proteases evolved for the purpose of injuring lung tissue, the elastin-rich connective tissue framework of the lungs appears to be particularly susceptible to the action of elastolytic proteases. Assuming that neutrophil elastase most likely plays a role in the migration of neutrophils toward a site of inflammation and degradation of proteins from invading organisms or other products of the inflammatory response, it is the role of inhibitors of this protease to protect normal tissues from its effects. In alpha-1 antitrypsin deficiency we find an experiment of nature that disrupts this protease-anti-protease balance, resulting in an increased risk of destructive lung disease.

Epigenetic therapy activates type I interferon signaling in murine ovarian cancer to reduce immunosuppression and tumor burden.

Ovarian cancer is the most lethal of all gynecological cancers, and there is an urgent unmet need to develop new therapies. Epithelial ovarian cancer (EOC) is characterized by an immune suppressive microenvironment, and response of ovarian cancers to immune therapies has thus far been disappointing. We now find, in a mouse model of EOC, that clinically relevant doses of DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi, respectively) reduce the immune suppressive microenvironment through type I IFN signaling and improve response to immune checkpoint therapy. These data indicate that the type I IFN response is required for effective in vivo antitumorigenic actions of the DNMTi 5-azacytidine (AZA). Through type I IFN signaling, AZA increases the numbers of CD45+ immune cells and the percentage of active CD8+ T and natural killer (NK) cells in the tumor microenvironment, while reducing tumor burden and extending survival. AZA also increases viral defense gene expression in both tumor and immune cells, and reduces the percentage of macrophages and myeloid-derived suppressor cells in the tumor microenvironment. The addition of an HDACi to AZA enhances the modulation of the immune microenvironment, specifically increasing T and NK cell activation and reducing macrophages over AZA treatment alone, while further increasing the survival of the mice. Finally, a triple combination of DNMTi/HDACi plus the immune checkpoint inhibitor α-PD-1 provides the best antitumor effect and longest overall survival, and may be an attractive candidate for future clinical trials in ovarian cancer.

A CXCL1 Paracrine Network Links Cancer Chemoresistance and Metastasis

Metastasis and chemoresistance in cancer are linked phenomena, but the molecular basis for this link is unknown. We uncovered a network of paracrine signals between carcinoma, myeloid, and endothelial cells that drives both processes in breast cancer. Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites. CXCL1/2 attract CD11b(+)Gr1(+) myeloid cells into the tumor, which produce chemokines including S100A8/9 that enhance cancer cell survival. Although chemotherapeutic agents kill cancer cells, these treatments trigger a parallel stromal reaction leading to TNF-α production by endothelial and other stromal cells. TNF-α via NF-kB heightens the CXCL1/2 expression in cancer cells, thus amplifying the CXCL1/2-S100A8/9 loop and causing chemoresistance. CXCR2 blockers break this cycle, augmenting the efficacy of chemotherapy against breast tumors and particularly against metastasis. This network of endothelial-carcinoma-myeloid signaling interactions provides a mechanism linking chemoresistance and metastasis, with opportunities for intervention.

Radiation and Dual Checkpoint Blockade Activates Non-Redundant Immune Mechanisms in Cancer

Immune checkpoint inhibitors result in impressive clinical responses, but optimal results will require combination with each other and other therapies. This raises fundamental questions about mechanisms of non-redundancy and resistance. Here we report major tumour regressions in a subset of patients with metastatic melanoma treated with an anti-CTLA4 antibody (anti-CTLA4) and radiation, and reproduced this effect in mouse models. Although combined treatment improved responses in irradiated and unirradiated tumours, resistance was common. Unbiased analyses of mice revealed that resistance was due to upregulation of PD-L1 on melanoma cells and associated with T-cell exhaustion. Accordingly, optimal response in melanoma and other cancer types requires radiation, anti-CTLA4 and anti-PD-L1/PD-1. Anti-CTLA4 predominantly inhibits T-regulatory cells (Treg cells), thereby increasing the CD8 T-cell to Treg (CD8/Treg) ratio. Radiation enhances the diversity of the T-cell receptor (TCR) repertoire of intratumoral T cells. Together, anti-CTLA4 promotes expansion of T cells, while radiation shapes the TCR repertoire of the expanded peripheral clones. Addition of PD-L1 blockade reverses T-cell exhaustion to mitigate depression in the CD8/Treg ratio and further encourages oligoclonal T-cell expansion. Similarly to results from mice, patients on our clinical trial with melanoma showing high PD-L1 did not respond to radiation plus anti-CTLA4, demonstrated persistent T-cell exhaustion, and rapidly progressed. Thus, PD-L1 on melanoma cells allows tumours to escape anti-CTLA4-based therapy, and the combination of radiation, anti-CTLA4 and anti-PD-L1 promotes response and immunity through distinct mechanisms.

Matrix Remodeling Promotes Pulmonary Hypertension through Feedback Mechanoactivation of the YAP/TAZ-miR-130/301 Circuit.

Pulmonary hypertension (PH) is a deadly vascular disease with enigmatic molecular origins. We found that vascular extracellular matrix (ECM) remodeling and stiffening are early and pervasive processes that promote PH. In multiple pulmonary vascular cell types, such ECM stiffening induced the microRNA-130/301 family via activation of the co-transcription factors YAP and TAZ. MicroRNA-130/301 controlled a PPAR?-APOE-LRP8 axis, promoting collagen deposition and LOX-dependent remodeling and further upregulating YAP/TAZ via a mechanoactive feedback loop. In turn, ECM remodeling controlled pulmonary vascular cell crosstalk via such mechanotransduction, modulation of secreted vasoactive effectors, and regulation of associated microRNA pathways. In vivo, pharmacologic inhibition of microRNA-130/301, APOE, or LOX activity ameliorated ECM remodeling and PH. Thus, ECM remodeling, as controlled by the YAP/TAZ-miR-130/301 feedback circuit, is an early PH trigger and offers combinatorial therapeutic targets for this devastating disease.

Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function

Thyroid hormone (TH) is critical for the maintenance of cellular homeostasis during stress responses, but its role in lung fibrosis is unknown. Here we found that the activity and expression of iodothyronine deiodinase 2 (DIO2), an enzyme that activates TH, were higher in lungs from patients with idiopathic pulmonary fibrosis than in control individuals and were correlated with disease severity. We also found that Dio2-knockout mice exhibited enhanced bleomycin-induced lung fibrosis. Aerosolized TH delivery increased survival and resolved fibrosis in two models of pulmonary fibrosis in mice (intratracheal bleomycin and inducible TGF-β1). Sobetirome, a TH mimetic, also blunted bleomycin-induced lung fibrosis. After bleomycin-induced injury, TH promoted mitochondrial biogenesis, improved mitochondrial bioenergetics and attenuated mitochondria-regulated apoptosis in alveolar epithelial cells both in vivo and in vitro. TH did not blunt fibrosis in Ppargc1a- or Pink1-knockout mice, suggesting dependence on these pathways. We conclude that the antifibrotic properties of TH are associated with protection of alveolar epithelial cells and restoration of mitochondrial function and that TH may thus represent a potential therapy for pulmonary fibrosis.

CX3CR1 is required for airway inflammation by promoting T helper cell survival and maintenance in inflamed lung

Allergic asthma is a T helper type 2 (T(H)2)-dominated disease of the lung. In people with asthma, a fraction of CD4(+) T cells express the CX3CL1 receptor, CX3CR1, and CX3CL1 expression is increased in airway smooth muscle, lung endothelium and epithelium upon allergen challenge. Here we found that untreated CX3CR1-deficient mice or wild-type (WT) mice treated with CX3CR1-blocking reagents show reduced lung disease upon allergen sensitization and challenge. Transfer of WT CD4(+) T cells into CX3CR1-deficient mice restored the cardinal features of asthma, and CX3CR1-blocking reagents prevented airway inflammation in CX3CR1-deficient recipients injected with WT T(H)2 cells. We found that CX3CR1 signaling promoted T(H)2 survival in the inflamed lungs, and injection of B cell leukemia/lymphoma-2 protein (BCl-2)-transduced CX3CR1-deficient T(H)2 cells into CX3CR1-deficient mice restored asthma. CX3CR1-induced survival was also observed for T(H)1 cells upon airway inflammation but not under homeostatic conditions or upon peripheral inflammation. Therefore, CX3CR1 and CX3CL1 may represent attractive therapeutic targets in asthma.

Neutrophils Suppress Intraluminal NK Cell-Mediated Tumor Cell Clearance and Enhance Extravasation of Disseminated Carcinoma Cells.

UNLABELLED Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion-metastasis cascade remain poorly understood. Here, we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b(+)/Ly6G(+) neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL1β and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their cross-talk with host cells and disseminating carcinoma cells. SIGNIFICANCE This study provides important insights into the systemic contributions of neutrophils to cancer metastasis by identifying how neutrophils facilitate intermediate steps of the invasion-metastasis cascade. We demonstrate that neutrophils suppress natural killer cell activity and increase extravasation of tumor cells. Cancer Discov; 6(6); 630-49. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 561.

Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose.

In subunit vaccines, strong CD8(+) T-cell responses are desired, yet they are elusive at reasonable adjuvant doses. We show that targeting adjuvant to the lymph node (LN) via ultrasmall polymeric nanoparticles (NPs), which rapidly drain to the LN after intradermal injection, greatly enhances adjuvant efficacy at low doses. Coupling CpG-B or CpG-C oligonucleotides to NPs led to better dual-targeting of adjuvant and antigen (codelivered on separate NPs) in cross-presenting dendritic cells compared with free adjuvant. This led to enhanced dendritic cell maturation and T helper 1 (Th1)-cytokine secretion, in turn driving stronger effector CD8(+) T-cell activation with enhanced cytolytic profiles and, importantly, more powerful memory recall. With only 4 μg CpG, NP-CpG-B could substantially protect mice from syngeneic tumor challenge, even after 4 mo of vaccination, compared with free CpG-B. Together, these results show that nanocarriers can enhance vaccine efficacy at a low adjuvant dose for inducing potent and long-lived cellular immunity.

ImmTACs for targeted cancer therapy: Why, what, how, and which.

Overcoming immunosuppression and activating a cytotoxic T cell response has the potential to halt the progression of cancer and, in some circumstances, eradicate it. Designing therapeutic interventions that achieve this goal has proven challenging, but now a greater understanding of the complexities of immune responses is beginning to produce some notable breakthroughs. ImmTACs (immune-mobilising monoclonal TCRs against cancer) are a new class of bispecific reagents, based on soluble monoclonal T cell receptors, which have been engineered to possess extremely high affinity for cognate tumour antigen. In this way, ImmTACs overcome the problem of low affinity tumour-specific T cells imposed by thymic selection and provide access to the large number of antigens presented as peptide-HLA complexes. Once bound to tumour cells the anti-CD3 effector end of the ImmTAC drives recruitment of polyclonal T cells to the tumour site, leading to a potent redirected T cell response and tumour cell destruction. Extensive in vitro testing coupled with promising early clinical data has provided an enhanced appreciation of ImmTAC function in vivo and indicates their potential therapeutic benefit in terms of a durable response and ultimately the breaking of T cell tolerance. This review introduces ImmTACs in the context of immunotherapy, and outlines their design, construction and mechanism of action, as well as examining target selection and aspects of preclinical safety testing.

CCL2/monocyte chemoattractant protein-1 mediates enhanced transmigration of human immunodeficiency virus (HIV)-infected leukocytes across the blood-brain barrier: a potential mechanism of HIV-CNS invasion and NeuroAIDS.

Encephalitis and dementia associated with acquired immunodeficiency syndrome (AIDS) are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood-brain barrier (BBB) disruption, and eventual loss of neurons. Little is known about whether human immunodeficiency virus 1 (HIV-1) infection of leukocytes affects their ability to transmigrate in response to chemokines and to alter BBB integrity. We now demonstrate that HIV infection of human leukocytes results in their increased transmigration across our tissue culture model of the human BBB in response to the chemokine CCL2, as well as in disruption of the BBB, as evidenced by enhanced permeability, reduction of tight junction proteins, and expression of matrix metalloproteinases (MMP)-2 and MMP-9. HIV-infected cells added to our model did not transmigrate in the absence of CCL2, nor did this condition alter BBB integrity. The chemokines CXCL10/interferon-gamma-inducible protein of 10 kDa, CCL3/macrophage inflammatory protein-1alpha, or CCL5/RANTES (regulated on activation normal T-cell expressed and secreted) did not enhance HIV-infected leukocyte transmigration or BBB permeability. The increased capacity of HIV-infected leukocytes to transmigrate in response to CCL2 correlated with their increased expression of CCR2, the chemokine receptor for CCL2. These data suggest that CCL2, but not other chemokines, plays a key role in infiltration of HIV-infected leukocytes into the CNS and the subsequent pathology characteristic of NeuroAIDS.

Lung dendritic cells imprint T cell lung homing and promote lung immunity through the chemokine receptor CCR4

T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC-activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC-imprinted T cells protect against influenza more effectively than do gut and skin DC-imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC-activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC-activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4.

Combination cancer immunotherapies tailored to the tumour microenvironment

Evidence suggests that cancer immunotherapy will be a major part of the combination treatment plan for many patients with many cancer types in the near future. There are many types of immune processes involving different antitumour and tumour-promoting leucocytes, and tumour cells use many strategies to evade the immune response. The tumour microenvironment can help determine which immune suppressive pathways become activated to restrain antitumour immunity. This includes immune checkpoint receptors on effector T-cells and myeloid cells, and release of inhibitory cytokines and metabolites. Therapeutic approaches that target these pathways, particularly immune-checkpoint receptors, can induce durable antitumour responses in patients with advanced-stage cancers, including melanoma. Nevertheless, many patients do not have a good response to monotherapy approaches and alternative strategies are required to achieve optimal therapeutic benefit. These strategies include eliminating the bulk of tumour cells to provoke tumour-antigen release and antigen-presenting cell (APC) function, using adjuvants to enhance APC function, and using agents that enhance effector-cell activity. In this Review, we discuss the stratification of the tumour microenvironment according to tumour-infiltrating lymphocytes and PD-L1 expression in the tumour, and how this stratification enables the design of optimal combination cancer therapies tailored to target different tumour microenvironments.

Activation of liver X receptors and retinoid X receptors prevents bacterial-induced macrophage apoptosis.

Microbe-macrophage interactions play a central role in the pathogenesis of many infections. The ability of some bacterial pathogens to induce macrophage apoptosis has been suggested to contribute to their ability to elude innate immune responses and successfully colonize the host. Here, we provide evidence that activation of liver X receptors (LXRs) and retinoid X receptors (RXRs) inhibits apoptotic responses of macrophages to macrophage colony-stimulating factor (M-CSF) withdrawal and several inducers of apoptosis. In addition, combined activation of LXR and RXR protected macrophages from apoptosis caused by infection with Bacillus anthracis, Escherichia coli, and Salmonella typhimurium. Expression-profiling studies demonstrated that LXR and RXR agonists induced the expression of antiapoptotic regulators, including AIM/CT2, Bcl-X(L), and Birc1a. Conversely, LXR and RXR agonists inhibited expression of proapoptotic regulators and effectors, including caspases 1, 4/11, 7, and 12; Fas ligand; and Dnase1l3. The combination of LXR and RXR agonists was more effective than either agonist alone at inhibiting apoptosis in response to various inducers of apoptosis, and it acted synergistically to induce expression of AIM/CT2. Inhibition of AIM/CT2 expression in response to LXR/RXR agonists partially reversed their antiapoptotic effects. These findings reveal unexpected roles of LXRs and RXRs in the control of macrophage survival and raise the possibility that LXR/RXR agonists may be exploited to enhance innate immunity to bacterial pathogens that induce apoptotic programs as a strategy for evading host responses.

Calreticulin exposure dictates the immunogenicity of cancer cell death

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy.

Specific killing of multiple myeloma cells by (-)-epigallocatechin-3-gallate extracted from green tea: biologic activity and therapeutic implications.

Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, is an antioxidant with chemopreventive and chemotherapeutic actions. Based on its ability to modulate growth factor-mediated cell proliferation, we evaluated its efficacy in multiple myeloma (MM). EGCG induced both dose- and time-dependent growth arrest and subsequent apoptotic cell death in MM cell lines including IL-6-dependent cells and primary patient cells, without significant effect on the growth of peripheral blood mononuclear cells (PBMCs) and normal fibroblasts. Treatment with EGCG also led to significant apoptosis in human myeloma cells grown as tumors in SCID mice. EGCG interacts with the 67-kDa laminin receptor 1 (LR1), which is significantly elevated in myeloma cell lines and patient samples relative to normal PBMCs. RNAi-mediated inhibition of LR1 resulted in abrogation of EGCG-induced apoptosis in myeloma cells, indicating that LR1 plays an important role in mediating EGCG activity in MM while sparing PBMCs. Evaluation of changes in gene expression profile indicates that EGCG treatment activates distinct pathways of growth arrest and apoptosis in MM cells by inducing the expression of death-associated protein kinase 2, the initiators and mediators of death receptor-dependent apoptosis (Fas ligand, Fas, and caspase 4), p53-like proteins (p73, p63), positive regulators of apoptosis and NF-kappaB activation (CARD10, CARD14), and cyclin-dependent kinase inhibitors (p16 and p18). Expression of related genes at the protein level were also confirmed by Western blot analysis. These data demonstrate potent and specific antimyeloma activity of EGCG and provide the rationale for its clinical evaluation.

A local macrophage chemokine network sustains protective tissue-resident memory CD4 T cells

CD8 tissue-resident memory T (TRM) cells provide efficient local control of viral infection, but the role of CD4 TRM is less clear. Here, by using parabiotic mice, we show that a preexisting pool of CD4 TRM cells in the genital mucosa was required for full protection from a lethal herpes simplex virus 2 (HSV-2) infection. Chemokines secreted by a local network of macrophages maintained vaginal CD4 TRM in memory lymphocyte clusters (MLCs), independently of circulating memory T cells. CD4 TRM cells within the MLCs were enriched in clones that expanded in response to HSV-2. Our results highlight the need for vaccine strategies that enable establishment of TRM cells for protection from a sexually transmitted virus and provide insights as to how such a pool might be established.

Chemosensitization of acute myeloid leukemia (AML) following mobilization by the CXCR4 antagonist AMD3100.

The CXCR4-SDF-1 axis plays a central role in the trafficking and retention of normal and malignant stem cells in the bone marrow (BM) microenvironment. Here, we used a mouse model of acute promyelocytic leukemia (APL) and a small molecule competitive antagonist of CXCR4, AMD3100, to examine the interaction of mouse APL cells with the BM microenvironment. APL cells from a murine cathepsin G-PML-RARalpha knockin mouse were genetically modified with firefly luciferase (APL(luc)) to allow tracking by bioluminescence imaging. Coculture of APL(luc) cells with M2-10B4 stromal cells protected the leukemia cells from chemotherapy-induced apoptosis in vitro. Upon injection into syngeneic recipients, APL(luc) cells rapidly migrated to the BM followed by egress to the spleen then to the peripheral blood with death due to leukostasis by day 15. Administration of AMD3100 to leukemic mice induced a 1.6-fold increase in total leukocytes and a 9-fold increase of circulating APL blast counts, which peak at 3 hours and return to baseline by 12 hours. Treatment of leukemic mice with chemotherapy plus AMD3100 resulted in decreased tumor burden and improved overall survival compared with mice treated with chemotherapy alone. These studies provide a proof-of-principle for directing therapy to the critical tethers that promote AML-niche interactions.

Alkylating agent melphalan augments the efficacy of adoptive immunotherapy using tumor-specific CD4+ T cells.

In recent years, the immune-potentiating effects of some widely used chemotherapeutic agents have been increasingly appreciated. This provides a rationale for combining conventional chemotherapy with immunotherapy strategies to achieve durable therapeutic benefits. Previous studies have implicated the immunomodulatory effects of melphalan, an alkylating agent commonly used to treat multiple myeloma, but the underlying mechanisms remain obscure. In the present study, we investigated the impact of melphalan on endogenous immune cells as well as adoptively transferred tumor-specific CD4(+) T cells in tumor-bearing mice. We showed that melphalan treatment resulted in a rapid burst of inflammatory cytokines and chemokines during the cellular recovery phase after melphalan-induced myelodepletion and leukodepletion. After melphalan treatment, tumor cells exhibited characteristics of immunogenic cell death, including membrane translocation of the endoplasmic reticulum-resident calreticulin and extracellular release of high-mobility group box 1. Additionally, there was enhanced tumor Ag uptake by dendritic cells in the tumor-draining lymph node. Consistent with these immunomodulatory effects, melphalan treatment of tumor-bearing mice led to the activation of the endogenous CD8(+) T cells and, more importantly, effectively drove the clonal expansion and effector differentiation of adoptively transferred tumor-specific CD4(+) T cells. Notably, the combination of melphalan and CD4(+) T cell adoptive cell therapy was more efficacious than either treatment alone in prolonging the survival of mice with advanced B cell lymphomas or colorectal tumors. These findings provide mechanistic insights into melphalan's immunostimulatory effects and demonstrate the therapeutic potential of combining melphalan with adoptive cell therapy utilizing antitumor CD4(+) T cells.

Cancer-associated fibroblasts promote hepatocellular carcinoma metastasis through chemokine-activated hedgehog and TGF-β pathways.

Fibroblasts are rich in the surrounding microenvironment of hepatocellular carcinoma (HCC) because most HCCs occur in fibrotic or cirrhotic livers. However, the role of cancer-associated fibroblasts (CAFs) in HCC metastasis remains obscure. Here, we reported that CAFs promote the migration and invasion of HCC cells in vitro and facilitate the HCC metastasis to the bone, brain and lung in NOD/SCID mice. The RayBio human chemokine antibody array revealed that CAFs secret higher levels of CCL2, CCL5, CCL7 and CXCL16 than peri-tumor fibroblasts. CCL2 and CCL5 increase the migration but not the invasion of HCC cells, while CCL7 and CXCL16 promote both migration and invasion of HCC cells. Moreover, CCL2 and CCL5 stimulate the activation of the hedgehog (Hh) pathway, while CCL7 and CXCL16 enhance the activity of the transforming growth factor-β (TGF-β) pathway in HCC cells. The neutralizing antibodies of chemokines notably attenuate the effect of CAFs on HCC metastasis and compromised the activation of Hh and TGF-β pathways in HCC cells. In summary, CAF-secreted CCL2, CCL5, CCL7 and CXCL16 promote HCC metastasis through the coordinate activation of Hh and TGF-β pathways in HCC cells.

Disruption of the immune-checkpoint VISTA gene imparts a proinflammatory phenotype with predisposition to the development of autoimmunity.

V domain-containing Ig suppressor of T-cell activation (VISTA) is a negative checkpoint regulator that suppresses T cell-mediated immune responses. Previous studies using a VISTA-neutralizing monoclonal antibody show that VISTA blockade enhances T-cell activation. The current study describes a comprehensive characterization of mice in which the gene for VISTA has been deleted. Despite the apparent normal hematopoietic development in young mice, VISTA genetic deficiency leads to a gradual accumulation of spontaneously activated T cells, accompanied by the production of a spectrum of inflammatory cytokines and chemokines. Enhanced T-cell responsiveness was also observed upon immunization with neoantigen. Despite the presence of multiorgan chronic inflammation, aged VISTA-deficient mice did not develop systemic or organ-specific autoimmune disease. Interbreeding of the VISTA-deficient mice with 2D2 T-cell receptor transgenic mice, which are predisposed to the development of experimental autoimmune encephalomyelitis, drastically enhanced disease incidence and intensity. Disease development is correlated with the increase in the activation of encephalitogenic T cells in the periphery and enhanced infiltration into the CNS. Taken together, our data suggest that VISTA is a negative checkpoint regulator whose loss of function lowers the threshold for T-cell activation, allowing for an enhanced proinflammatory phenotype and an increase in the frequency and intensity of autoimmunity under susceptible conditions.

Ketamine reduces LPS-induced HMGB1 via activation of the Nrf2/HO-1 pathway and NF-κB suppression.

BACKGROUND Ketamine, as an anesthetic agent, has an anti-inflammatory effect. In the present study, we investigated whether ketamine inhibits release of high mobility group box 1 (HMGB1), a late-phase cytokine of sepsis, in lipopolysaccharide (LPS)-stimulated macrophages through heme oxygenase-1 (HO-1) induction. METHODS Macrophages were preincubated with various concentrations of ketamine and then treated with LPS (1 μg/mL). The cell culture supernatants were collected to measure inflammatory mediators (HMGB1, nitric oxide, tumor necrosis factor-α, and interleukin 1β) by enzyme-linked immunosorbent assay. Moreover, HO-1 protein expression, the phosphorylation and degradation of IκB-α, and the nuclear translocation of nuclear factor E2-related factor 2 and nuclear factor κB (NF-κB) p65 were tested by Western blot analysis. In addition, to further identify the role of HO-1 in this process, tin protoporphyrin (SnPP), an HO-1 inhibitor, was used. RESULTS Ketamine treatment dose-dependently attenuated the increased levels of proinflammatory mediators (HMGB1, nitric oxide, tumor necrosis factor α, and interleukin 1β) and increased the HO-1 protein expression in LPS-activated macrophages. Furthermore, ketamine suppressed the phosphorylation and degradation of IκB-α as well as the LPS-stimulated nuclear translocation of NF-κB p65 in macrophages. In addition, the present study also demonstrated that ketamine induced HO-1 expression through the nuclear translocation of nuclear factor E2-related factor 2 in macrophages. The effects of ketamine on LPS-induced proinflammatory cytokines production were partially reversed by the HO inhibitor tin protoporphyrin (SnPP). CONCLUSION Ketamine inhibits the release of HMGB1 in LPS-stimulated macrophages, and this effect is at least partly mediated by the activation of the Nrf2/HO-1 pathway and NF-κB suppression.

CD4+ T cell-mediated cytotoxicity eliminates primary tumor cells in metastatic melanoma through high MHC class II expression and can be enhanced by inhibitory receptor blockade

Metastatic melanoma is a rapidly progressing disease with high mortality rate and limited treatment options. Immunotherapy based on tumor-targeting cytotoxic T cell responses represents a promising strategy. To assist in its development, we examined the possibility and efficacy of using CD4+ cytotoxic T cells. The regulatory mechanisms controlling CD4+ T cell-mediated cytotoxicity were also investigated. We found that naturally occurring granzyme B and perforin-expressing CD4+ cytotoxic T cells can be recovered from metastatic melanoma patients at significantly elevated frequencies compared to those from healthy controls. These CD4+ cytotoxic T cells were also capable of killing autologous tumor cells harvested from metastatic melanoma, independent of CD8+ T cells or any other cell types. However, several restricting factors were observed. First, the cytolytic activity by CD4+ T cells required high MHC class II expression on melanoma cells, which was not satisfied in a subset of melanomas. Second, the granzyme B and perforin release by activated CD4+ cytotoxic T cells was reduced after coculturing with autologous melanoma cells, characterized by low LAMP-1 expression and low granzyme B and perforin secretion in the supernatant. This suggested that inhibitory mechanisms were present to suppress CD4+ cytotoxic T cells. Indeed, blockade of PD-1 and CTLA-4 had increased the cytolytic activity of CD4+ T cells but was only effective in MHC class II high but not MHC class II low melanomas. Together, our study showed that CD4+ T cell-mediated cytotoxicity could eliminate primary melanoma cells but the efficacy depended on MHC class II expression.

CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein–coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase–independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

Immune activation and HIV persistence: implications for curative approaches to HIV infection.

Despite complete or near-complete suppression of human immunodeficiency virus (HIV) replication with combination antiretroviral therapy, both HIV and chronic inflammation/immune dysfunction persist indefinitely. Untangling the association between the virus and the host immune environment during therapy might lead to novel interventions aimed at either curing the infection or preventing the development of inflammation-associated end-organ disease. Chronic inflammation and immune dysfunction might lead to HIV persistence by causing virus production, generating new target cells, enabling infecting of activated and resting target cells, altering the migration patterns of susceptible target cells, increasing the proliferation of infected cells, and preventing normal HIV-specific clearance mechanisms from function. Chronic HIV production or replication might contribute to persistent inflammation and immune dysfunction. The rapidly evolving data on these issues strongly suggest that a vicious cycle might exist in which HIV persistence causes inflammation that in turn contributes to HIV persistence.

Induction of stem-like cells with malignant properties by chronic exposure of human lung epithelial cells to single-walled carbon nanotubes

BACKGROUND Carbon nanotubes (CNT) hold great promise to create new and better products for commercial and biomedical applications, but their long-term adverse health effects are a major concern. The objective of this study was to address human lung cancer risks associated with chronic pulmonary exposure to single-walled (SW) CNT through the fundamental understanding of cellular and molecular processes leading to carcinogenesis. We hypothesized that the acquisition of cancer stem cells (CSC), a subpopulation that drive tumor initiation and progression, may contribute to CNT carcinogenesis. METHODS Non-tumorigenic human lung epithelial cells were chronically exposed to well-dispersed SWCNT for a period of 6 months at the physiologically relevant concentration of 0.02 μg/cm2 surface area dose. Chronic SWCNT-exposed cells were evaluated for the presence of CSC-like cells under CSC-selective conditions of tumor spheres and side population (SP). CSC-like cells were isolated using fluorescence-activated cell sorting and were assessed for aggressive behaviors, including acquired apoptosis resistance and increased cell migration and invasion in vitro, and tumor-initiating capability in vivo. Non-small cell lung cancer cells served as a positive control. RESULTS We demonstrated for the first time the existence of CSC-like cells in all clones of chronic SWCNT-exposed lung epithelial cells. These CSC-like cells, in contrary to their non-CSC counterpart, possessed all biological features of lung CSC that are central to irreversible malignant transformation, self-renewal, aggressive cancer behaviors, and in vivo tumorigenesis. These cells also displayed aberrant stem cell markers, notably Nanog, SOX-2, SOX-17 and E-cadherin. Restored expression of tumor suppressor p53 abrogated CSC properties of CSC-like cells. Furthermore, we identified specific stem cell surface markers CD24low and CD133high that are associated with SWCNT-induced CSC formation and tumorigenesis. CONCLUSIONS Our findings provide new and compelling evidence for the acquisition of CSC-like cells induced by chronic SWCNT exposure, which are likely to be a major driving force for SWCNT tumorigenesis. Thus, our study supports prudent adoption of prevention strategies and implementation of exposure control for SWCNT. We also suggest that the detection of CSC and associated surface markers may provide an effective screening tool for prediction of the carcinogenic potential of SWCNT and related nanoparticles.

Nuclear Factor-�B Affects Tumor Progression in a Mouse Model of Malignant Pleural Effusion

We developed a novel mouse model of malignant pleural effusion (MPE) by injecting Lewis lung cancer (LLC) cells directly into the pleural space of syngeneic C57B/6 mice. The pleural effusions in this model share common cellular and biochemical features with human MPEs. Implantation and growth of pleural tumors triggers a host inflammatory response characterized by a mixed inflammatory cell influx into the pleural fluid. LLC cells exhibited high basal nuclear factor (NF)-κB activity in vitro and in vivo, which we used to drive expression of a NF-κB–dependent green fluorescent protein-firefly luciferase fusion reporter construct. NF-κB–dependent reporter expression allowed intravital tracing of pleural tumors. Inhibition of NF-κB in LLC cells did not affect cell viability in culture; however, injection of LLC cells expressing a dominant NF-κB inhibitor resulted in decreased tumor burden, decreased pleural effusion volume, and decreased pleural effusion TNF-α levels. These studies indicate that tumor NF-κB a...

Strong association between respiratory viral infection early after hematopoietic stem cell transplantation and the development of life-threatening acute and chronic alloimmune lung syndromes.

Alloimmune lung syndromes (allo-LS), including idiopathic pneumonia syndrome, bronchiolitis obliterans syndrome, and bronchiolitis obliterans organizing pneumonia, are severe complications after hematopoietic stem cell transplantation (HSCT). In our cohort of 110 pediatric patients, 30 had allo-LS (27.3%), 18 with idiopathic pneumonia syndrome and 12 with bronchiolitis obliterans syndrome. Multivariate analysis showed that respiratory viral infection early after HSCT is an important predictor for the development of allo-LS (P <.0001). This was true for all viruses tested. In multivariate analysis, allo-LS was the only predictor for higher mortality (P = .04). Paradoxically, prolonged administration of immunosuppressive agents because of acute graft-versus-host disease had a protective effect on the development of allo-LS (P = .004). We hypothesize that early infection of the respiratory tract with a common cold virus makes the lungs a target for alloimmunity.

CD28 and ITK signals regulate autoreactive T cell trafficking

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

Guidance of B cells by the orphan G protein-coupled receptor EBI2 shapes humoral immune responses.

Humoral immunity depends on both rapid and long-term antibody production against invading pathogens. This is achieved by the generation of spatially distinct extrafollicular plasmablast and follicular germinal center (GC) B cell populations, but the signals that guide responding B cells to these alternative compartments have not been fully elucidated. Here, we show that expression of the orphan G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) by activated B cells was essential for their movement to extrafollicular sites and induction of early plasmablast responses. Conversely, downregulation of EBI2 enabled B cells to access the center of follicles and promoted efficient GC formation. EBI2 therefore provides a previously uncharacterized dimension to B cell migration that is crucial for coordinating rapid versus long-term antibody responses.

Oncogenic mTOR signaling recruits myeloid-derived suppressor cells to promote tumor initiation

Myeloid-derived suppressor cells (MDSCs) play critical roles in primary and metastatic cancer progression. MDSC regulation is widely variable even among patients harbouring the same type of malignancy, and the mechanisms governing such heterogeneity are largely unknown. Here, integrating human tumour genomics and syngeneic mammary tumour models, we demonstrate that mTOR signalling in cancer cells dictates a mammary tumour's ability to stimulate MDSC accumulation through regulating G-CSF. Inhibiting this pathway or its activators (for example, FGFR) impairs tumour progression, which is partially rescued by restoring MDSCs or G-CSF. Tumour-initiating cells (TICs) exhibit elevated G-CSF. MDSCs reciprocally increase TIC frequency through activating Notch in tumour cells, forming a feedforward loop. Analyses of primary breast cancers and patient-derived xenografts corroborate these mechanisms in patients. These findings establish a non-canonical oncogenic role of mTOR signalling in recruiting pro-tumorigenic MDSCs and show how defined cancer subsets may evolve to promote and depend on a distinct immune microenvironment.

Tissue-specific alterations in the glucocorticoid sensitivity of immune cells following repeated social defeat in mice

Endogenous glucocorticoids (GC) play an important role in the termination of the inflammatory response following infection and tissue injury. However, recent findings indicate that stress can impair the anti-inflammatory capacities of these hormones. Lipopolysaccharide (LPS)-stimulated splenocytes of mice that were repeatedly subjected to social disruption (SDR) stress were less sensitive to the immunosuppressive effects of corticosterone (CORT) as demonstrated by an increased production of pro-inflammatory cytokines and enhanced cell survival. Myeloid cells expressing the marker CD11b were shown to play a key role in this process. Here we investigated the role of the bone marrow as a potential source of the GC-insensitive cells. The study revealed that LPS-stimulated bone marrow cells, in the absence of experimental stress, were virtually GC-resistant and retained high levels of cell viability after treatment with CORT. Recurrent exposure to the acute stressor over a period of 2, 4 or 6 days led to an increase in the GC sensitivity of the bone marrow cells. This increase in GC sensitivity was associated with enhanced mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), an increase in the number of myeloid progenitors, and a decrease in the proportion of mature CD11b+ cells. The changes in the cellular composition of the bone marrow were accompanied by an increase in splenic CD11b+ cell numbers. Simultaneous assessment of the GC sensitivity in bone marrow and spleen revealed a significant negative correlation between both tissues suggesting that social stress causes the redistribution of GC-insensitive myeloid cells from the bone marrow to the spleen.

Metformin reverses established lung fibrosis in a bleomycin model

Fibrosis is a pathological result of a dysfunctional repair response to tissue injury and occurs in a number of organs, including the lungs1. Cellular metabolism regulates tissue repair and remodelling responses to injury2-4. AMPK is a critical sensor of cellular bioenergetics and controls the switch from anabolic to catabolic metabolism5. However, the role of AMPK in fibrosis is not well understood. Here, we demonstrate that in humans with idiopathic pulmonary fibrosis (IPF) and in an experimental mouse model of lung fibrosis, AMPK activity is lower in fibrotic regions associated with metabolically active and apoptosis-resistant myofibroblasts. Pharmacological activation of AMPK in myofibroblasts from lungs of humans with IPF display lower fibrotic activity, along with enhanced mitochondrial biogenesis and normalization of sensitivity to apoptosis. In a bleomycin model of lung fibrosis in mice, metformin therapeutically accelerates the resolution of well-established fibrosis in an AMPK-dependent manner. These studies implicate deficient AMPK activation in non-resolving, pathologic fibrotic processes, and support a role for metformin (or other AMPK activators) to reverse established fibrosis by facilitating deactivation and apoptosis of myofibroblasts.

Suppressor of cytokine signaling 3 inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages.

HIV-1 replication within macrophages of the CNS often results in cognitive and motor impairment, which is known as HIV-associated dementia (HAD) in its most severe form. IFN-beta suppresses viral replication within these cells during early CNS infection, but the effect is transient. HIV-1 eventually overcomes this protective innate immune response to resume replication through an unknown mechanism, initiating the progression toward HAD. In this article, we show that Suppressor of Cytokine Signaling (SOCS)3, a molecular inhibitor of IFN signaling, may allow HIV-1 to evade innate immunity within the CNS. We found that SOCS3 is elevated in an in vivo SIV/macaque model of HAD and that the pattern of expression correlates with recurrence of viral replication and onset of CNS disease. In vitro, the HIV-1 regulatory protein transactivator of transcription induces SOCS3 in human and murine macrophages in a NF-kappaB-dependent manner. SOCS3 expression attenuates the response of macrophages to IFN-beta at proximal levels of pathway activation and downstream antiviral gene expression and consequently overcomes the inhibitory effect of IFN-beta on HIV-1 replication. These studies indicate that SOCS3 expression, induced by stimuli present in the HIV-1-infected brain, such as transactivator of transcription, inhibits antiviral IFN-beta signaling to enhance HIV-1 replication in macrophages. This consequence of SOCS3 expression in vitro, supported by a correlation with increased viral load and onset of CNS disease in vivo, suggests that SOCS3 may allow HIV-1 to evade the protective innate immune response within the CNS, allowing the recurrence of viral replication and, ultimately, promoting progression toward HAD.

Interleukin-17 mRNA expression in blood and CSF mononuclear cells is augmented in multiple sclerosis.

Myelin-directed autoimmunity is considered to play a key role in the pathogenesis of multiple sclerosis (MS). Increased production of both pro- and anti-inflammatory cytokines is a common finding in MS. Interleukin-17 (IL-17) is a recently described cytokine produced in humans almost exclusively by activated memory T cells, which can induce the production of proinflammatory cytokines and chemokines from parenchymal cells and macrophages. In situ hybridisation with synthetic oligonucleotide probes was adopted to detect and enumerate IL-17 mRNA expressing mononuclear cells (MNC) in blood and cerebrospinal fluid (CSF) from patients with MS and control individuals. Numbers of IL-17 mRNA expressing blood MNC were higher in patients with MS and acute aseptic meningoencephalitis (AM) compared to healthy individuals. Higher numbers of IL-17 mRNA expressing blood MNC were detected in MS patients examined during clinical exacerbation compared to remission. Patients with MS had higher numbers of IL-17 mRNA expressing MNC in CSF compared to blood. This increase in numbers of IL-17 mRNA expressing MNC in CSF was not observed in patients with AM. Our results thus demonstrate increased numbers of IL-17 mRNA expressing MNC in MS with higher numbers in CSF than blood, and with the highest numbers in blood during clinical exacerbations.

Ablation and regeneration of tolerance-inducing medullary thymic epithelial cells after cyclosporine, cyclophosphamide, and dexamethasone treatment.

Immunosuppressive drugs and cytotoxic chemotherapy agents are designed to kill or suppress autoreactive, alloaggressive, or hyperinflammatory T cells, or disseminated malignancies. However, they also cause severe immunological side effects ranging from interrupted thymopoiesis and general immunodeficiency to, paradoxically, autoimmunity. Consistent with the cross-talk between thymocytes and stromal cells, we now show that these common therapeutic agents have major effects on murine thymic epithelial cells (TEC), crucially required to rebuild immunity posttreatment. We show that the immunosuppressant cyclosporine A, which has been linked to a thymus-dependent autoimmune syndrome in some patients, causes extensive loss of autoimmune regulator (Aire(+)) tolerance-inducing MHC class II(high) medullary TEC (mTEC(high)). Post-cyclosporine A, Aire expression was restored within 7 days. Full recovery of the mTEC(high) subset occurred within 10 days and was linked to a decrease in a relatively resistant MHC class II(low) mTEC subset (mTEC(low)), consistent with a previously described precursor-product relationship. Cyclophosphamide and dexamethasone caused more extensive ablation of thymocytes and stromal cells but again severely depleted tolerance-inducing mTEC(high). Together, these data show that Aire(+) mTECs are highly sensitive to damage and that mTEC regeneration follows a conserved pattern regardless of the treatment regimen used.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells.

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity

Patients with asthma, a major public health problem, are at high risk for serious disease from influenza virus infection, but the pathogenic mechanisms by which influenza A causes airway disease and asthma are not fully known. We show here in a mouse model that influenza infection acutely induced airway hyper-reactivity (AHR), a cardinal feature of asthma, independently of T helper type 2 (TH2) cells and adaptive immunity. Instead, influenza infection induced AHR through a previously unknown pathway that required the interleukin 13 (IL-13)–IL-33 axis and cells of the non-T cell, non-B cell innate lymphoid type called 'natural helper cells'. Infection with influenza A virus, which activates the NLRP3 inflammasome, resulted in much more production of IL-33 by alveolar macrophages, which in turn activated natural helper cells producing substantial IL-13.

IL-17 promotes tumor development through the induction of tumor promoting microenvironments at tumor sites and myeloid-derived suppressor cells.

The role of immune responses in tumor development is a central issue for tumor biology and immunology. IL-17 is an important cytokine for inflammatory and autoimmune diseases. Although IL-17-producing cells are detected in cancer patients and tumor-bearing mice, the role of IL-17 in tumor development is controversial, and mechanisms remain to be fully elucidated. In the current study, we found that the development of tumors was inhibited in IL-17R-deficient mice. A defect in IFN-gammaR increased tumor growth, whereas tumor growth was inhibited in mice that were deficient in both IL-17R and IFN-gammaR compared with wild-type animals. Further experiments showed that neutralization of IL-17 by Abs inhibited tumor growth in wild-type mice, whereas systemic administration of IL-17 promoted tumor growth. The IL-17R deficiency increased CD8 T cell infiltration, whereas it reduced the infiltration of myeloid-derived suppressor cells (MDSCs) in tumors. In contrast, administration of IL-17 inhibited CD8 T cell infiltration and increased MDSCs in tumors. Further analysis indicated that IL-17 was required for the development and tumor-promoting activity of MDSCs in tumor-bearing mice. These data demonstrate that IL-17-mediated responses promote tumor development through the induction of tumor-promoting microenvironments at tumor sites. IL-17-mediated regulation of MDSCs is a primary mechanism for its tumor-promoting effects. The study provides novel insights into the role of IL-17 in tumor development and has major implications for targeting IL-17 in treatment of tumors.

Key questions about the checkpoint blockade-are microRNAs an answer?

The introduction of immune-checkpoint blockade in the cancer therapy led to a paradigm change of the management of late stage cancers. There are already multiple FDA approved checkpoint inhibitors and many other agents are undergoing phase 2 and early phase 3 clinical trials. The therapeutic indication of immune checkpoint inhibitors expanded in the last years, but still remains unclear who can benefit. MicroRNAs are small RNAs with no coding potential. By complementary pairing to the 3' untranslated region of messenger RNA, microRNAs exert posttranscriptional control of protein expression. A network of microRNAs directly and indirectly controls the expression of checkpoint receptors and several microRNAs can target multiple checkpoint molecules, mimicking the therapeutic effect of a combined immune checkpoint blockade. In this review, we will describe the microRNAs that control the expression of immune checkpoints and we will present four specific issues of the immune checkpoint therapy in cancer: (1) imprecise therapeutic indication, (2) difficult response evaluation, (3) numerous immunologic adverse-events, and (4) the absence of response to immune therapy. Finally, we propose microRNAs as possible solutions for these pitfalls. We consider that in the near future microRNAs could become important therapeutic partners of the immune checkpoint therapy.

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice

Whilst data recognise both myeloid cell accumulation during choroidal neovascularisation (CNV) as well as complement activation, none of the data has presented a clear explanation for the angiogenic drive that promotes pathological angiogenesis. One possibility that is a pre-eminent drive is a specific and early conditioning and activation of the myeloid cell infiltrate. Using a laser-induced CNV murine model, we have identified that disruption of retinal pigment epithelium (RPE) and Bruch's membrane resulted in an early recruitment of macrophages derived from monocytes and microglia, prior to angiogenesis and contemporaneous with lesional complement activation. Early recruited CD11b(+) cells expressed a definitive gene signature of selective inflammatory mediators particularly a pronounced Arg-1 expression. Accumulating macrophages from retina and peripheral blood were activated at the site of injury, displaying enhanced VEGF expression, and notably prior to exaggerated VEGF expression from RPE, or earliest stages of angiogenesis. All of these initial events, including distinct VEGF (+) Arg-1(+) myeloid cells, subsided when CNV was established and at the time RPE-VEGF expression was maximal. Depletion of inflammatory CCR2-positive monocytes confirmed origin of infiltrating monocyte Arg-1 expression, as following depletion Arg-1 signal was lost and CNV suppressed. Furthermore, our in vitro data supported a myeloid cell uptake of damaged RPE or its derivatives as a mechanism generating VEGF (+) Arg-1(+) phenotype in vivo. Our results reveal a potential early driver initiating angiogenesis via myeloid-derived VEGF drive following uptake of damaged RPE and deliver an explanation of why CNV develops during any of the stages of macular degeneration and can be explored further for therapeutic gain.

CNS repair requires both effector and regulatory T cells with distinct temporal and spatial profiles.

Monocyte-derived macrophages (mo-MΦs) and T cells have been shown to contribute to spinal cord repair. Recently, the remote brain choroid plexus epithelium (CP) was identified as a portal for monocyte recruitment, and its activation for leukocyte trafficking was found to be IFN-γ-dependent. Here, we addressed how the need for effector T cells can be reconciled with the role of inflammation-resolving immune cells in the repair process. Using an acute spinal cord injury model, we show that in mice deficient in IFN-γ-producing T cells, the CP was not activated, and recruitment of inflammation-resolving mo-MΦ to the spinal cord parenchyma was limited. We further demonstrate that mo-MΦ locally regulated recruitment of thymic-derived Foxp3(+) regulatory T (Treg) cells to the injured spinal cord parenchyma at the subacute/chronic phase. Importantly, an ablation protocol that resulted in reduced Tregs at this stage interfered with tissue remodeling, in contrast to Treg transient ablation, restricted to the 4 d period before the injury, which favored repair. The enhanced functional recovery observed following such a controlled decrease of Tregs suggests that reduced systemic immunosuppression at the time of the insult can enhance CNS repair. Overall, our data highlight a dynamic immune cell network needed for repair, acting in discrete compartments and stages, and involving effector and regulatory T cells, interconnected by mo-MΦ. Any of these populations may be detrimental to the repair process if their level or activity become dysregulated. Accordingly, therapeutic interventions must be both temporally and spatially controlled.

Loss of immune escape mutations during persistent HCV infection in pregnancy enhances replication of vertically transmitted viruses

Globally, about 1% of pregnant women are persistently infected with the hepatitis C virus (HCV). Mother-to-child transmission of HCV occurs in 3-5% of pregnancies and accounts for most new childhood infections. HCV-specific CD8(+) cytotoxic T lymphocytes (CTLs) are vital in the clearance of acute HCV infections, but in the 60-80% of infections that persist, these cells become functionally exhausted or select for mutant viruses that escape T cell recognition. Increased HCV replication during pregnancy suggests that maternofetal immune tolerance mechanisms may further impair HCV-specific CTLs, limiting their selective pressure on persistent viruses. To assess this possibility, we characterized circulating viral quasispecies during and after consecutive pregnancies in two women. This revealed a loss of some escape mutations in HLA class I epitopes during pregnancy that was associated with emergence of more fit viruses. CTL selective pressure was reimposed after childbirth, at which point escape mutations in these epitopes again predominated in the quasispecies and viral load dropped sharply. Importantly, the viruses transmitted perinatally were those with enhanced fitness due to reversion of escape mutations. Our findings indicate that the immunoregulatory changes of pregnancy reduce CTL selective pressure on HCV class I epitopes, thereby facilitating vertical transmission of viruses with optimized replicative fitness.

BCL-6 represses genes that function in lymphocyte differentiation, inflammation, and cell cycle control.

BCL-6, a transcriptional repressor frequently translocated in lymphomas, regulates germinal center B cell differentiation and inflammation. DNA microarray screening identified genes repressed by BCL-6, including many lymphocyte activation genes, suggesting that BCL-6 modulates B cell receptor signals. BCL-6 repression of two chemokine genes, MIP-1alpha and IP-10, may also attenuate inflammatory responses. Blimp-1, another BCL-6 target, is important for plasmacytic differentiation. Since BCL-6 expression is silenced in plasma cells, repression of blimp-1 by BCL-6 may control plasmacytic differentiation. Indeed, inhibition of BCL-6 function initiated changes indicative of plasmacytic differentiation, including decreased expression of c-Myc and increased expression of the cell cycle inhibitor p27kip1. These data suggest that malignant transformation by BCL-6 involves inhibition of differentiation and enhanced proliferation.

Role of the CXCR4/SDF-1 chemokine axis in circulating neutrophil homeostasis.

The bone marrow is the primary site for neutrophil production and release into the circulation. Because the CXC chemokine receptor-4/stromal derived factor-1 (CXCR4/SDF-1) axis plays a central role in the interactions of hematopoietic stem cells, lymphocytes, and developing neutrophils in the marrow, we investigated whether reciprocal CXCR4-dependent mechanisms might be involved in neutrophil release and subsequent return to the marrow following circulation. Neutralizing antibody to CXCR4 reduced marrow retention of infused neutrophils (45.7% +/- 0.5% to 6.9% +/- 0.5%) and was found to mobilize neutrophils from marrow (34.4% +/- 4.4%). Neutrophil CXCR4 expression and SDF-1-induced calcium flux decreased with maturation and activation of the cells, corresponding to the decreased marrow homing associated with these characteristics in vivo. Infusion of the inflammatory mediator and CXCR2 ligand KC led to mobilization of neutrophils from marrow by itself and was augmented 3-fold by low doses of CXCR4-blocking antibody that otherwise had no mobilizing effect. Examination of KC and SDF-1 calcium signaling demonstrated that the effect of KC may, in part, be due to heterologous desensitization to SDF-1. These results suggest that the CXCR4/SDF-1 axis is critical in circulating neutrophil homeostasis and that it may participate in the rapid release of neutrophils from the marrow during inflammation through a novel interaction with inflammatory CXC chemokines.

Finding the right niche: B-cell migration in the early phases of T-dependent antibody responses.

Humoral immune responses depend on B cells encountering antigen, interacting with helper T cells, proliferating and differentiating into low-affinity plasma cells or, after organizing into a germinal center (GC), high-affinity plasma cells and memory B cells. Remarkably, each of these events occurs in association with distinct stromal cells in separate subcompartments of the lymphoid tissue. B cells must migrate from niche to niche in a rapid and highly regulated manner to successfully mount a response. The chemokine, CXCL13, plays a central role in guiding B cells to follicles whereas T-zone chemokines guide activated B cells to the T zone. Sphingosine-1-phosphate (S1P) promotes cell egress from the tissue, as well as marginal-zone B-cell positioning in the spleen. Recent studies have identified a role for the orphan receptor, EBV-induced molecule 2 (EBI2; GPR183), in guiding activated B cells to inter and outer follicular niche(s) and down-regulation of this receptor is essential for organizing cells into GCs. In this review, we discuss current understanding of the roles played by chemokines, S1P and EBI2 in the migration events that underlie humoral immune responses.

Beyond melanoma: inhibiting the PD-1/PD-L1 pathway in solid tumors.

Immune checkpoint inhibitors have been identified as breakthrough treatment in melanoma given its dramatic response to PD-1/PD-L1 blockade. This is likely to extend to many other cancers as hundreds of clinical trials are being conducted or proposed using this exciting modality of therapy in a variety of malignancies. While immune checkpoint inhibitors have been extensively studied in melanoma and more recently in lung cancer, little is known regarding immune checkpoint blockade in other cancers. This review will focus on the tumor immune microenvironment, the expression of PD-1/PD-L1 and the effect of immune modulation using PD-1 or PD-L1 inhibitors in patients with head and neck, prostate, urothelial, renal, breast, gastrointestinal and lung cancers.

Exploiting lymphatic transport and complement activation in nanoparticle vaccines

Antigen targeting and adjuvancy schemes that respectively facilitate delivery of antigen to dendritic cells and elicit their activation have been explored in vaccine development. Here we investigate whether nanoparticles can be used as a vaccine platform by targeting lymph node–residing dendritic cells via interstitial flow and activating these cells by in situ complement activation. After intradermal injection, interstitial flow transported ultra-small nanoparticles (25 nm) highly efficiently into lymphatic capillaries and their draining lymph nodes, targeting half of the lymph node–residing dendritic cells, whereas 100-nm nanoparticles were only 10% as efficient. The surface chemistry of these nanoparticles activated the complement cascade, generating a danger signal in situ and potently activating dendritic cells. Using nanoparticles conjugated to the model antigen ovalbumin, we demonstrate generation of humoral and cellular immunity in mice in a size- and complement-dependent manner.

Augmenting Antitumor Immune Responses with Epigenetic Modifying Agents

Epigenetic silencing of immune-related genes is a striking feature of the cancer genome that occurs in the process of tumorigenesis. This phenomena impacts antigen processing and antigen presentation by tumor cells and facilitates evasion of immunosurveillance. Further modulation of the tumor microenvironment by altered expression of immunosuppressive cytokines impairs antigen-presenting cells and cytolytic T-cell function. The potential reversal of immunosuppression by epigenetic modulation is therefore a promising and versatile therapeutic approach to reinstate endogenous immune recognition and tumor lysis. Pre-clinical studies have identified multiple elements of the immune system that can be modulated by epigenetic mechanisms and result in improved antigen presentation, effector T-cell function, and breakdown of suppressor mechanisms. Recent clinical studies are utilizing epigenetic therapies prior to, or in combination with, immune therapies to improve clinical outcomes.

Bortezomib enhances dendritic cell (DC)-mediated induction of immunity to human myeloma via exposure of cell surface heat shock protein 90 on dying tumor cells: therapeutic implications.

Most anticancer chemotherapies are immunosuppressive and induce nonimmunogenic tumor cell death. Bortezomib, a specific inhibitor of 26S proteasome, has shown clinical activity in several human tumors, including myeloma. Here we show that the uptake of human myeloma cells by dendritic cells (DCs) after tumor cell death by bortezomib, but not gamma irradiation or steroids, leads to the induction of antitumor immunity, including against primary tumor cells, without the need for any additional adjuvants. The delivery of activating signal from bortezomib-killed tumor cells to DCs depends on cell-cell contact between DCs and dying tumor cells and is mediated by bortezomib-induced exposure of heat shock protein 90 (hsp90) on the surface of dying cells. The combination of bortezomib and geldanamycin (an hsp90 inhibitor) leads to greater apoptosis of tumor cells but abrogates their immunogenicity. These data identify drug-induced exposure of endogenous heat shock proteins on the surface of dying cells as a mechanism of immunogenic death of human tumors. Specific targeting of bortezomib to tumors may enhance their immunogenicity and the induction of antitumor immunity.

Oncolytic adenovirus armed with IL-24 Inhibits the growth of breast cancer in vitro and in vivo

BACKGROUND Interleukin-24 (IL-24) is a cytokine that belongs to the IL-10 family. It can selectively induce cancer cell apoptosis which has been utilized as a cancer gene therapy strategy. METHODS A recombinant type five adenovirus containing IL-24 gene (designated CNHK600-IL24) was constructed, whose replication is activated only in tumor cells. The replication of CNHK600-IL24 in breast tumor cells and fibroblasts were assessed by TCID50 and MTT assay; the secretion of IL-24 was measured by ELISA and western blotting. The in vivo anti-tumor effect of CNHK600-IL24 was investigated in nude mice carrying orthotopic or metastatic breast tumor. RESULTS We observed that CNHK600-IL24 could replicate efficiently and resulted in high level IL-24 expression and massive cell death in human breast cancer cell MDA-MB-231 but not in normal fibroblast cell MRC-5. In addition, orthotopic breast tumor growth in the nude mice model was significantly suppressed when CNHK600-IL24 was administered. In the metastatic model generated by tail vein injection, CNHK600-IL24 virotherapy significantly improved survival compared with the same virus expressing EGFP (median survival CNHK600-IL24, 55 days vs. CNHK600-EGFP, 41 day, p < 0.05 Mantal-Cox test). A similar phenomenon was observed in the metastatic model achieved by left ventricular injection as suggested by in vivo luminescence imaging of tumor growth. CONCLUSION The oncolytic adenovirus armed with IL-24, which exhibited enhanced anti-tumor activity and improved survival, is a promising candidate for virotherapy of breast cancer.

Isolated liver perfusion for non-resectable liver tumours: a review.

Many treatments have been proposed for non-resectable primary or secondary hepatic cancer but the results have generally been disappointing. Isolated Hepatic Perfusion (IHP) was first attempted four decades ago but it gained acceptance only recently, after spectacular tumour responses were obtained by isolated limb perfusion with melphalan and tumour necrosis factor (TNF) for melanomas and sarcomas. Surgical isolation of the liver is a technically demanding operation that allows the safe administration of high doses of chemotherapeutics and TNF. Percutaneous techniques using balloon occlusion catheters are simpler but result in higher leakage rates from the perfusion circuit into the systemic circulation. Several phase I-II trials indicate that IHP can yield high tumour response rates, even when there is resistance to systemic chemotherapy. However, no significant advantage in overall survival has been demonstrated so far. IHP offers unique pharmacokinetic advantages for locoregional chemotherapy and biotherapy. It might also allow gene therapy with limited systemic exposure and toxicity. At present, IHP nevertheless remains an experimental treatment modality which should therefore be used in controlled trials only.

Latent membrane protein 1 of Epstein–Barr virus coordinately regulates proliferation with control of apoptosis

Latent membrane protein 1 (LMP1), an oncoprotein encoded by Epstein–Barr virus (EBV), is an integral membrane protein, which acts like a constitutively active receptor. LMP1 is critical for some facet of EBV's induction and maintenance of proliferation of infected B cells. It, in part, mimics signaling by the CD40 receptor and has been implicated in regulating proliferation, survival, or both properties of EBV-infected cells. We established a conditional LMP1 allele in the context of the intact EBV genome to define the immediate-early cellular target genes regulated by LMP1 in order to assess its contributions to infected human B cells. The functional analysis of this conditional system indicated that LMP1 specifically induces mitogenic B-cell activation through c-myc and Jun/AP1 family members and confirms its direct role in upregulating expression of multiple genes with opposing activities involved in cell survival. LMP1's signals were found to be essential for the G1/S transition in human B cells; cells lacking LMP1's signals are cell cycle arrested and survive quiescently. LMP1's activities are therefore not required to maintain survival in nonproliferating cells. LMP1 does induce both pro- and antiapoptotic genes whose balance seems to permit survival during LMP1's induction and maintenance of proliferation.

The Immune Response to Melanoma Is Limited by Thymic Selection of Self-Antigens

The expression of melanoma-associated antigens (MAA) being limited to normal melanocytes and melanomas, MAAs are ideal targets for immunotherapy and melanoma vaccines. As MAAs are derived from self, immune responses to these may be limited by thymic tolerance. The extent to which self-tolerance prevents efficient immune responses to MAAs remains unknown. The autoimmune regulator (AIRE) controls the expression of tissue-specific self-antigens in thymic epithelial cells (TECs). The level of antigens expressed in the TECs determines the fate of auto-reactive thymocytes. Deficiency in AIRE leads in both humans (APECED patients) and mice to enlarged autoreactive immune repertoires. Here we show increased IgG levels to melanoma cells in APECED patients correlating with autoimmune skin features. Similarly, the enlarged T cell repertoire in AIRE(-/-) mice enables them to mount anti-MAA and anti-melanoma responses as shown by increased anti-melanoma antibodies, and enhanced CD4(+) and MAA-specific CD8(+) T cell responses after melanoma challenge. We show that thymic expression of gp100 is under the control of AIRE, leading to increased gp100-specific CD8(+) T cell frequencies in AIRE(-/-) mice. TRP-2 (tyrosinase-related protein), on the other hand, is absent from TECs and consequently TRP-2 specific CD8(+) T cells were found in both AIRE(-/-) and AIRE(+/+) mice. This study emphasizes the importance of investigating thymic expression of self-antigens prior to their inclusion in vaccination and immunotherapy strategies.

Neutrophils support lung colonization of metastasis-initiating breast cancer cells

Despite progress in the development of drugs that efficiently target cancer cells, treatments for metastatic tumours are often ineffective. The now well-established dependency of cancer cells on their microenvironment suggests that targeting the non-cancer-cell component of the tumour might form a basis for the development of novel therapeutic approaches. However, the as-yet poorly characterized contribution of host responses during tumour growth and metastatic progression represents a limitation to exploiting this approach. Here we identify neutrophils as the main component and driver of metastatic establishment within the (pre-)metastatic lung microenvironment in mouse breast cancer models. Neutrophils have a fundamental role in inflammatory responses and their contribution to tumorigenesis is still controversial. Using various strategies to block neutrophil recruitment to the pre-metastatic site, we demonstrate that neutrophils specifically support metastatic initiation. Importantly, we find that neutrophil-derived leukotrienes aid the colonization of distant tissues by selectively expanding the sub-pool of cancer cells that retain high tumorigenic potential. Genetic or pharmacological inhibition of the leukotriene-generating enzyme arachidonate 5-lipoxygenase (Alox5) abrogates neutrophil pro-metastatic activity and consequently reduces metastasis. Our results reveal the efficacy of using targeted therapy against a specific tumour microenvironment component and indicate that neutrophil Alox5 inhibition may limit metastatic progression.

The inflammasome component NLRP3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells.

The inflammasome is a proteolysis complex that generates the active forms of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Inflammasome activation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3(-/-) mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended on CD8(+) T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3(-/-) hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSC). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3(-/-) MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3(-/-) mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T-cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling.

Acute administration of recombinant Angiopoietin-1 ameliorates multiple-organ dysfunction syndrome and improves survival in murine sepsis.

INTRODUCTION Endothelial activation leading to vascular barrier breakdown plays an essential role in the pathophysiology of multiple-organ dysfunction syndrome (MODS) in sepsis. Increasing evidence suggests that the function of the vessel-protective factor Angiopoietin-1 (Ang-1), a ligand of the endothelial-specific Tie2 receptor, is inhibited by its antagonist Angiopoietin-2 (Ang-2) during sepsis. In order to reverse the effects of the sepsis-induced suppression of Ang-1 and elevation of Ang-2 we aimed to investigate whether an intravenous injection of recombinant human (rh) Ang-1 protects against MODS in murine sepsis. METHODS Polymicrobiological abdominal sepsis was induced by cecal ligation and puncture (CLP). Mice were treated with either 1 μg of intravenous rhAng-1 or control buffer immediately after CLP induction and every 8h thereafter. Sham-operated animals served as time-matched controls. RESULTS Compared to buffer-treated controls, rhAng-1 treated septic mice showed significant improvements in several hematologic and biochemical indicators of MODS. Moreover, rhAng-1 stabilized endothelial barrier function, as evidenced by inhibition of protein leakage from lung capillaries into the alveolar compartment. Histological analysis revealed that rhAng-1 treatment attenuated leukocyte infiltration in lungs and kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression in rhAng-1 treated mice. Finally, the protective effects of rhAng-1 treatment were reflected by an improved survival time in a lethal CLP model. CONCLUSIONS In a clinically relevant murine sepsis model, intravenous rhAng-1 treatment alone is sufficient to significantly improve a variety of sepsis-associated organ dysfunctions and survival time, most likely by preserving endothelial barrier function. Further studies are needed to pave the road for clinical application of this therapy concept.

Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis.

Inflammasome-mediated IL-1beta production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17-producing CD4(+) T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1beta plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI(-/-)) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1beta production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI(-/-) mice. Furthermore, the bacterial load was enhanced in IL-17-defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1beta production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1beta-mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.

The modulation of human natural killer cell activity by prostaglandins.

Prostaglandins (PGs) have been implicated as a regulator of tumor growth in mice and humans. Since natural killer cell (NK) cytotoxicity may be an important component of immune surveillance against cancer, it is appropriate to study whether the amount of PGs produced by tumors may be sufficient to suppress NK activity. Accordingly, the effect of various PGs on the NK activity of human peripheral mononuclear cells was investigated. The percentage cytotoxicity was measured by the release of Cr51 from labeled K562 and other target cells. At very high concentrations of PG (10(-6) M), suppression was seen with PGE2, PGD2, PGA2, and PGF2 alpha. However, at concentrations of PG in the physiologic range (10(-8) M), significant suppression was seen with PGE2 and PGD2 only. The percentage suppression with PGE2 ranged from 77% to 9.5% over a range of concentrations from 10(-5) to 10(-9) M (45% at 10(-8) M). Significant suppression was observed at 10(-8) M PGE2 with 4 different targets and at effector:target ratios varying from 50:1 to 12.5:1. To assess whether the suppressive effect of PGE2 was directed at the effector and/or target cell, K562 cells or effector cells were pretreated with PGE2. Significant suppression was seen with effector cell pretreatment but not with target cell pretreatment. Finally, the suppressive effects of supernatants obtained from tumor cell lines (polyoma virus-transformed murine fibroblast cell line, PY3T3) was determined. The marked suppressive effect of the supernatant could be attributed to its content of PGE. Thus, it appears that the production of PGE by tumor cells may be an important modulator of human NK activity.

mir-1-mediated paracrine effect of cancer-associated fibroblasts on lung cancer cell proliferation and chemoresistance.

Lung cancer is the leading cause of cancer-related mortality in humans worldwide. Moreover, the overall 5-year survival rate is only 15%. Pathologically almost 80% of all lung cancer cases are non-small cell lung cancer (NSCLC). Cancer-associated fibroblasts (CAFs) have been found to exist in a large number of NSCLCs. CAFs have been proven to promote tumor progression, metastasis and resistance to therapy through paracrine effects in most solid tumors. In the present study, firstly we isolated CAFs from patient tissues and demonstrated that they promoted cell proliferation and chemoresistance to cisplatin in the lung cancer cell lines A549 and 95D in a paracrine manner. Secondly, using ELISA and quantative PCR, we found that a higher amount of stromal cell-derived factor 1 (SDF-1) existed in the CAFs rather than that observed in the normal fibroblasts (NFs). Thirdly, we detected that SDF-1 facilitated lung cancer cell proliferation and drug resistance via the CXCR4-mediated signaling pathway which involved NF-κB and Bcl-xL. Moreover, we also confirmed that the expression level of SDF-1 in the CAFs was negatively regulated by microRNA mir-1 through microRNA overexpression and quantitative PCR. Overall, our data provide one explanation for the effects of CAFs on lung cancer cells. Meanwhile, our results also suggest CAFs as a potential therapeutic target in tumor treatment.

Response to self antigen imprints regulatory memory in tissues

Immune homeostasis in tissues is achieved through a delicate balance between pathogenic T-cell responses directed at tissue-specific antigens and the ability of the tissue to inhibit these responses. The mechanisms by which tissues and the immune system communicate to establish and maintain immune homeostasis are currently unknown. Clinical evidence suggests that chronic or repeated exposure to self antigen within tissues leads to an attenuation of pathological autoimmune responses, possibly as a means to mitigate inflammatory damage and preserve function. Many human organ-specific autoimmune diseases are characterized by the initial presentation of the disease being the most severe, with subsequent flares being of lesser severity and duration. In fact, these diseases often spontaneously resolve, despite persistent tissue autoantigen expression. In the practice of antigen-specific immunotherapy, allergens or self antigens are repeatedly injected in the skin, with a diminution of the inflammatory response occurring after each successive exposure. Although these findings indicate that tissues acquire the ability to attenuate autoimmune reactions upon repeated responses to antigens, the mechanism by which this occurs is unknown. Here we show that upon expression of self antigen in a peripheral tissue, thymus-derived regulatory T cells (Treg cells) become activated, proliferate and differentiate into more potent suppressors, which mediate resolution of organ-specific autoimmunity in mice. After resolution of the inflammatory response, activated Treg cells are maintained in the target tissue and are primed to attenuate subsequent autoimmune reactions when antigen is re-expressed. Thus, Treg cells function to confer ‘regulatory memory’ to the target tissue. These findings provide a framework for understanding how Treg cells respond when exposed to self antigen in peripheral tissues and offer mechanistic insight into how tissues regulate autoimmunity.

Influence of DNA on the activities and inhibition of neutrophil serine proteases in cystic fibrosis sputum.

Uncontrolled proteolysis by neutrophil serine proteases (NSPs) in lung secretions is a hallmark of cystic fibrosis (CF). We have shown that the active neutrophil elastase, protease 3, and cathepsin G in CF sputum resist inhibition in part by exogenous protease inhibitors. This resistance may be due to their binding to neutrophil extracellular traps (NETs) secreted by the activated neutrophils in CF sputum and to genomic DNA released from senescent and dead neutrophils. Treating CF sputum with DNase dramatically increases its elastase activity, which can then be stoichiometrically inhibited by exogenous elastase inhibitors. However, DNase treatment does not increase the activities of protease 3 and cathepsin G, indicating their different distribution and/or binding in CF sputum. Purified blood neutrophils secrete NETs when stimulated by the opportunistic CF bacteria Pseudomonas aeruginosa and Staphylococcus aureus. The activities of the three proteases were unchanged in these conditions, but subsequent DNase treatment produced a dramatic increase in all three proteolytic activities. Neutrophils activated with a calcium ionophore did not secrete NETs but released huge amounts of active proteases whose activities were not modified by DNase. We conclude that NETs are reservoirs of active proteases that protect them from inhibition and maintain them in a rapidly mobilizable status. Combining the effects of protease inhibitors with that of DNA-degrading agents could counter the deleterious proteolytic effects of NSPs in CF lung secretions.

Inhibition of fatty acid oxidation modulates immunosuppressive functions of myeloid-derived suppressor cells and enhances cancer therapies

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies.

Cancer-Associated Fibroblasts Neutralize the Anti-tumor Effect of CSF1 Receptor Blockade by Inducing PMN-MDSC Infiltration of Tumors.

Tumor-associated macrophages (TAM) contribute to all aspects of tumor progression. Use of CSF1R inhibitors to target TAM is therapeutically appealing, but has had very limited anti-tumor effects. Here, we have identified the mechanism that limited the effect of CSF1R targeted therapy. We demonstrated that carcinoma-associated fibroblasts (CAF) are major sources of chemokines that recruit granulocytes to tumors. CSF1 produced by tumor cells caused HDAC2-mediated downregulation of granulocyte-specific chemokine expression in CAF, which limited migration of these cells to tumors. Treatment with CSF1R inhibitors disrupted this crosstalk and triggered a profound increase in granulocyte recruitment to tumors. Combining CSF1R inhibitor with a CXCR2 antagonist blocked granulocyte infiltration of tumors and showed strong anti-tumor effects.

Chronic obstructive pulmonary disease and neutrophil infiltration: role of cigarette smoke and cyclooxygenase products.

Cigarette smoke is the main cause of chronic obstructive pulmonary disease (COPD), where it can contribute to the observed airway inflammation. PGE(2) is produced within human airways, and both pro- and anti-inflammatory activities have been reported. We quantitated PGE(2) concentrations in induced sputum supernatants from different groups of subjects and correlated the obtained values to neutrophil infiltration as well as to the expression of cyclooxygenase-2 (COX-2). Cigarette smoke extract (CSE) was used to evaluate the effect of smoking on COX-2 and PGE(2) receptor expression as well as on PGE(2) release in neutrophils and alveolar macrophages (AM) obtained from normal donors. The effects of PGE(2) and of PGE receptor agonists and antagonists were evaluated on the adhesion of neutrophil to a human bronchial epithelial cell line (16HBE). PGE(2) levels, COX-2 expression, and neutrophil infiltration were significantly higher in normal smokers and COPD smokers (P < 0.0001) compared with controls and COPD former smokers. Induced sputum supernatant caused neutrophil adhesion to 16HBE that was significantly reduced, in COPD smokers only, by PGE(2) immunoprecipitation. In vitro experiments confirmed that CSE increased PGE(2) release and COX-2 and PGE(2) receptor expression in neutrophils and AM; PGE(2) enhanced the adhesion of neutrophils to 16HBE, and a specific E-prostanoid 4 (EP(4)) receptor antagonist blunted its effect. These results suggest that CSE promote the induction of COX-2 and contributes to the proinflammatory effects of PGE(2) in the airways of COPD subjects.

Selective elimination of high constitutive activity or chemokine binding in the human herpesvirus 8 encoded seven transmembrane oncogene ORF74.

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines, e.g. growth-related oncogene-alpha, and inhibited by angiostatic chemokines e.g. interferon-gamma-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity.

NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.

The early events leading to the development of rheumatoid arthritis (RA) remain unclear, but formation of autoantibodies to citrullinated protein antigens (ACPAs) is considered a key pathogenic event. Neutrophils isolated from patients with various autoimmune diseases display enhanced neutrophil extracellular trap (NET) formation, a phenomenon that exposes autoantigens in the context of immunostimulatory molecules. We investigated whether aberrant NETosis occurs in RA, determined its triggers, and examined its deleterious inflammatory consequences. Enhanced NETosis was observed in circulating and RA synovial fluid neutrophils compared to neutrophils from healthy controls and from patients with osteoarthritis (OA). Further, netting neutrophils infiltrated RA synovial tissue, rheumatoid nodules, and skin. NETosis correlated with ACPA presence and levels and with systemic inflammatory markers. RA sera and immunoglobulin fractions from RA patients with high levels of ACPA and/or rheumatoid factor significantly enhanced NETosis, and the NETs induced by these autoantibodies displayed distinct protein content. Indeed, during NETosis, neutrophils externalized the citrullinated autoantigens implicated in RA pathogenesis, and anti-citrullinated vimentin antibodies potently induced NET formation. Moreover, the inflammatory cytokines interleukin-17A (IL-17A) and tumor necrosis factor-α (TNF-α) induced NETosis in RA neutrophils. In turn, NETs significantly augmented inflammatory responses in RA and OA synovial fibroblasts, including induction of IL-6, IL-8, chemokines, and adhesion molecules. These observations implicate accelerated NETosis in RA pathogenesis, through externalization of citrullinated autoantigens and immunostimulatory molecules that may promote aberrant adaptive and innate immune responses in the joint and in the periphery, and perpetuate pathogenic mechanisms in this disease.

The precursor form of IL-1alpha is an intracrine proinflammatory activator of transcription.

Although most cytokines are studied for biological effects after engagement of their specific cell surface membrane receptors, increasing evidence suggests that some function in the nucleus. In the present study, the precursor form of IL-1alpha was overexpressed in various cells and assessed for activity in the presence of saturating concentrations of IL-1 receptor antagonist to prevent receptor signaling. Initially diffusely present in the cytoplasm of resting cells, IL-1alpha translocated to the to nucleus after activation by endotoxin, a Toll-like receptor ligand. The IL-1alpha precursor, but not the C-terminal mature form, activated the transcriptional machinery in the GAL4 system by 90-fold; a 50-fold increase was observed using only the IL-1alpha propiece, suggesting that transcriptional activation was localized to the N terminus where the nuclear localization sequence resides. Under conditions of IL-1 receptor blockade, intracellular overexpression of the precursor and propiece forms of IL-1alpha were sufficient to activate NF-kappaB and AP-1. Stable transfectants overproducing precursor IL-1alpha released the cytokines IL-8 and IL-6 but also exhibited a significantly lower threshold of activation to subpicomolar concentrations of tumor necrosis factor alpha or IFN-gamma. Thus, intracellular functions of IL-1alpha might play an unforeseen role in the genesis of inflammation. During disease-driven events, the cytosolic precursor moves to the nucleus, where it augments transcription of proinflammatory genes. Because this mechanism of action is not affected by extracellular inhibitors, reducing intracellular functions of IL-1alpha might prove beneficial in some inflammatory conditions.

A JOURNAL OF NEUROLOGY

Brain glial cells, five times more prevalent than neurons, have recently received attention for their potential involvement in epileptic seizures. Microglia and astrocytes, associated with inflammatory innate immune responses, are responsible for surveillance of brain damage that frequently results in seizures. Thus, an intriguing suggestion has been put forward that seizures may be facilitated and perhaps triggered by brain immune responses. Indeed, recent evidence strongly implicates innate immune responses in lowering seizure threshold in experimental models of epilepsy, yet, there is no proof that they can play an independent role in initiating seizures in vivo. Here, we show that cortical innate immune responses alone produce profound increases of brain excitability resulting in focal seizures. We found that cortical application of lipopolysaccharide, binding to toll-like receptor 4 (TLR4), triples evoked field potential amplitudes and produces focal epileptiform discharges. These effects are prevented by pre-application of interleukin-1 receptor antagonist. Our results demonstrate how the innate immune response may participate in acute seizures, increasing neuronal excitability through interleukin-1 release in response to TLR4 detection of the danger signals associated with infections of the central nervous system and with brain injury. These results suggest an important role of innate immunity in epileptogenesis and focus on glial inhibition, through pharmacological blockade of TLR4 and the pro-inflammatory mediators released by activated glia, in the study and treatment of seizure disorders in humans.

Early life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues

It is unclear how the immune response in early life becomes appropriately stimulated to provide protection while also avoiding excessive activation as a result of diverse new antigens. T cells are integral to adaptive immunity; mouse studies indicate that tissue localization of T cell subsets is important for both protective immunity and immunoregulation. In humans, however, the early development and function of T cells in tissues remain unexplored. We present here an analysis of lymphoid and mucosal tissue T cells derived from pediatric organ donors in the first two years of life, as compared to adult organ donors, revealing early compartmentalization of T cell differentiation and regulation. Whereas adult tissues contain a predominance of memory T cells, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (T(EM)) found only in the lungs and small intestine. Additionally, regulatory T (T(reg)) cells comprise a high proportion (30-40%) of CD4(+) T cells in pediatric tissues but are present at much lower frequencies (1-10%) in adult tissues. Pediatric tissue T(reg) cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest T(reg):T(EM) cell ratios, which suggests control in situ of immune responses in early life.

Regulation of myeloid cell function through the CD200 receptor.

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-gamma- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.

Suppression of interferon-mediated anti-HBV response by single CpG methylation in the 5′-UTR of TRIM22

Objective Interferons (IFNs) mediate direct antiviral activity. They play a crucial role in the early host immune response against viral infections. However, IFN therapy for HBV infection is less effective than for other viral infections. Design We explored the cellular targets of HBV in response to IFNs using proteome-wide screening. Results Using LC-MS/MS, we identified proteins downregulated and upregulated by IFN treatment in HBV X protein (HBx)-stable and control cells. We found several IFN-stimulated genes downregulated by HBx, including TRIM22, which is known as an antiretroviral protein. We demonstrated that HBx suppresses the transcription of TRIM22 through a single CpG methylation in its 5′-UTR, which further reduces the IFN regulatory factor-1 binding affinity, thereby suppressing the IFN-stimulated induction of TRIM22. Conclusions We verified our findings using a mouse model, primary human hepatocytes and human liver tissues. Our data elucidate a mechanism by which HBV evades the host innate immune system.

High-Density Lipoproteins Exert Pro-inflammatory Effects on Macrophages via Passive Cholesterol Depletion and PKC-NF-κB/STAT1-IRF1 Signaling.

Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.

Programmed death-1–induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection

Viral replication and microbial translocation from the gut to the blood during HIV infection lead to hyperimmune activation, which contributes to the decline in CD4+ T cell numbers during HIV infection. Programmed death-1 (PD-1) and interleukin-10 (IL-10) are both upregulated during HIV infection. Blocking interactions between PD-1 and programmed death ligand-1 (PD-L1) and between IL-10 and IL-10 receptor (IL-10R) results in viral clearance and improves T cell function in animal models of chronic viral infections. Here we show that high amounts of microbial products and inflammatory cytokines in the plasma of HIV-infected subjects lead to upregulation of PD-1 expression on monocytes that correlates with high plasma concentrations of IL-10. Triggering of PD-1 expressed on monocytes by PD-L1 expressed on various cell types induced IL-10 production and led to reversible CD4+ T cell dysfunction. We describe a new function for PD-1 whereby microbial products inhibit T cell expansion and function by upregulating PD-1 levels and IL-10 production by monocytes after binding of PD-1 by PD-L1.

Expression profiles of Toll-like receptors in non-small cell lung cancer and idiopathic pulmonary fibrosis.

Patients with idiopathic pulmonary fibrosis (IPF) have a higher incidence             of lung cancer. The role of Toll-like receptors (TLRs), a key component of the             innate immunity, in interstitial lung diseases (ILDs) and lung cancer pathogenesis             is not clarified. TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA expression was quantitatively             measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR)             in bronchoalveolar lavage fluid (BALF) of 16 IPF patients, 16 non-small cell lung             cancer (NSCLC) patients and 9 control subjects. TLR2, TLR3, TLR4 and TLR9 protein             expression was assessed on BALF T-lymphocytes using flow cytometry. TLR3 mRNA             expression was significantly higher in NSCLC compared to IPF (p=0.023) and controls             (p=0.001). TLR7 mRNA expression levels were significantly higher in both NSCLC             and IPF groups compared to controls (p=0.029, p=0.009). TLR9 expression at the             mRNA level was significantly higher in both NSCLC and IPF groups compared to controls             (p=0.01, p=0.001). Finally, TLR2 mRNA expression was significantly higher in IPF             patients compared to controls (p=0.042). Flow cytometry revealed decreased TLR3             and TLR9 expression in IPF patients compared to the NSCLC group (p=0.02, p=0.014)             and decreased TLR9 expression in IPF compared with the controls (p=0.04). TLR2             protein expression was significantly higher in IPF patients compared to NSCLC             (p=0.04). Increased expression of endosomal TLRs in NSCLC patients and elevated             expression of TLR2 in pulmonary fibrosis are the main results of this study. These             results do not provide support for a common TLR pathway hypothesis between NSCLC             and IPF.