Neuroprotection for Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Early to intermediate AMD is characterized by the accumulation of lipid- and protein-rich drusen. Late stages of the disease are characterized by the development of choroidal neovascularization, termed “exudative” or “neovascular AMD,” or retinal pigment epithelium (RPE) cell and photoreceptor death, termed “geographic atrophy” (GA) in advanced nonexudative AMD. Although we have effective treatments for exudative AMD in the form of anti-VEGF agents, they have no role for patients with GA. Neuroprotection strategies have emerged as a possible way to slow photoreceptor degeneration and vision loss in patients with GA. These approaches include reduction of oxidative stress, modulation of the visual cycle, reduction of toxic molecules, inhibition of pathologic protein activity, prevention of cellular apoptosis or programmed necrosis (necroptosis), inhibition of inflammation, direct activation of neurotrophic factors, delivery of umbilical tissue–derived cells, and RPE replacement. Despite active investigation in this area and significant promise based on preclinical studies, many clinical studies have not yielded successful results. We discuss selected past and current neuroprotection trials for AMD, highlight the lessons learned from these past studies, and discuss our perspective regarding remaining questions that must be answered before neuroprotection can be successfully applied in the field of AMD research.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Therapies that inhibit VEGF have been revolutionary for patients with the exudative or wet form of advanced AMD. Nonetheless, anti-VEGF therapies have no role in treating advanced nonexudative or dry advanced AMD or patients with exudative AMD who respond to anti-VEGF treatments but ultimately develop macular atrophy. Thus, there is a significant clinical need to develop novel treatments to slow or prevent photoreceptor death.
The pathogenesis of advanced AMD is complex. Past studies relying on cellular and animal models with confirmatory studies in humans have suggested that there are numerous pathways involved in AMD pathogenesis, including but not limited to altered lipid homeostasis, dysregulated immune activation, increased oxidative stress, and impaired metabolism. 1 Despite significant efforts, there is no clear consensus regarding the predominant causative pathologic process or processes that ultimately lead to photoreceptor death. It is becoming increasingly apparent that AMD is a multifactorial disease whose pathogenesis likely involves perturbations in numerous molecular and cellular pathways that ultimately lead to photoreceptor degeneration. Because photoreceptors are the lightsensitive cells of the retina that are responsible for phototransduction, photoreceptor death inevitably causes blindness.
Neuroprotection has emerged as a strategy for delaying photoreceptor death and preserving vision. One advantage of the broad strategy of neuroprotection is that it may have the ability to slow photoreceptor degeneration regardless of the underlying causative pathway and may even be generalizable to other retinal neurodegenerative diseases. For the purposes of this review, we define "neuroprotection" to be any therapeutic strategy intended to enhance photoreceptor survival, regardless of whether they target a primary causative or secondary/contributory pathologic process. Moreover, we focus on pharmacologic strategies rather than nonpharmacologic interventions, such as laser. Other therapeutic strategies, such as those intended to inhibit pathologic complement activation, which likely involve other retinal cells, have been reviewed. 2 In this review, we highlight selected past and ongoing clinical studies investigating neuroprotection as a strategy for treating AMD, provide our perspective regarding the lessons learned from these studies, and discuss remaining areas of important research that must be explored before neuroprotection can become a reality for AMD therapy.

Past and Current Neuroprotection Trials
There are numerous past and ongoing neuroprotection trials in AMD. Broadly, these approaches can be divided into their overall strategy: reduction of oxidative stress, modulation of the visual cycle, reduction of toxic molecules, inhibition of pathologic protein activity, inhibition of cellular death pathways such as apoptosis or programed necrosis (necroptosis), inhibition of inflammation, direct activation of neurotrophic factors, delivery of umbilical tissueederived cells, and retinal pigment epithelium (RPE) replacement (Fig 1). Selected trials are highlighted in Table 1.

Systemic Natural Antioxidants
One neuroprotective approach is to reduce exposure of photoreceptors to oxidative stress. The initial evidence that targeting oxidative stress may protect against advanced AMD was from the Age-Related Eye Disease Study (AREDS). In AREDS, post-hoc analysis of a specific cohort of patients with intermediate AMD was found to have reduced odds of progression to advanced neovascular AMD if they were randomized to the combination of numerous antioxidants (vitamin C, vitamin E, beta-carotene) plus zinc compared with those randomized to placebo. 3e6 The subsequent Age-Related Eye Disease Study 2 (AREDS2) replaced beta-carotene with other antioxidants, lutein and zeaxanthin, because beta-carotene was found to be associated with increased rate of lung cancer among smokers in large population studies. There was no difference in the rate of progression to advanced AMD in those with the new or old formulation. 7 The AREDS2 supplements are widely used in patients with intermediate nonexudative AMD, although the durability and magnitude of effect may be small, and this initial finding has never been replicated in a subsequent randomized placebo-controlled phase III trial.
Other systemic antioxidants, such as alpha lipoic acid (ALA), have been tested as well. However, high-dose ALA was found to be intolerable in older subjects because of flushing sensation and gastrointestinal side effects. 8 Moreover, a randomized clinical trial showed that ALA had no beneficial effect on GA or visual acuity (VA) in patients with advanced nonexudative AMD. 9 Forms of lipoylcarnitine esters may be more bioavailable and more suitable for testing in AMD.
A prespecified ancillary study of the Vitamin D and Omega-3 Trial showed that randomization to vitamin D and omega-3 fatty acids, which are thought to have possible antioxidative properties, did not lead to any significant reduction in either AMD incidence or AMD progression. 10

Local Antioxidants
Local delivery of antioxidants to the retina has also been tested. This strategy has the theoretical benefit of increased bioavailability to the eye with fewer systemic adverse effects. OT-551 (Othera Pharmaceuticals) is a lipophilic, disubstituted hydroxylamine that is administrated as an eye drop. Both OT-551 and its active metabolite, TEMPOL-H, can terminate free radical reactions in vitro and thereby exert an antioxidant effect with the potential result of protecting photoreceptors from oxidative damage. Although OT-551 was safe and tolerable when dosed 4 times daily for up to 2 years, it did not reduce GA growth or reduce the extent of VA loss in patients with AMD. 11,12 Another agent that has shown promise is elamipretide (Stealth BioTherapeutics), a peptide that enhances mitochondrial function and reduces formation of reactive oxygen species (ROS) in vitro. Elamipretide is being investigated for numerous diseases, especially those in which mitochondrial dysfunction is suspected to play a pathogenic role. The Phase 1 ReCLAIM study suggested that subcutaneous elamipretide is tolerable and may improve visual function in patients with noncentral GA or those with high-risk drusen. 13,14 These initial results have led to the Phase II ReCLAIM-2 trial (NCT03891875). Unfortunately, the drug failed on the primary endpoints of lowluminance VA and GA progression, though the secondary endpoints of potentially less ellipsoid zone loss was encouraging to the study investigators.
The small peptide integrin regulator risuteganib (Allegro Ophthalmics), which reduces ROS and enhances mitochondrial function in preclinical studies, has also shown promise. In a recent Phase IIa study, patients randomized to risuteganib had significant improvement in VA compared with control patients. 15 We should be cautious in interpreting these successes given the inherent limitations of early-phase studies.

Modulation of the Visual Cycle
The visual cycle is necessary for regeneration of functional visual pigment for phototransduction. Over a lifetime, continuous activity of the visual cycle can lead to accumulation of by-products such as N-retinylidene-N-retinylethanolamine, a bisretinoid that is suspected to have a cytotoxic effect and contribute to photoreceptor degeneration in diseases such as AMD. Accordingly, another neuroprotective strategy for AMD is modulation of the visual cycle, thereby reducing formation of toxic by-products. Fenretinide (ReVision Therapeutics) is a retinol-binding protein (RBP) antagonist. Retinol-binding proteins are essential for transporting vitamin A in the systemic circulation. Retinol-binding protein antagonism can reduce systemic vitamin A availability and, thus, reduces activity of the visual cycle by limiting substrate availability. Despite this theoretical effect, oral fenretinide was not found to have any significant effect on GA lesion growth in a Phase II clinical trial. 16 Use of fenretinide has also been associated with impaired dark adaptation and dyschromatopsia, both in this study in AMD patients and in the oncology literature. 16,17 Other inhibitors of specific RBPs, such as LBS-008 (also known as "tinlarebant"; Belite Bio), an antagonist of RBP4, are still under investigation. In 2020, Belite Bio announced positive safety results from their Phase I clinical trial of LBS-008 and are planning to initiate studies investigating the potential role of LBS-008 for treating dry AMD and Stargardt disease.
Another strategy for modulating the visual cycle has been attempted with emixustat (Kubota Pharmaceutical Holdings), a small-molecule inhibitor of retinoid isomerohydrolase (RPE65), an enzyme of the visual cycle. Although initial studies were promising, the subsequent Phase IIb/III SEATTLE study did not show any significant effect of emixustat on GA growth rate. 18,19 Similar to patients taking fenretinide, a large proportion of patients on emixustat have delayed dark adaption and dyschromatopsia. 19,20 Finally, the approach of disrupting the visual cycle via administration of a chemically modified form of vitamin A, ALK-001 (Alkeus Pharmaceuticals), has been attempted. This C20 deuterated vitamin A forms toxic by-products more slowly than normal vitamin A and thereby leads to fewer cytotoxic effects. ALK-001 has shown promising results for treating the inherited retinal degenerative disease, Stargardt disease, and is currently being tested for patients with AMD in the Phase III SAGA study (NCT03845582).

Reduction of Other Toxic Molecules
Visual cycle by-products are not the only molecules that may cause cellular toxicity. The amyloid pathway involves cleavage of amyloid precursor protein into smaller fragments, including beta-amyloid. Accumulation of betaamyloid in the brain has been shown to be neurotoxic in neurodegenerative diseases such as Alzheimer disease (AD), although the exact pathophysiological mechanism by which this occurs remains unclear. Beta-amyloid is also a component of drusen found in AMD patients and is known to activate the complement cascade.
In a Phase II study, GSK933776 (GlaxoSmithKline), an anti-beta-amyloid monoclonal antibody, was found to have no clinically meaningful effect on slowing VA loss or reducing GA growth. 21 Phase II evaluation of another antibeta-amyloid antibody, RN6G (Pfizer), was registered but terminated early because of an "organizational decision" (NCT01577381). The dipeptide GAL-101 (formerly known as MRZ-99030/EG30; Galimedix Therapeutics), which prevents formation of toxic beta-amyloid oligomers, has also been hypothesized to have a possible neuroprotective role for AMD. GAL-101 eye drops were shown to be safe and tolerable in patients with glaucoma in a Phase I study (NCT01714960).
Although not yet registered, Galimedix Therapeutics reported plans to pursue a Phase II study of GAL-101 in patients with nonexudative AMD.

Inhibition of Pathologic Protein Activity
High-temperature requirement A1 (HTRA1) is a trimeric serine protease that is widely expressed in the retina and known to cleave multiple substrates including extracellular matrix proteins. Its association with AMD was established on the basis of genetic studies showing an association between AMD and a locus on chromosome 10q26, which contains the HTRA1 gene. 22 Although it has not been definitively proven that the HTRA1 gene is the gene within this locus that promotes AMD, HTRA1 activity has been suggested to be pathologic and contribute to AMD pathogenesis based on preclinical studies. In line with this, intravitreal delivery of FHTR2163/RO7171009/RG6147, an anti-HTRA1 antibody fragment (Genentech), was shown to be safe and well tolerated in Phase I 23 and is now being evaluated as a therapy for GA in the Phase II GALLEGO study (NCT03972709).

Inhibition of Cellular Death
Another approach for preventing photoreceptor death is to inhibit activation of cellular death pathways, such as the Fas/Fas ligand interaction, which initiates a downstream cascade that ultimately triggers apoptotic cellular death or necroptotic cell death depending on the balance of complex intracellular signaling pathways. 24e32 Intrinsic intracellular pathways can also trigger apoptosis or necrosis. Other cellular death pathways, such as ferroptosis and pyroptosis, have been speculated to play roles in photoreceptor degeneration. A schematic of cell death pathways is depicted in Figure 2. ONL1204 (ONL Therapeutics) is a small-molecule Fas inhibitor intended to protect photoreceptors from apoptosis triggered by pathologic pathways. Based on promising preclinical studies, a Phase I study testing its safety and tolerability in patients with GA is under way (NCT04744662).
Although further exploration in human studies is necessary, preclinical studies suggest that photoreceptors are affected by all these cell death pathways in a redundant and complementary fashion. There is complexity not only in that cone and rod photoreceptors may be affected by different predominant cell death pathways but also in that different cell death pathways may be triggered by different inciting stimuli. Thus, it may be necessary to target multiple death pathways simultaneously to achieve the desired clinical effect. Agents that target upstream initiators of cellular death pathways versus those that target end effectors have different advantages and disadvantages and need to be further explored.

Anti-Inflammatory Agents
Preclinical studies suggest that inflammation may contribute to photoreceptor degeneration. Thus, some efforts have been directed toward evaluating possible anti-inflammatory agents for the purpose of neuroprotection. Although better known for its role as a tetracycline antibiotic, doxycycline (Oracea) is also used to treat inflammatory disorders such as rosacea. The Phase II/III TOGA study was intended to evaluate the possible role for doxycycline in slowing enlargement of GA area (NCT01782989); although this study was initiated in 2013 and was listed as completed as of November 2020, no results have yet been published. There have also been small Phase I/II studies investigating the possible role of intravitreal or subconjunctival delivery of sirolimus, an inhibitor of mammalian target of rapamycin, to slow GA. Intravitreal sirolimus was found to have possible adverse effects, 33 whereas subconjunctival sirolimus was found to be well tolerated but without significant benefit. 34

Neurotrophic Factors
There are many endogenous molecules that are known to support the growth, survival, and differentiation of neurons, such as ciliary neurotrophic factor (CNTF), bone-derived neurotrophic factor, and glial cell line-derived neurotrophic factor, to name a few. These factors have been shown to be neuroprotective in numerous animal models of disease. As such, delivery of these factors may enhance photoreceptor survival in AMD.
NT-501/Renexus (Neurotech Pharmaceuticals) is an intraocular implant that delivers CNTF to the vitreous space via encapsulated cell technology. Subgroup analysis of an early study provided evidence that NT-501 may have a small benefit for some patients with GA. 35 Notably, this beneficial effect on VA was seen only in patients with VA 20/63 or better at baseline. Because CNTF failed in neuroprotection trials for patients with retinitis pigmentosa, 36 more recent studies are now focused on evaluating NT-501 for treating macular telangiectasia (NCT03316300, NCT03071965). It is also important to note that CNTF supplementation may have side effects in patients, such as miosis and suppressed electroretinogram responses, which may interfere with other aspects of visual function despite possibly promoting photoreceptor survival.
Other molecules have been found to have possible neuroprotective effect despite having a different primary effect. For example, brimonidine is an alpha-2-adrenergic agonist that is commonly prescribed for lowering intraocular pressure (IOP) in patients with glaucoma. Beyond its effect of lowering IOP, brimonidine is thought to be neuroprotective for retinal ganglion cells (RGCs) based on studies in animal models. 37,38 A possible neuroprotective effect for brimonidine is also supported by findings of the Low-Pressure Glaucoma Treatment Study, in which the investigators found that despite achieving similar control of IOP, glaucoma patients randomized to brimonidine had less disease progression compared with patients randomized to a different IOP-lowering drop (timolol). 39 One interpretation of this difference in effect, although not conclusive, is that brimonidine may also have a neuroprotective effect beyond its effect on IOP.
In view of this possibility, some investigators have tested whether brimonidine may also be neuroprotective for photoreceptors. The Brimonidine Drug Delivery System (Brimo DDS, Allergan, an AbbVie company) is an intravitreal implant designed as a depot formulation of brimonidine that can be administered via intravitreal injection and deliver brimonidine into the vitreous humor over a period of several months. A Phase II study in patients with GA found that there may be less progression in GA lesion area in patients randomized to Brimo DDS, although this difference was statistically significant only at month 3 and not at later time points. 40 Despite limitations of this early study, such as insufficient statistical power, use of the last-observationcarried-forward method for missing data, and lower completion rate in the implant group (76%) versus the sham group (91%), further studies are under way.
In addition to their role in treating psychiatric illness, serotonin A1 receptor (5-HT1A) agonists are thought to have a potential neuroprotective role in the eye. Despite promising preclinical studies in animal models, the Phase III GATE study showed that AL-8309B/tandospirone (Alcon) eye drops had no beneficial effect on GA growth in patients with AMD. 41 Another therapy that is well known for its other effects but may also have neuroprotective properties are corticosteroids. The fluocinolone acetonide intravitreal implant (Iluvien, Alimera Sciences) releases fluocinolone at a continuous low dose to the retina. Iluvien is approved for treatment of diabetic macular edema. 42 A clinical trial examining the possibility that Iluvien may treat GA was registered but later terminated before completing enrollment (NCT00695318).

Human Umbilical TissueeDerived Cell Delivery
Another approach for neuroprotection is subretinal delivery of human umbilical tissue-delivered cells (palucorcel or CNTO-2467, Janssen Pharmaceuticals). Although the mechanism by which these cells protect photoreceptors remains under active investigation, preclinical models show promising results. Initial human studies showed an unacceptably high rate of retinal perforations and retinal detachments when delivering these cells to the subretinal space. 43 The surgical technique has since been altered to make it safer, although the Phase II study still showed no significant decrease in GA area, retardation of GA growth rate, or improvement in VA in patients treated with palucorcel. 44

Retinal Pigment Epithelium Replacement
A different neuroprotective approach is based on the observation that RPE degeneration is observed in association with photoreceptor death in AMD. As such, some have hypothesized that partial restoration of the RPE may enhance photoreceptor resilience. OpRegen (Lineage Cell Therapeutics) is stem cellebased therapy that involves allogeneic RPE transplantation into the retina. It is currently being evaluated in a Phase I/IIa study (NCT02286089).
Another therapy involving allogeneic RPE transplantation is ASP7317 (Astellas), which is also still in Phase I (NCT03178149). Other devices, including the CPCB-RPE1 (Regenerative Patch Technologies) and PF-05206388 (Moorfields Eye Hospital) implants, attempt to maintain the polarized orientation of RPE cells by immobilizing the RPE cells to a scaffold/membrane. These implants are in the early safety/tolerability phase of study (NCT02590692 and NCT01691261, respectively). Although still in their infancy, these strategies of RPE transplantation would be seminal if they are successful, because they would support the idea that RPE degeneration causes photoreceptor degeneration.

Lessons Learned
Despite numerous attempts to achieve neuroprotection for AMD, many clinical studies have not been successful. Despite these disappointments, there are many lessons that can be gleaned from these past studies with numerous remaining questions to be addressed by further research. Key points that we have learned from past research are summarized in Table 2.

AMD as a Complex, Multifactorial Disease
There have been extensive investigations intended to better understand the pathologic mechanisms involved in AMD. Cumulatively, these studies have revealed numerous complex cellular and molecular processes that contribute to RPE and photoreceptor dysfunction, ultimately culminating in irreversible cell death and blindness. Despite numerous attempts to achieve neuroprotection with various strategies, many of these trials have not been fruitful. These failures support the notion that AMD is a complex, multifactorial disease, whose progression likely involves multiple pathophysiological processes that, in synergy, lead to observed cellular and tissue dysfunction.
One remaining question is the temporal relationship between RPE dysfunction and photoreceptor degeneration. This understanding would better guide treatments toward efforts to maintain RPE function or to preserve photoreceptor survival. Histopathological studies examining postmortem eyes from patients with GA have suggested that photoreceptor loss, especially rod photoreceptors, is observed early in absence of obvious morphological changes in Bruch's membrane or RPE. 45 In contrast, others speculate that accumulation of lipids and injury to RPE may precede photoreceptor degeneration. 46,47 Further studies are necessary to clarify this relationship.

The Value of Cellular and Animal Models
Past and current clinical trials are based on preclinical studies relying on cellular and animal models. Although in vitro and in vivo systems have many advantages, these systems are models of human disease and rarely recapitulate all features of a disease's complex pathogenesis. Models of AMD are no exception. The failures to translate many preclinical studies of AMD into novel therapeutics may reflect the imperfections of our present models.
One facile approach for studying AMD pathogenesis is using in vitro models. Many studies have evaluated factors involved in photoreceptor and RPE survival by using immortalized cell lines, such as 661W cells 48 or ARPE-19 cells. 49 These cell lines proliferate rapidly, allowing for ease of experimentation. However, these immortalized cell lines recapitulate only some features of their corresponding epithelial cell type. As such, findings at the in vitro level do not always translate to the tissue or organismal level. Another approach has been to study RPE cells derived from induced pluripotent stem cells (iPSCs) or iPSC-RPE cells. Because iPSC-RPE cells can be generated from adult somatic cells of patients with ocular diseases, they allow for analysis at the patient-level to elucidate how gene mutations contribute to disease pathophysiology, a valuable tool, especially for inherited retinal degenerative diseases. 50 One major limitation of all in vitro models is that they fail to account for the retina's highly specialized structure. The complex interactions between photoreceptors and other retinal neurons with supporting cells, including RPE and choroid, are impossible to recapitulate in vitro.
Animal models, especially mouse models, are relied on extensively in biomedical science, and research in AMD is no exception. In contrast with in vitro models, these tools have the advantage of being able to model tissue-and organismal-level pathology. However, to date, there is no mouse model that develops all features of AMD in a progressive, stepwise fashion, resembling the disease process in human patients. Instead, there have been multiple mouse models identified that recapitulate certain features of AMD, such as development of (1) drusen or drusen-like deposits, (2) changes in Bruch's membrane, (3) immune dysregulation in the retina, (4) oxidative damage in the retina, (5) photoreceptor and RPE cell death resembling GA, and (6) choroidal neovascularization. The details of these murine models, many of which involve genetic mutations and environmental factors, are the subject of other excellent reviews. 51,52 As an example of how animal models can still be used to help our understanding of human disease despite their limitations, we previously studied mutant mice lacking the genes for the chemokine CCL2 and the chemokine receptor CX3CR1 on a genetic background with the rd8 mutation of the Crb1 gene (Cx3cr1 -/-Ccl2 -/-Crb1 rd8/rd8 ). With age, these mice develop focal lesions and photoreceptor degeneration, which has now been shown to be due primarily to the rd8 mutation of the Crb1 gene rather than Cx3cr3/Ccl2 deletion. 53,54 We found that these mutant mice had higher gene expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3) in the retina compared with mice with the normal/wild-type Crb1 gene (Fig 3A), whereas there was no difference in gene expression of RIPK1 (Fig 3B). Receptorinteracting serine/threonine-protein kinase 1 and RIPK3 are known to be important for initiating the cell death pathway known as necroptosis. Moreover, when we also delete the gene coding for RIPK3 from these mice, there is significantly less accumulation of retinal pathology at 3 and 6 months of life (Fig 3C, D). Taken together, within the limitations of this model, our findings suggest that necroptosis may be involved in driving retinal pathology in mice and, thus, may have a role in photoreceptor death in AMD. These findings provide support for further investigation of necroptosis as a possible contributor to AMD pathology.
Other groups have also identified mouse models that recapitulate aspects of AMD pathobiology. Work by Toomey et al 55 and Landowski et al 56 highlighted the multifactorial contributions of immune regulation, diet, and age on retinal pathology. Prior genetic-wide association studies revealed a strong association between the Y402H variant of the gene for complement factor H (CFH), a regulator of the alternative complement pathway, and AMD. 57 Bowes Rickman's group identified that mice with monoallelic CFH deletion or with expression of the H402 human pathologic variant of CFH develop drusen-like, retinal deposits at advanced age when challenged with a high-fat diet. The contribution of impaired cholesterol homeostasis to AMD pathology was further supported by seminal work by the Apte and Grimm labs: mutant mice lacking the cholesterol efflux transporters ATP-binding  1. AMD is a complex, multifactorial disease that likely involves contributions from multiple pathologic processes that lead to photoreceptor death and blindness. 2. Cell-based models have their value but have inherent limitations because of their inability to capture the complexity of the human retina. 3. Rodent animal models, although helpful, have major limitations in their ability to model human disease given their lack of macula in addition to being rod photoreceptor-dominant, nocturnal animals. 4. Anti-VEGF agents have taught us that multiple diseases with heterogeneous underlying etiologies can be managed successfully with a common approach if they share a critical, common process. 5. Lessons from diverse neurodegenerative diseases, including beyond the eye, may be applicable to AMD and may lead unifying therapies that have widespread effects.
cassette transporter A1 and G1 specifically from macrophages, photoreceptors, or RPE all develop lipid-rich retinal deposits and associated retinal/RPE dysfunction mimicking AMD. 58e60 These mouse models highlight a possible role for immune dysregulation and lipid dyshomeotasis in AMD pathogenesis and may have utility in future studies to test novel therapeutic strategies. Although in vivo models have their value, the fact that they exhibit single features of disease in isolation challenges how relevant they may be for understanding the disease process in humans. Moreover, differences in retinal anatomy and structure when comparing humans with model organisms such as mice, including differences in types of photoreceptors (i.e., rod vs. cone photoreceptors) present in the retina, the underlying spatial distribution of the photoreceptors (i.e., presence or absence of fovea), and their nocturnal versus diurnal lifestyles, further complicate matters. Identification of new models or improvements of current models may be necessary to improve our understanding of complex AMD pathogenesis.

The Right Drug for the Right Patient
Clinical classification systems for AMD have been developed for large clinical studies, such as AREDS. 61 These classification systems imply that AMD progresses stepwise, although not necessarily linearly. However, it remains possible that AMD is not a monolithic disease. In fact, given the highly heterogeneous clinical presentation and clinical features of AMD, there may be multiple subtypes of AMD with different prognoses and underlying pathologic processes. This complexity has been highlighted by large population studies showing that patients with AMD typically have a combination of multiple genetic risk factors along with lifestyle factors influencing propensity for disease. 62 Advanced imaging techniques such as swept-source OCT and OCT angiography may be able to provide further insight regarding interindividual differences by identifying subclinical features of disease and may inform identification of AMD subclassifications. 63 Figure 3. Necroptosis may drive retinal pathology in a mouse model of AMD-like pathology. A, Mice lacking the genes for the chemokine receptor Cx3cr1 and the chemokine Ccl2 on a background strain with the rd8 mutation of the Crb1 gene (Cx3cr1/Ccl2 DKO Crb1 rd8/rd8 ; DKO ¼ double knockout) have higher retinal messenger RNA (mRNA) expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3) compared with wild-type (WT) mice, (B) whereas there was no difference in expression of RIPK1. C, D, Cx3cr1/Ccl2 DKO Crb1 rd8/rd8 mice that also have the gene for RIPK3 deleted have fewer focal lesions in the retina compared with Cx3cr1/Ccl2 DKO Crb1 rd8/rd8 mice that have intact RIPK3 at 3 months (3M) and 6 months (6M) of age (C, representative images with quantification of independent images in D). Graphs show mean þ standard error of the mean (A, B, D). *P < 0.05, **P < 0.01.
Another avenue for distinguishing subpopulations of patients with AMD who may respond differently to different therapies center around identifying biomarkers. As mentioned earlier, significant efforts have been undertaken to identify genetic mutations associated with AMD. 22,64,65 This research has identified multiple loci within genes coding for proteins with diverse functions. Because certain genetic mutations may be associated with a less robust response to anti-VEGF treatments or to antioxidant/vitamin supplementation, 5,66 it follows that patients with different underlying genetic mutations may have different underlying pathophysiology and thus might require different treatments for optimal management. Moreover, given a possible contribution of inflammation and immune dysregulation in AMD pathogenesis, others have focused on identifying inflammatory biomarkers, including both general serum markers of inflammation and markers of complement activation. 57,67 Finally, because dysregulation of lipid metabolism is also thought to contribute to AMD pathogenesis, others have investigated whether patients with AMD also have systemic perturbations in lipid homeostasis. 68,69 Given significant between-study heterogeneity related to both inflammatory and lipid biomarkers, more research is necessary to elucidate the clinical implications of the published findings. Nonetheless, we speculate that using biomarkers will likely be necessary for guiding precision-medicine efforts to tailor AMD treatments to a patient's underlying pathophysiology. The myriad biomarkers of AMD that have been investigated are reviewed extensively by Lambert et al. 70 The Right Drug at the Right Time Despite promising results from preclinical studies for numerous drugs that target various pathways thought to be involved in AMD pathogenesis, many of these drugs have failed to show efficacy in slowing GA progression in human clinical trials. One explanation for these failures is a true lack of clinical efficacy of the drugs. However, it remains possible that they are being tested in patients at too late a stage of AMD. In other words, it is possible that these drugs may have improved efficacy if they were used in early to intermediate AMD before the development of GA, although this poses a challenge because not all patients with early or intermediate AMD progress to advanced disease. Further studies are necessary not only to test this hypothesis but also to identify the characteristics of patients who may be at higher risk of progression to advanced disease and thus may benefit from early therapies.

Lessons Learned from Other Cells
The use of neuroprotective strategies has been explored extensively in other fields, including in neurodegenerative diseases within the central nervous system, such as for AD, Parkinson's disease, and neurodegeneration of other retinal cells, such as in glaucoma. We are not aware of any blockbuster successes in neuroprotection to date, but research in these other areas may have parallels to efforts in AMD that warrant consideration.
Work in the field of AD research may provide some lessons regarding properties of an ideal neuroprotective therapy for AMD. The hallmark pathological feature of AD is accumulation of extracellular plaques composed of betaamyloid and intracellular neurofibrillary tangles containing tau. Although there are multiple pathologic pathways involved in AD pathogenesis, 2 key contributors are amyloid precursor protein mis-metabolism leading to amyloid deposition and tau hyperphosphorylation leading to tau misfolding and propagation. 71 As a result, anti-amyloid and anti-tau strategies have been explored to reduce the extent of plaque and neurofibrillary tangle accumulation and possibly to slow onset of or to delay dementia. Nonetheless, despite significant efforts including numerous randomized clinical trials, none of these strategies have been successful to date. Of note, many of these therapies have succeeded in achieving their intended effect of reducing plaque or tau accumulation but without the patients having any clinical benefit. The anti-amyloid beta antibody aducanumab (Aduhelm; Biogen) was recently approved by the Food and Drug Administration under an accelerated pathway, but it was approved amid significant controversy and has not been widely implemented, with some even calling for the Food and Drug Administration to withdraw its approval. 72e74 Because many insurance companies have refused to cover its cost, and the Centers for Medicare and Medicaid Services announced that they would cover its cost only in qualifying clinical trials, Biogen recently withdrew its marketing application for aducanumab in Europe.
These failures bring to the forefront the idea that solely treating one pathological feature of disease may not be sufficient to achieve a desired clinical effect. Although they have different underlying compositions, drusen, the sine qua non of AMD, are like beta-amyloid plaques in that they accumulate with age but are found at much higher concentrations in patients with disease. The failures of prior trials in AD suggest that therapies that solely target drusen biogenesis may not be sufficient to prevent vision loss in patients with AMD. This phenomenon has been observed in the past with prior trials demonstrating that laser photocoagulation of drusen leads to their regression but without significant reduction in risk of developing vision loss. 75 Moreover, it is important to use patient-centered end points in future clinical trials in addition to molecular/imaging end points to ensure that novel therapies achieve not only a statistically significant but also a clinically significant benefit. Work in the field of Parkinson's disease may be informative in guiding how to approach neuroprotection in AMD. Parkinson's disease is a disease of progressive loss of dopamine-producing neurons in the substantia nigra, leading to cardinal signs of resting tremor, bradykinesia, rigidity, and postural instability. The exact cellular mechanisms leading to neuronal death remain elusive, with some proposing that alpha-synuclein misfolding, mitochondrial dysfunction, impaired protein clearance, and neuroinflammation may contribute. 76,77 Because the exact cause of neuronal death has not been established, current therapies center around enhancing dopaminergic signaling by administering dopamine precursors, dopamine agonists, or agents that slow dopamine metabolism. These therapies control motor symptoms but are not disease modifying insofar as they do not address the pathologic process causing dopaminergic neuron death and have limited durability. 78 Applying these lessons to AMD, we speculate that a successful treatment for AMD will likely have to address the underlying pathologic process or even multiple pathologic processes to achieve the desired effect. Moreover, given growing lifespans as a result of innovations in health care, it will be increasingly important to consider the long-term durability of treatments as well.
Basic research in the field of glaucoma also offers some insight. By using single-cell RNA sequencing, some investigators have identified that there are subpopulations of RGCs that are more vulnerable to degeneration in mouse models of axonal degeneration. 79 These vulnerable RGC subpopulations have unique genetic profiles. Beyond identifying novel molecular targets for neuroprotection, these findings may be relevant in that there may be photoreceptor subpopulations that are uniquely vulnerable. Finally, if there are common pathways involved in neurodegeneration of different tissues, it may be possible to develop unifying strategies for neuroprotection that can be used for multiple neurodegenerative diseases.

Our Perspective
One major limitation of the neuroprotection clinical trials to date is that they provide limited insight into the primary or causative cellular death pathways in AMD. Although there is not yet clear consensus regarding the predominant pathogenic pathways underlying AMD, our work and that of others in neurodegeneration have highlighted that there are redundant complementary cellular death pathways (e.g., apoptosis and receptor interacting protein kinaseeregulated necrosis) that may be involved in photoreceptor degeneration. Mechanistic studies have revealed that blocking an individual cellular death pathway may activate another and vice versa. 27,28,80e83 Furthermore, we have seen that different cellular death pathways may predominate in different cell types. 28e30, 81,82,84 Likewise, different upstream stimuli may lead to activation of different cellular death pathways. 31,32,85e87 For example, when ROS are the predominant cell death stimulus, ferroptosis (iron-dependent death) is the predominant pathway, 87e92 whereas when tumor necrosis factor alpha and FAS/FAS ligand are the predominant stimuli, apoptosis and receptor interacting protein kinaseedependent necrosis are the predominant pathways. 24,25,31,32,86,93e96 To make things more complex, infiltrating immune cells also contribute to neurodegeneration via the inflammasome and cross-talk with several other pathways. 80,85,97e106 On the basis of these findings, we speculate that successful future approaches must target multiple pathways; optimal neuroprotection may require inhibition of upstream and downstream targets simultaneously because cellular death activation is stimulus dependent. This multifactorial approach is not new in medicine. In many other diseases, such as cancer, human immunodeficiency virus, and tuberculosis, combinatorial therapies are the rule rather than the exception. 107e109 The key challenges surrounding neuroprotection as a strategy for treating AMD are summarized in Table 3.

Conclusions
Age-related macular degeneration is a significant cause of blindness worldwide. Neuroprotection has emerged as a new approach for preventing photoreceptor degeneration and subsequent blindness. Although clinical studies to date have not yielded significant results, neuroprotection holds promise for prevention of vision loss caused by photoreceptor death. More research and different approaches are necessary before neuroprotection can emerge as the new frontier for AMD. We remain hopeful that as we gain a more complete understanding of the complexity of AMD, successful clinical results will emerge in the form of novel neuroprotection strategies to treat patients who have AMD and similar conditions. Yakuhin Ltd., Santen Pharmaceutical Co. Ltd., AMO Japan Co. Ltd., Senju Pharmaceutical Co. Ltd., Otsuka Pharmaceutical Co. Ltd. Dr Demetrios Vavvas, MD, PhD, an Editor of the Journal, was recused from the peer-review process of this article and had no access to information regarding its peer review.
HUMAN SUBJECTS: No human subjects were included in this study. All research adhered to the tenets of the Declaration of Helsinki. The requirement for informed consent was waived because of the retrospective nature of the study.
No animal subjects were used in this study.


Macrophages: shapes and functions

Macrophages are cells of the innate immune system and represent an important component of the first-line defense against pathogens and tumor cells. Here, their diverse functions in inflammation and tumor defense are described, and the mechanisms, tools, and activation pathways and states applied are presented. The main focus is on the role and origin of reactive oxygen species (ROS), the important signal pathways TLR/NF-κB, and the M1/M2 polarization of macrophages. Graphical abstract

Introduction
This lecture text has been written to introduce the nature and function of macrophages to students of biochemistry, pharmacy, chemistry, and similar subjects. Macrophages are important cells of the innate or nonspecific immune system present in all vertebrates. Like all immune cells, macrophages are derived from a pluripotent hematopoietic stem cell in the bone marrow. From this, on the one hand, lymphocytes and natural killer (NK) cells and certain dendritic cells develop via a lymphatic precursor cell. On the other hand, erythrocytes, platelets, and precursors of granulocytes and macrophages develop via a myeloid progenitor cell; monocytes, neutrophils, basophils, or eosinophils arise from the aforementioned precursors. Monocytes that migrate from the circulation into tissue mature into macrophages (Figs. 1, 2). In the circulation, the normal range of monocytes is between 2% and 8% of the white blood cell count, corresponding to 200-800 monocytes per microliter of blood. Monocytes are round cells with a kidney-shaped nucleus and are 25-30 µm in size. Upon maturation into macrophages, they assume different shapes depending on their functionality (see "M1/M2 macrophage polarization") and usually become even larger. Macrophages can be  which represent the cellular arsenal of the immune system (with kind permission [75]) fetal liver. In adulthood, in the absence of stimulatory factors, circulating monocytes do not substantially contribute to the tissue content of macrophages. Other tissues, such as the heart and gastrointestinal tract, recruit monocytic precursors to a greater extent where immigrating monocytes differentiate into resident macrophages. In adult life, resident macrophages in many tissues represent a mix of cells originating during development and from circulating monocytic precursors. In adult humans, however, due to the extended life span, the majority of tissue macrophages seems to be recruited from the circulation [4,24,46]. Macrophages function in the defense against pathogens and in the clearance of old, senescent, or dead cells, but also fulfill important functions in tissue homeostasis and repair, e.g., wound healing and muscle regeneration [25]. Various macrophage populations exist that play distinct and non-redundant roles in fibrosis, tissue repair, and regeneration [26,69]. It still remains to be established, however, whether resident or recruited macrophages represent functionally different subsets or are able to assume all possible states depending on the cellular/ tissue microenvironment.
Macrophages are an important part of the first line of defense. During infection, inflammation, or tissue injury, they can follow chemotactic signals to migrate to the damaged tissue/inflammatory site, where they ingest pathogens and cell debris through a process called phagocytosis and digest them in phagolysosomes, which results from the fusion of phagosomes with lysosomes. Lysosomes are membrane-enclosed organelles containing a set of hydrolases capable of breaking down the diverse classes of macromolecules. This degradation not only contributes to pathogen removal and clearance of an inflammatory site, for example, but also facilitates the presentation of resulting peptides to cells of the specific immune system, like T or B lymphocytes. This happens via presentation on major histocompatibility complex II (MHC II) molecules. Thereby, an antigen-specific immune response can be initiated. In addition, phagolysosome-associated enzymes facilitate the production of large amounts of reactive oxygen species (ROS) that function to effectively kill ingested pathogens.
Through the action of stimulatory factors, e.g., in the case of tissue injury, large numbers of inflammatory monocytes, macrophage precursors, are recruited from the bone marrow via chemokine gradients and various adhesion molecules. In such pathophysiological situations, the migrating (recruited) macrophages far outnumber the tissue-resident macrophages [9,22]. Consequently, growth factors and cytokines released in the local tissue microenvironment force recruited and resident macrophages to proliferate and undergo substantial phenotypic and functional changes [34].
Depending on microenvironmental stimuli and the nature of the pathogen/antigen structure a macrophage encounters, differentiation into M1 or M2 macrophages could be favored. The predominant differentiation profile determines the functional orientation of the macrophages.
In this article emphasis is put on the (I) macrophage's effector functions, (II) ROS produced, (III) the signaling pathways engaged, and (IV) the role of macrophage subpopulations in health and disease.

Macrophage effector functions
As mentioned earlier, phagocytosis is an outstanding ability of macrophages and represents one of their main effector mechanisms. Macrophages effectively internalize particulate structures, with the uptake of pathogens being of particular relevance. The recognition and binding of such structures takes place through their opsonization by means of complement factors or antibodies. For this purpose, macrophages are equipped with specific receptors, some of which also represent macrophage-specific surface markers (immune cell surface markers are classified as clusters of differentiation, CDs). These are mainly the representatives of the complement receptors, CR1-CR4, as well as the so-called Fcγ receptors, FcγRI, FcγRII, and FcγRIII ( Table 2). The Fcγ receptors bind with different affinity to the Fc tail of the immunoglobulin G (IgG) molecule, which is located opposite to the antigen-binding domains of IgG and, thus, still accessible when IgGs have specifically bound to, e.g., the surface of pathogens, thereby enabling their phagocytosis. This process is known as antibody-dependent cellular phagocytosis (ADCP). Ideally, this process leads to ROS-mediated killing and enzymatic digestion of pathogens or other particulate matter in the phagolysosome. Peptide fragments consisting of 12-25 amino acids that originate from protein (pathogen) digestion can be loaded onto major histocompatibility complex (MHC) class II molecules when endoplasmic reticulum (ER)-derived vesicles containing newly synthesized MHC molecules fuse with phagolysosomes. Peptide (antigen)-loaded MHC class II molecules are then transported to the cell surface ( Fig. 2), where they serve to activate cells of the specific immune system, in particular T helper cells (T H cells), in an antigen-specific way.
Immune cell recruitment and activation represent two other major macrophage effector functions. One mechanism by which macrophages, whether recruited in response to a stimulus or tissue-resident ones, attract and activate other immune cells is the production and release of soluble factors which include proinflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNFα), IL-6, IL-12, as well as chemokines like C-X-C motif chemokine ligand 8 (CXCL8, IL-8). This happens as soon as macrophages encounter pathogens/damage. Whereas IL-1β and TNFα cause, among other things, endothelial activation, increased vascular permeability, and activation of lymphocytes, IL-12 leads to the activation of natural killer (NK) cells, a type of cytotoxic lymphocyte critical to the innate immune system. NK cells function to eliminate virus-infected cells and cells infected by other intracellular pathogens, and tumor cells. Macrophages have typical receptor equipment for the effective recognition of pathogens. These so-called pattern recognition receptors (PRR), whose best-known representatives belong to the Toll-like receptors (TLR), recognize conserved structures that are characteristic of different classes of pathogens. Such typically pathogenic and therefore "foreign" structures are also referred to as pathogenassociated molecular patterns (PAMPS) and include, among others, double-stranded RNA, unmethylated CpG DNA, lipoproteins, and lipopolysaccharides (LPS) (Fig. 3).
Activation of the TLRs by binding PAMPs triggers the probably most typical signaling pathway of the nonspecific defense, the NF-κB signaling pathway, which ultimately leads to the production/release of the aforementioned cytokines (Fig. 4). Macrophage activation via TLRs, but also by, e.g., the macrophage-activating cytokine, interferon-γ, leads to increased phagocytotic activity and pathogen killing. The increased production of ROS/reactive nitrogen species (RNS) plays an important role in the effective killing of phagocytosed pathogens.

Generation of ROS/RNS
The production of antimicrobial reactive oxygen and nitrogen species (ROS/RNS) is an important microbicidal mechanism of phagocytic cells. Congenital enzyme defects associated with a lack of or reduced activity of ROS/RNSforming enzymes manifest themselves in the incomplete killing of pathogens or tumor cells and affected patients  suffer from recurrent infections or cancer, respectively. ROS are generated intracellularly as by-products during various electron-transfer reactions, especially in the mitochondrial respiratory chain. However, receptor-mediated stimulation of ROS production is the key to important effector functions of macrophages. Various receptors, including complement receptors, TLRs, and receptors for bacterial peptides (e.g., the fMLF receptor) induce the production of ROS/ RNS in phagolysosomes. Here, the assembly of the NADPH oxidase plays a dominant role. Its subunits p22 and gp91, which are located in the cell membrane and therefore in the phagolysosome, become assembled with the initially cytosolically located subunits (p40, p47, and p67) which provides the active NADPH oxidase. The formation of the active NADPH oxidase leads to an increase in oxygen consumption, which is referred to as "oxidative burst". This creates superoxide anion radical ( O ⋅− 2 ) in the lumen of the phagolysosome, which is converted into H 2 O 2 by superoxide dismutase (SOD) [59]. Further reactions lead to the formation of toxic ROS, including OH ⋅ and OCl − from H 2 O 2 . The antimicrobial repertoire of macrophages also includes toxic nitrogen oxides, especially NO ⋅ . In cells that produce both O ⋅− 2 and NO ⋅ , such as macrophages, the near diffusion-limited reaction between NO ⋅ and O ⋅− 2 is likely to occur and yields peroxynitrite ( ONOO − ) [17]. The peroxynitrite ion itself is not highly reactive; however, its acid form, peroxynitrous acid (ONOOH) is very unstable and reactive. Therefore, it is the formation of peroxynitrous acid that is considered as the mechanism underlying superoxide anion radical-mediated toxicity when Fenton chemistry is not at work [17]. NO ⋅ is produced in macrophages by the inducible NO synthase (iNOS) which in comparison to the forms constitutively expressed in other tissues, nNOS and eNOS, produces much larger amounts of NO ⋅ . Increased expression/activity of iNOS and the resulting increased production of NO ⋅ have been associated with many pathophysiological processes, including trauma, inflammation, ischemia/hypoxia, and a severe drop in blood pressure as observed during septic shock. Mechanistically, NO ⋅ is formed by the oxidation of l-arginine which results in citrulline in addition to NO ⋅ as shown below: For this purpose, arginine can be withdrawn from the serum, where it is present at almost 100 µM concentrations, with the kidney being the most important source.

Macrophage cell signaling
The acquisition/exercise of the various effector functions of macrophages requires the specific activation of signaling pathways in the cells. TLR ligands, cytokines, immunocomplexes, hypoxia and CD4-positive T lymphocytes, provisional extracellular matrix (ECM) and collagen remodeling and tissue structure are important stimuli that lead to the activation of macrophages [50], e.g., via Jak/STAT, NF-κB, and redox signaling.

Cytokines and JAK/STAT signaling
Cytokines represent a large group of regulatory proteins that act primarily on the immune cell differentiation, activation, and function. This also applies to macrophages. Exposure to certain cytokines results in an adequate response of macrophages to various challenges such as infection, tissue damage, tumor, or alloantigen. This is achieved by a subtle balancing of production, release, or activation of a large set of individual cytokines whose effects are defined by complex synergistic or antagonistic interactions. Besides their actions on macrophages and other immune cells, cytokines potently affect other cells and tissues.
Members of the heterologous group of cytokines bind to different types of specific receptors, among them receptor kinases (e.g., TGF-β1 receptor) and tyrosine kinase-linked receptors that lack intrinsic kinase activity. The latter group of receptors is also referred to as cytokine receptors (e.g., IL-1β and TNFα receptor).
Ligand binding to these receptors by a wide variety of cytokines or growth factors generally provokes di-or trimerization of monomeric subunits, which in turn induces rapid activation of kinases associated with the receptor, typically members of the JAK (Janus kinase) family of tyrosine kinases. Phosphorylation of the receptor itself enables recruitment and binding via SH2 (src homology 2) domains of members of the STAT (signal transducers and activators of transcription) family of transcription factors to the receptor. This allows JAKs to phosphorylate STATs, which subsequently form dimers that are capable of entering the nucleus and specifically initiate transcription of target genes, which include cytokines, SOCS1-3, and transcription factors such as GATA3, C-Maf, c-myc, NFAT1/NFAT2 [30,33].

TLR/NF-κB signaling
The NF-κB family of transcription factors plays a crucial role in inflammatory and apoptotic responses. Family members include NF-κB1 p50, NF-κB2 p52, RELA (also called p65), RELB, and c-REL1 which as homodimers or heterodimers mediate transcription of NF-κB target genes [66]. They are retained in the cytoplasm by interaction with the inhibitory molecule IκB. Two NF-κB activation cascades can be discriminated. In the classical (canonical) activation pathway, in response to various signals IκB becomes phosphorylated by serine protein kinases, mainly IKK2 but also IKK1. IκB is then degraded in the proteasome, facilitating the translocation into the nucleus of p50/p65 NFκB. This heterodimer is the most abundant complex, often referred to as being "NF-κB". It initiates and enhances the expression of target genes including iNOS, the chemoattractant cytokine, IL-8, the inflammatory TNFα, IL-1β, and IL-6, and adhesion molecules (ICAM1) and chemokines (IP-10, MCP-1, MIP-1; Fig. 5, for details see [45]). In the alternative activation cascade, IKK1 mediates processing of p100 to p52, the latter forming heterodimers with relB which upon transfer into the nucleus initiate transcription of NF-κB target genes. C-Rel is of special importance for, e.g., the transcriptional induction of IL-12 p40 in macrophages [38].
Some stimuli preferentially activate the alternative pathway; others, including lymphotoxin-β (LTβ) and LPS, activate both cascades [10,11]. It is supposed that the two NF-κB cascades are activated in a sequential manner: the early response mediated by p65 containing dimers, the latter then continues a response with relB-containing complexes to sustain the NF-κB activation [54].
Oxidative stress, viral proteins, growth factors (angiotensin II), mitogens, the inflammatory cytokines IL-1β and TNFα via IL1 /IL-18 receptors or TNF receptors, respectively, NOD-like receptors, and TLR ligands activate IKKβ [31]. Activation of all these receptors also feeds into other signaling pathways that interfere, directly or indirectly, with the NF-κB signaling pathway [14]. Upon activation by LPS, TLR4 through Myd88 activates NF-κB, but in addition activates mitogen-activated protein kinase (MAPK), IRF5 pathways, and via endosomal TLR4 signaling, IRF3 and to TLR leads to the association of adapter molecules such as MyD88, Toll-interacting Protein (TOLLIP), the protein kinase IRAK, and TRAF6 (TNF-receptor associated factor 6). TRAF6 activates IκB kinases 1 and 2 (IKK-1/2) via the kinase TAK1 (TGF-β activated kinase). These kinases phosphorylate IκB, which leads to the degradation of the inhibitory protein and releases NF-κB as a dimer. NF-κB migrates into the cell nucleus and induces a transcriptional activation of proinflammatory and immunomodulatory genes ( [76] with kind permission) the production of the strong antiviral interferons become induced [14]. Autocrine effects of any cytokines released from activated macrophages further add to the complexity of resulting responses.
In humans, at least ten different TLRs exist which differ in their specificity towards distinct and characteristic pathogenic structures, like LPS, double-stranded RNA or unmethylated CpG DNA, so-called pathogen-associated molecular patterns. For this reason, TLRs are also called PRRs (pattern recognition receptors). The first TLR to be identified is TLR4, which is activated by LPS (gram-negative bacteria). LPS is bound to the macrophage surface via LBP and is bound by TLR4 in this complex (Fig. 4).

Redox signaling
Accumulating evidence supports a signaling role for ROS that are generated by macrophages during oxidative burst.
In the last few years the view has hardened that ROS-at least when present in certain concentrations-can be viewed as a second messenger. In full agreement with this function, ROS show certain characteristics such as stimulus-dependent transient induction (e.g., via the activation of NADPH oxidase), specific function (interfering specifically with signaling components like, e.g., kinases), and the rapid termination of the signal by antioxidant enzymes/antioxidants (catalase, superoxide dismutase, glutathione peroxidases) [18]. The modulation of the intracellular concentration of calcium [Ca 2+ ] i is a general mechanism involved here because many intracellular signaling pathways actually depend on it.
ROS have been shown to induce the activation of NF-κB, although the underlying mechanisms still remain to be elucidated fully [32]. Reversible S-glutathiolation has been established as one mechanism underlying the regulation of, e.g., the transcription factor activator protein 1 (AP1) [19].
It is well established that ROS produced by macrophages ( O ⋅− 2 and the species subsequently formed therefrom) contributes to the oxidation of low-density lipoproteins (LDL). Oxidized LDL is rapidly taken up by macrophages leading to their transformation into "foam cells", which are associated with an increased risk for atherosclerosis and complications that are related to this. Notably, oxidized LDL itself has been shown to interfere with many signaling pathways in macrophages [41].

M1/M2 macrophage polarization
Monocytes/macrophages are characterized by a strong plasticity and heterogeneity. Dependent on the surrounding micromilieu and the immunological context, macrophages can acquire distinct functional characteristics through a process called polarization. The polarization leads to maturation and formation of "classically activated" M1 or "alternatively activated" M2 macrophages, respectively, which represent major functional macrophage categories. The alternatively activated macrophages have been further subcategorized ( Table 3). The M1 macrophages have been associated with enhanced phagocytotic activity, antigen-presenting capacity, and increased synthesis and release of proinflammatory cytokines. The cytokine interferon-γ (IFNγ) was the first identified substance converting resting macrophages into M1. Substances known to cause/promote the formation of M1 macrophages now include bacterial LPS and autocrineacting IFNβ.
M1 macrophages exert strong cytotoxic activity against infected cells, remove pathogens during infection, and mediate resistance against infections. This is achieved by enhanced production and secretion of proinflammatory/ activating cytokines, adhesion molecules, chemokines, and cyclooxygenase-2 (Cox2) as well as by increased production of O ⋅− 2 due to activation of NADPH oxidase [45,64]. M1 macrophages may cause inflammation and tissue damage by these mechanisms, particularly if resolution of inflammation and initiation of tissue repair is not initiated or not initiated in time.
The polarization towards M2 cells, on the other hand, is mediated by the cytokines IL-4 and IL-13. M2 macrophages may promote angiogenesis and neovascularization, stromal activation and remodeling, tissue repair, fibrosis, and is fairly anti-inflammatory in general. In support of this view, IL-4 has been shown to inhibit oxidative burst and inflammatory cytokine expression, e.g., of IL-1β.
In the context of cancer, M2 macrophages have been associated with tumor progression and immunosuppression, thereby negatively affecting the prognosis of patients.
Different functional states of macrophages have been associated with cell shape changes [50]. Vice versa, by applying a micropatterning approach, macrophage cell shape could be directly controlled, demonstrating that elongation per se, without any exogeneous cytokines is able to induce the expression of M2 makers (Table 3) such as arginase-1 or CD206 [50].
It is becoming increasingly clear that the M1/M2 polarization also affects the macrophage metabolism. Metabolic disorders such as type 2 diabetes mellitus (T2DM) are therefore associated with drastic consequences for macrophage polarization and function. Classically activated, proinflammatory M1 macrophages depend to a large extent on glycolysis and produce lactate as the tricarboxylic acid cycle is blocked at two steps. Alternatively activated M2 macrophages prefer β-oxidation and oxidative phosphorylation to synthesize ATP (for an excellent review, see [61]). It is well established that continuous and excessive inflammatory stimuli, e.g., in various inflammatory and metabolic diseases, including T2DM, lead to macrophage activation. In the situation of T2DM, a particulate activation state arises, which is defined by expression of specific markers that include macrophage scavenger receptor (MSR1), ATP-binding cassette subfamily A member 1 (ABCA1), perilipin-2 (PLIN2), in addition with CD11b and CD36 [5,39,61].
Macrophages are also considered as key modulators in cancer progression. Infiltration of macrophages in and around tumors (tumor-associated macrophages, TAMs) is regularly observed and their presence is associated with poor prognosis in solid tumors [13,23]. The interaction of TAMs with either tumor cells or the tumor microenvironment drives macrophage polarization. For a long time it was assumed that TAMs originate from circulating monocytes [48]. In a murine mammary carcinoma model, tumor growth was accompanied by loss of resident cells and repopulation of the TAMs by monocytes [20]. TAMs generally exhibit an M2 phenotype [2]. In contrast, macrophages of embryonic origin persisted in pancreatic ductal adenocarcinoma and significantly contributed to disease progression and fibrosis [74]. Although they have no cytotoxic activity themselves, they produce growth factors for tumor cells and exhibit immunosuppressive activity [2]. M2 macrophages may promote angiogenesis and neovascularization, remodeling of stroma and the tumor niche, and dissemination of tumor cells all contributing to tumor progression. Fast-growing solid tumors typically have only insufficient oxygen supply resulting in hypoxic zones, especially in the tumor center or in perinecrotic areas. Mechanistically, this hypoxia via induction of, e.g., IL4 and IL-10, contributes to the recruitment and M2 polarization of macrophages. TAMs, therefore, preferentially localize in these hypoxic areas of a tumor [35]. The M1 phenotype functions in phagocytosis and lysis of tumor cells [43]. Through their enhanced antigen-presentation ability, M1 macrophages enable cytotoxic leukocyte functions. Increased production and release of inflammatory cytokines such as TNFα, IL-6, or IL-12 contribute to these effects and may support tumor cell apoptosis [43].
There is ample evidence that the location of macrophages in and around the tumor is critical to the polarization and, thus, function of macrophages. Location-related signals enforce distinct macrophage functions. In this sense, M2 macrophages promote tumor cell motility and invasion, favor metastasis, and stimulate neoangiogenesis [28,42,43,57]. Most notably, the tumor-related distribution pattern of macrophages has been identified as an independent prognostic factor in gastric cancer [44].

Macrophages as therapeutic targets
The involvement of macrophages/TAMs in the aforementioned processes such as cellular homeostasis, repair mechanisms, regeneration, and angiogenesis make macrophages a promising target, e.g., in tumor therapy. Of high relevance for patients with cancer is the fact that TAMs impair the effectiveness of chemotherapeutic agents, radiation, and angiogenesis inhibitors [13,15,60]. As stated earlier, the tumor environment, including tissue fibrosis, hypoxia, availability of nutrients, and lymphocyte-derived factors all determine the macrophage phenotype [13]. TAMs are thought to generally display an M2-like phenotype [2]. However, recent data suggest that TAMs show an elevated degree of heterogeneity. This could be observed both between different patients with tumors and between different tumor lesions in a patient. Accordingly, TAMs can acquire a whole spectrum of phenotypic and functional profiles in response to environmental stimuli [70]. Accordingly, specific TAM subpopulations support oncogenesis via diverse mechanism which include stimulation of angiogenesis, matrix degradation, and resistance to therapy; others seems to support the efficacy of various anticancer (immuno)therapies [1,48,70]. TAMs that are located in perivascular or hypoxic areas exhibit a rather M2-like phenotype and show proangiogenic, profibrotic, and immunosuppressive properties [40]. In contrast, TAMs located at the tumor invasion site often show a proinflammatory M1-like phenotype and display rather tumoricidal properties [72].
It was assumed that TAMs are predominantly derived from circulating monocytes [53,65], but up to 50% of TAMs in murine models of lung, brain, and pancreatic cancer have been shown to originate from tissue-resident macrophages [3,47,74]. A promising therapy is to stop the replenishment of TAMs from circulating monocytes. As the latter strongly depends on CCL2-CCR2 signal transduction for their mobilization from the bone marrow and the recruitment to the tumor tissue, CCR2 blockade actually improved the efficacy of chemotherapy, radiation therapy, and immunotherapy [8,21,37,55,62]. In a similar approach, these effects are associated with increased T cell infiltration, a finding that was also observed in some preclinical models. Therefore, further combinations with checkpoint immunotherapy are currently under investigation.
With another strategy, namely the inhibition of the CSF1/ CSF1R axis, the apoptotic death of TAMs is counteracted. Indeed, CSF1R inhibition improved T cell responses in combination with chemotherapy or radiation therapy in several animal models [12,51,71].

The dark side of macrophages
Because of the great heterogeneity and plasticity that macrophages exhibit it is not surprising that they are involved in a multitude of functions and processes ranging from the defense against pathogens, the clearance of old, senescent, or dead cells, maintenance of tissue homeostasis and mediating fibrosis, tissue repair, and regeneration, tumor growth, and immunosuppression. In the traditional view the M2 macrophage phenotype is pro-regenerative and associated with positive wound healing outcomes, whereas the M1 phenotype is proinflammatory and associated with pathogenesis. According to today's understanding, this is more complex: both M1 and M2 macrophages play different, but equally vital, roles in organizing/mediating tissue repair [49]. As explained earlier, for the therapy of cancer, the immunosuppressive, tumor growth-promoting M2 phenotype should be overcome and the inflammatory tumoricidal M1 phenotype should be supported. Any missing, delayed, or incomplete termination of an inflammatory reaction against pathogens, cell debris, or the like can lead to fatal adverse effects from macrophages. Failure to appropriately terminate an inflammatory response, in the worst case, can lead to an excessive recruitment/activation of immune cells and the excessive production and release of cytokines, also referred to as "cytokine storm" or "cytokine release syndrome" (CRS) [67]. As currently defined, cytokine storm is a condition associated with elevated systemic cytokine levels, systemic clinical signs of inflammation, and severe secondary tissue damage [16]. As excellently reviewed in [16], a cytokine storm can be induced by pathogenic invasive infectious disease [58,73] or as a side effect of chimeric antigen receptor T-cell (CAR-T) cell therapy [27,52], a recent cancer immunotherapy approach. Depending on the triggering agent, the induced cytokine profile can be of different composition [67]. While, e.g., CRS caused by SARS leads to the predominant release of IL-1β, IL-6, IL-12, IFNγ, IP10, and MCP-1 [6], COVID-19-associated CRS [56] has been characterized by increased release of IL-2, IL-7, IL-10, G-CSF, IP10, MCP-1, MIP-1α, and TNFα [29]. If pathogens are involved, typically PRRs and NF-κB signaling play an essential role. Therefore, macrophages and macrophage-derived cytokines such as MCP-1, MIP-1α, TNFα, and IL-8 regularly contribute to the development of a cytokine storm.

B lymphocytes
A type of white blood cell of the lymphocyte subtype, upon activation by an antigen, a naïve or memory B cell proliferates and differentiates into an antibody-secreting effector cell, known as a plasma cell. CD molecules Cell surface molecules, which are expressed on various types of cells in the immune system that are designated by the cluster of differentiation or CD number. C-Maf (Proto-Oncogene C-Maf), a DNAbinding, leucine zipper-containing transcription factor that acts as a transcriptional activator or repressor; among its related pathways is NFAT-dependent transcription in lymphocytes. c-myc (MYC Proto-Oncogene), a member of basic helix-loop-helix (BHLH) transcription factors. Complement (Also referred to as complement cascade), a system of serum and cell surface-bound proteins that is a part of the immune system and functions to enhance (complement) the ability of antibodies and phagocytic cells to remove microbes and damaged cells from an organism, (Cytokine-inducible SH2-containing protein), a family of proteins involved in inhibiting the JAK-STAT signaling pathway. T lymphocytes A type of white blood cell of the lymphocyte subtype; CD8 + T cells, also known as cytotoxic T cells, are able to directly kill virus-infected cells, as well as cancer cells. CD4 + T cells function as "helper cells": they recognize antigen peptides presented by MHC II and function by further activating memory B cells and cytotoxic T cells, which leads to a larger immune response.
Funding Open Access funding enabled and organized by Projekt DEAL.
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