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Mast Cells Play an Important Role in Chlamydia pneumoniae Lung Infection by Facilitating Immune Cell Recruitment into the Airway
Norika Chiba, Kenichi Shimada, Shuang Chen, Heather D. Jones, Randa Alsabeh, Anatoly V. Slepenkin, Ellena Peterson, Timothy R. Crother and Moshe Arditi
J Immunol April 15, 2015, 194 (8) 3840-3851; DOI: https://doi.org/10.4049/jimmunol.1402685
Norika Chiba
*Division of Pediatric Infectious Diseases and Immunology, Department of Biomedical Sciences, Cedars-Sinai Medical Center and David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90048;
Kenichi Shimada
Heather D. Jones
†Division of Pulmonary and Critical Care Medicine, Department of Medicine, Cedars-Sinai Medical Center and David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90048;
Randa Alsabeh
‡Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center and David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90048; and
Anatoly V. Slepenkin
§Department of Pathology, University of California Irvine, Irvine, CA 92697
Ellena Peterson
Timothy R. Crother
Moshe Arditi
PDF + SI
Mast cells are known as central players in allergy and anaphylaxis, and they play a pivotal role in host defense against certain pathogens. Chlamydia pneumoniae is an important human pathogen, but it is unclear what role mast cells play during C. pneumoniae infection. We infected C57BL/6 (wild-type [WT]) and mast cell–deficient mice (KitW-sh/W-sh [Wsh]) with C. pneumoniae. Wsh mice showed improved survival compared with WT mice, with fewer cells in Wsh bronchoalveolar lavage fluid (BALF), despite similar levels of cytokines and chemokines. We also found a more rapid clearance of bacteria from the lungs of Wsh mice compared with WT mice. Cromolyn, a mast cell stabilizer, reduced BALF cells and bacterial burden similar to the levels seen in Wsh mice; conversely, Compound 48/80, a mast cell degranulator, increased the number of BALF cells and bacterial burden. Histology showed that WT lungs had diffuse inflammation, whereas Wsh mice had patchy accumulations of neutrophils and perivascular accumulations of lymphocytes. Infected Wsh mice had reduced amounts of matrix metalloprotease-9 in BALF and were resistant to epithelial integral membrane protein degradation, suggesting that barrier integrity remains intact in Wsh mice. Mast cell reconstitution in Wsh mice led to enhanced bacterial growth and normal epithelial integral membrane protein degradation, highlighting the specific role of mast cells in this model. These data suggest that mast cells play a detrimental role during C. pneumoniae infection by facilitating immune cell infiltration into the airspace and providing a more favorable replicative environment for C. pneumoniae.
Chlamydia pneumoniae, an obligate intracellular pathogen, is responsible for up to 10–20% of cases of community-acquired pneumonia and is associated with many chronic inflammatory disorders, including atherosclerosis, asthma, and Alzheimer's disease (1–6). Most people will become seropositive for C. pneumoniae, and the titer of anti–C. pneumoniae Ab increases with age, indicating continued exposure and immune response throughout life (1–7). Thus, understanding the mechanism of C. pneumoniae–induced inflammatory responses of the host remains an important endeavor. We and other researchers reported that C. pneumoniae is recognized by the pattern recognition receptors, such as TLR2 and TLR4, which signal through MyD88 and Trif, and the NOD-like receptors, Nod1 and Nod2, which signal through Rip2 (8–11). More recently, we and other investigators showed that C. pneumoniae activates the NLRP3 inflammasome directly (12–14), resulting in mature IL-1β production, which is critical for proper immune responses against C. pneumoniae infection (14, 15). In the airway, C. pneumoniae first infects alveolar macrophages and airway epithelial cells, which, in turn, secrete proinflammatory cytokines and chemokines that result in an influx of inflammatory cells, such as monocytes, macrophages, and neutrophils (10, 16, 17). One report observed that C. pneumoniae infection could induce human mast cells (MCs) to produce cytokines in vitro and that a pulmonary C. pneumoniae infection in mice led to MC degranulation in vivo (18).
MCs are best known for playing a key role in allergy, anaphylaxis, and host defense against helminth parasites by releasing chemical mediators, such as histamine, leukotrienes, and MC proteases (MCPTs). Recently, it was reported that MCs can recognize bacterial pathogens through TLRs and NOD-like receptors, produce proinflammatory cytokines (19–21), and play an important role in host defense against bacteria (12, 22–29). MCPTs, such as tryptase and chymase, degrade many proteins, including extracellular matrix (30, 31). In addition, chymase (MCPT4) was reported to activate matrix metalloprotease (MMP)-9 by cleaving a specific site of the catalytic domain of MMP-9 (32-34); chymase inhibitors reduce both the pro- and active forms of MMP-9 and attenuate its enzymatic activity in vivo (35, 36). MMP-9 is of particular interest in pulmonary infections because it is necessary for both neutrophil infiltration into the lung and airspace (35, 37) and intratracheal migration of dendritic cells (38, 39). Furthermore, MMP degrades claudins, adhesion molecules important in cell–cell tight junctions, during influenza-induced lung injury (39–41). In the lung, several claudin family members are expressed, including claudin-5 and -18, which are components of alveolar tight junctions (37, 38, 42); their degradation is important for paracellular permeability and cell transmigration (40, 43).
In the current study, we found that MCs were required for normal immune cell infiltration into the airspaces during a C. pneumoniae lung infection in mice. MC deficiency resulted in faster bacterial clearance and reduced lung inflammation. Prevention of MC degranulation phenocopied MC deficiency, whereas pharmacological induction of MC cell degranulation resulted in greater inflammatory responses to C. pneumoniae infection. MC-deficient mice had less MMP-9 secretion and activation, as well as less degradation of claudin-5 and -18, suggesting that MCs are required for the opening of tight junctions to allow immune cells to infiltrate into the airspaces during a bacterial infection. Finally, MC reconstitution in Wsh mice resulted in greater bacterial growth, increased MMP-9, and degradation of claudin-5. These data suggest an unappreciated role for MCs as gatekeepers to facilitate immune cell infiltration into the airspace by regulating tight junction opening during C. pneumoniae lung infection, where immune cell infiltration into host lungs is required for normal C. pneumoniae propagation. Thus, unlike most other infections in which MCs are protective, MCs appear to play an important role in the pathogenesis of C. pneumoniae infection in mice.
Animals and reagents
C57BL/6 (wild-type [WT]) and KitW-sh/W-sh (Wsh) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Eight- to ten-week-old female mice were used in all experiments in this study. All experiments were done according to Cedars-Sinai Medical Center Institutional Animal Care and Use Committee guidelines. Cromolyn and compound 48/80 were purchased from Sigma-Aldrich (St. Louis, MO).
C. pneumoniae infection and sample preparation
C. pneumoniae CM-1 (American Type Culture Collection, Manassas, VA) was propagated in HEp-2 cells, as previously described (8, 44). Both HEp-2 cells and C. pneumoniae aliquots were confirmed as being Mycoplasma-free by PCR. Mice were infected with either 2 × 106 or 5 × 106 infectious units (IFU) C. pneumoniae by inoculating intratracheally. Bronchoalveolar lavage fluid (BALF) was collected by injecting 0.5 ml PBS containing 5 mM EDTA. BALF was separated into supernatant and cells by centrifugation. Supernatant was used for cytokine and chemokine measurements and Western blotting. Cells were smeared on glass slides and stained by modified Wright–Giemsa staining (Diff-Quick; Fisher Scientific, Pittsburgh, PA) to determine subtypes of leukocytes by their cellular and nuclear characteristics. Lungs were homogenized with 1 ml ice-cold sucrose–phosphate–glutamate medium for cytokine and chemokine measurements and bacterial burden quantification.
Bacterial burden quantification
HEp-2 cells were infected with C. pneumoniae in lung specimens for bacterial burden quantification, as described previously (45, 46). Briefly, HEp-2 cells were incubated with diluted lung suspensions or cell lysates in the presence of 1 μg/ml cycloheximide and 10 μg/ml gentamicin containing RPMI 1640 medium. Centrifugation was performed for 1 h at 800 × g, and cells were placed in an incubator (37°C, 0.5% CO2). After 72 h of culture, cells were washed with PBS, fixed with methanol, and stained with FITC-conjugated anti-Chlamydia genus-specific mAb (Pathfinder Chlamydia Culture Confirmation System; Bio-Rad, Hercules, CA), according to the manufacturer's instructions. C. pneumoniae inclusions in cells were counted by fluorescence microscopic analysis.
MC reconstitution
Bone marrow cells from C57BL/6 mice were cultured in RPMI 1640 medium supplemented with 10% FBS and 20% WEHI-3–conditioned media for 6–8 wk to differentiate into bone marrow–derived MCs (BMMCs). BMMCs were confirmed as c-kit+ FcERIa+ by flow cytometry. Six-week-old Wsh mice received 5 × 106 BMMCs via tail vein injection. Twelve weeks after reconstitution, mice were sacrificed, and tissues and peritoneal lavages were collected and assessed for reconstitution of MC deficiency.
Flow cytometry analysis
Lungs were digested enzymatically to isolate leukocytes in the tissue for flow cytometric analysis, as previously described (17, 47). Briefly, lungs were digested at 37°C for 20 min in HANKS buffer containing 40 U/ml Liberase and 50 U/ml DNase I (both from Roche Diagnostics, Indianapolis, IN). Cells were filtered through a 70-μm cell strainer (BD Biosciences, San Jose, CA). Erythrocytes were obviated by RBC Lysis Buffer (eBioscience, San Diego, CA). Single cells were stained with the following Abs: F4/80 Ab, CD11c Ab, Ly6G Ab, CD11b Ab, triggering receptor expressed on myeloid cells 1 (TREM-1) Ab, CD4 Ab, CD44 Ab, c-kit Ab, IgE Ab, Lamp1 Ab, and CD62L Ab. For lung single-cell differentials, cell types were identified as neutrophils (CD11c− CD11b+ Ly6G+), alveolar macrophages (CD11b+ CD11c+ F4/80+), and T cells (CD3+) with either CD4 or CD8 positivity. MCs were identified as side scatterhigh and c-kit+ IgE+. MC degranulation was evaluated by surface LAMP1 staining. For intracellular Chlamydia staining, cells were permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences) and stained with anti-Chlamydia LPS mAb (Accurate Chemical and Scientific, Westbury, NY) and PE-conjugated anti mouse. Flow cytometric analysis was performed using a CyAn flow cytometer (Beckman Coulter) and analyzed using Summit software (Dako, Carpinteria, CA).
Evaluation of vascular permeability
Mice received 25 mg/kg Evans blue dye via tail vein injection 2 h before lung harvest. Lungs were homogenized with 1 ml PBS, centrifuged, and pellets were incubated with 500 ml formamide at 60°C for 18 h. The supernatant was measured at absorbencies of 620–740 nm. For airway permeability, mice were injected with Evans blue dye, as above, and sacrificed 2 h later, and BALF was obtained. The supernatant was measured at absorbencies of 620–740 nm.
Histopathological analysis
Lungs were fixed in formalin buffer and embedded in paraffin for histological analysis. Paraffin-embedded lungs were cut and stained with H&E. Immunohistochemistry was performed using anti-CD3 Ab (clone 2GV6) prediluted from Ventana Medical Systems (Tucson, AZ), anti-CD20 Ab (goat polyclonal; Santa Cruz Biotechnologies, Dallas, TX), and anti-Ly6G Ab (clone 1A8; eBioscience). Sections incubated with isotype-control Ab and HRP-conjugated secondary Ab were used as negative controls. Staining was developed with either 3,3′-Diaminobenzidine substrate alone or, in some cases, 3,3′-Diaminobenzidine substrate plus cobalt and nickel chloride (Roche Diagnostics). Images were taken using either a Keyence BZ-9000 microscope or an Olympus BX-51 microscope.
The concentration of cytokine in BALF and lung homogenates was determined using an OptEIA Mouse IL-6 ELISA Set (BD Biosciences) and Mouse IL-12p40, Mouse IFN-γ ELISA Kit (eBioscience), and DuoSet Mouse MIP-2 and Mouse KC (R&D Systems, Minneapolis, MN). These assays were performed according to the manufacturers' instructions.
Western blotting was performed as described previously (10, 48, 49). Briefly, lungs from C. pneumoniae–infected mice were homogenized and lysed in PLC lysis buffer containing proteinase inhibitors (Protease Inhibitor Cocktail; Sigma-Aldrich). Lung cell lysates and BALF were boiled in sample buffer, and proteins were separated with SDS-PAGE. Separated proteins were transferred to BioTrace polyvinylidene difluoride membrane (Pall, Pensacola, FL). Membranes were probed with anti-mouse claudin-5 Ab (mouse polyclonal), anti-human claudin-18 Ab (rabbit polyclonal; both from LifeSpan Biosciences, Seattle, WA), and anti-mouse MMP-9 Ab (rabbit polyclonal; Abcam, Cambridge, MA), followed by HRP-conjugated goat anti-mouse or anti-rabbit Ab (Thermo Fisher Scientific, Rockford, IL). Chemiluminescence was developed using an enhanced Immun-Star WesternC Kit and documented using ChemiDoc XRS+ (both from Bio-Rad). Densitometry was performed using Image Lab software (ver 3.0; Bio-Rad) and was normalized to β-actin.
Data are presented as mean ± SD. For survival studies, significance was evaluated by the Fisher exact test. Statistical significance was evaluated using the Mann–Whitney test. For experiments in which more than two samples were analyzed, one-way ANOVA with the Tukey post hoc test were used to assess statistical significance between groups.
MC–deficient mice are protected from C. pneumoniae lung infection–induced mortality
Although MCs were reported to be activated during C. pneumoniae infection, these data were obtained several weeks after inoculation (18). Therefore, we infected WT mice (2 × 106 IFU) with C. pneumoniae and sacrificed them 4 d postinfection. Using flow cytometry, we identified MCs in the lung and did not find a significant change in their numbers at this time (Supplemental Fig. 1A). However, using LAMP1 as a marker for degranulation (50, 51), we found that MCs appeared to be activated during C. pneumoniae infection, because LAMP1 surface staining increased in these MCs (Fig. 1A). To investigate the role that MCs might play during C. pneumoniae infection in mice, we used Wsh mice, which are deficient for MCs. WT and Wsh mice were infected with C. pneumoniae (5 × 106 IFU/mouse) intratracheally and monitored for mortality. All of the WT mice died from severe C. pneumoniae lung infection by 14 d postinfection, whereas ∼50% of the Wsh mice survived until the end of the experiment (day 31) (Fig. 1B), suggesting that the lack of MCs provide protection from C. pneumoniae infection. To understand this unexpected observation, we assessed the bacterial burden in the lung of C. pneumoniae–infected WT and Wsh mice at days 3, 5, and 14 post–C. pneumoniae infection (Fig. 1C). We used a lower dose of infection for the rest of these studies compared with the mortality experiment (2 × 106 versus 5 × 106 IFU). There was no significant difference in bacterial number between WT and Wsh mice at day 3, but at days 5 and 14 there was a significant decrease in C. pneumoniae IFU in Wsh mice compared with WT mice. We next investigated proinflammatory cytokines (IL-6, IL-12p40, and IFN-γ) that are known to be responsible for C. pneumoniae clearance. Wsh mice had increased IL-6 in the lung and increased IL-12p40 in BALF early in infection (day 3) (Fig. 1D). Similarly, IL-6 and IL-12p40 also were increased in the lungs at day 5 in Wsh mice compared with WT mice. However, this was reversed by day 14, and Wsh mice had significantly less IL-6 and IL-12p40 in both BALF and lung compared with WT mice. Interestingly, there was no difference in the amounts of IFN-γ between Wsh and WT mice at 3 and 5 d postinfection. IFN-γ is a key mediator of C. pneumoniae clearance and the prevention of persistent infection (12, 22–29). However, similar to IL-6 and IL-12p40, IFN-γ concentrations were significantly diminished in BALF at 14 d postinfection in Wsh mice. Although the reduction in cytokines at day 14 in Wsh mice was likely due to the decreased bacterial burden, we did not find this correlation at 5 d postinfection when Wsh mice had increased cytokines, yet a significant decrease in C. pneumoniae IFU (Fig. 1C, 1D).
Wsh mice are protected from C. pneumoniae–induced mortality and C. pneumoniae propagation in the lung compared with WT mice. (A) WT mice were infected with C. pneumoniae (2 × 106 IFU) or mock infected and sacrificed 4 d later. Single cells from the lung were prepared as described in Materials and Methods. MCs were identified as side scatterhigh c-kit+ IgE+ (Supplemental Fig. 1A). Surface expression levels of LAMP1 were assessed by flow cytometry and reported as the mean fluorescence intensity (MFI). (B–D) WT and Wsh mice were infected with C. pneumoniae intratracheally (5 × 106 IFU for mortality and 2 × 106 for others). (B) C. pneumoniae–induced mortality was observed by 31 d post–C. pneumoniae inoculation. (C) C. pneumoniae IFU in the lung from C. pneumoniae–infected WT and Wsh mice was quantified as described in Materials and Methods. (D) BALF was collected from euthanized mice, and the lung was perfused with cold PBS and homogenized. The experiment in (A) was performed one time (n = 5). The experiment in (B) was performed twice, and the data were pooled together (total n = 13 [WT]; n = 10 [Wsh]). The experiment in (C) and (D) was performed three times, and the data were pooled together (n = 10 [WT and Wsh] day 3; n = 7 [WT and Wsh] day 5; n = 7 [WT], n = 10 [Wsh] day 14). Cytokine concentration in BALF and lung homogenates was detected by ELISA. Data are mean ± SD. *p < 0.05, **p < 0.005, ***p < 0.001, Mann–Whitney test.
MCs enhance immune cell infiltration into the airway
We hypothesized that the number of macrophages and neutrophils would be increased in Wsh mice compared with WT mice because we observed that C. pneumoniae clearance was faster and IL-6 and IL-12p40 concentrations were elevated in Wsh mice compared with WT mice at an early time point in infection. However, there was no difference between Wsh and WT mice on day 3, but there was a significant reduction in the BALF total cell number in Wsh mice compared with WT mice at both 5 and 14 d postinfection (Fig. 2A). We next determined the inflammatory cell composition in BALF for Wsh and WT mice. We found that, on days 5 and 14, the reduction in cell numbers in BALF could be accounted for largely by the significant decrease in neutrophil numbers, along with a reduction in macrophages on day 14 (Fig. 2B, Supplemental Fig. 2A). Next, we analyzed immune cell infiltration in the lung tissue. Surprisingly, in contrast to BALF data, total cell number in the lung was significantly increased in Wsh mice compared with WT mice at day 3; a similar trend was noted at day 5 (Fig. 2C). Macrophage and neutrophil numbers were significantly increased in the lungs at 3 and 5 d postinfection in Wsh mice compared with WT mice (Fig. 2D, Supplemental Fig. 2B–D). There were no significant differences in the numbers of lymphocytes between Wsh and WT mice (Fig. 2D), including the number of regulatory T cells (Tregs) (Supplemental Fig. 2E). To corroborate the differences that we found between BALF cells and lung tissue cells in Wsh mice, we measured the concentration of neutrophilic chemokines, KC and MIP-2, in BALF and lungs of WT and Wsh mice (Fig. 2E). Although the number of neutrophils in BALF from Wsh mice was reduced, there was significantly more KC in both BALF and lung homogenates at 3 and 5 d postinfection, as well as in BALF at 14 d postinfection (Fig. 2E). There was no difference in MIP-2 concentrations between Wsh and WT mice on days 3 and 5 postinfection. To assess whether vascular permeability was affected, we injected Evans blue dye into C. pneumoniae–infected mice. Importantly, the vascular permeability was not altered in Wsh mice during C. pneumoniae infection (Supplemental Fig. 3A, 3B). Although there was no difference in the amount of Evans blue dye in the lungs of WT and Wsh mice, we also measured the amount of dye present in BALF. Similar to the cellular-infiltration data, there was less Evans blue dye in BALF from Wsh mice compared with WT controls (Supplemental Fig. 3C). Collectively, infiltrating immune cells into the airway were decreased in Wsh mice compared with WT mice, whereas immune cells in the lung were increased. Although pulmonary vascular permeability appeared to be normal, airway permeability was not. At the same time, although KC concentrations were increased in both BALF and lung homogenates in Wsh mice, this did not result in increased neutrophils in BALF.
Wsh mice displayed reduced inflammatory cell infiltration into the airspace compared with WT mice. WT (filled bars) and Wsh (open bars) mice were infected with 2 × 106 C. pneumoniae intratracheally. BALF was collected from euthanized mice on 3, 5, and 14 d post–C. pneumoniae propagation. Total (A) and differential (B) cell counts in BALF. Cell types in BALF were determined by modified Wright–Giemsa staining. (C and D) Single cells from lung were collected through enzymatic procedure and analyzed by flow cytometry. (E) Chemokine concentrations in BALF and lung homogenates were determined by ELISA. This experiment was performed three times, and the data were pooled (total n = 7 [WT and Wsh] day 0; n = 10 [WT] and n = 8 [Wsh] day 3; n = 9 [WT and Wsh] day 5; and n = 7 [WT] and n = 9 [Wsh] day 14). Data are mean ± SD. *p < 0.05, **p < 0.005, ***p < 0.001, Mann–Whitney test.
Chlamydial burden is significantly reduced in macrophages and neutrophils in MC-deficient mice
C. pneumoniae is an obligate intracellular bacteria and needs host cells to replicate. C. pneumoniae replicates mainly in macrophage/monocytes and neutrophils and, to a lesser degree, in epithelial cells (10, 49, 52, 53). We analyzed the presence of intracellular C. pneumoniae by flow cytometry to determine which cells were infected by C. pneumoniae in MC-deficient mice. On day 3, we did not find any significant differences in C. pneumoniae staining in BALF or lung cells between WT and MC-deficient mice (data not shown). This was not unexpected, because both mice had similar bacterial burdens at that time (Fig. 1B). Intracellular C. pneumoniae staining was mainly restricted to macrophages at this time (BALF and lungs). However, although intracellular macrophage C. pneumoniae staining was identical at day 3, macrophages in the lungs of WT mice had an increased C. pneumoniae burden (Fig. 3A) compared with Wsh mice. By day 5, there was a significant decrease in the amount of intracellular C. pneumoniae staining in Wsh macrophages and neutrophils in both BALF and lung cells compared with WT mice (Fig. 3B). These data suggest that C. pneumoniae infected and replicated in macrophages during the first 3 d and then infected newly infiltrating macrophages and neutrophils. However, perhaps with the reduction in macrophages and neutrophils infiltrating the airways of Wsh mice, C. pneumoniae replication was limited by the lack of sufficient numbers of host immune cells.
C. pneumoniae–positive macrophages and neutrophils were reduced in Wsh mice compared with WT mice. WT and Wsh mice were infected with 2 × 106 IFU C. pneumoniae intratracheally. Mice were sacrificed either 3 or 5 d post–C. pneumoniae infection. BALF cells and lung cells were collected at day 3 (A and B) or day 5 (B and D) and stained with anti–C. pneumoniae, anti-CD11c, anti CD11b, anti-F4/80, and anti-Ly6G for flow cytometric analysis, as before. (A) Representative graphs for C. pneumoniae staining in gated populations (see Supplemental Fig. 1 for gating strategy). (B) The percentage of C. pneumoniae–positive cells in BALF and lung at day 3. (C) Representative graphs for C. pneumoniae staining in gated populations on day 5. (D) The percentage of C. pneumoniae–positive cells (Cpn+) in BALF and lung at day 5. In (A) and (B), the experiment was performed twice (n = 5 [WT] and n = 6 [Wsh]). In (C) and (D), the experiment was performed twice (n = 4 [WT] and n = 4 [Wsh]). Data shown are a representative experiment. Data are mean ± SD. *p < 0.05, Mann–Whitney test.
MC degranulation is required for C. pneumoniae–induced immune cell infiltration into the airspace
Because MCs seemed to be required for proper immune cell infiltration into the airways of C. pneumoniae–infected mice, we next investigated the role of MC degranulation in this process. To address this question, we used cromolyn, a MC degranulation inhibitor (52, 54). WT mice were treated with cromolyn (3 mg/kg body weight) intratracheally 1 d prior to and 1 d after C. pneumoniae inoculation (Fig. 4). Similar to C. pneumoniae–infected Wsh mice, cromolyn-treated mice displayed reduced total BALF cell number and neutrophil number in BALF (Fig. 4A), as well as relatively increased total cell number in the lung (Fig. 4B). KC and MIP-2 were not significantly different between the groups that were treated or not with cromolyn (Fig. 4C). Strikingly, C. pneumoniae IFU was reduced significantly in cromolyn-treated mice (Fig. 4D).
Cromolyn reduces inflammatory cell infiltration into the airspace and bacterial number in the lung. WT mice were treated with cromolyn (3 mg/kg body weight) before and after C. pneumoniae infection (2 × 106 IFU) and sacrificed 5 d postinfection. (A) Total and differential cell counts in BALF. Cell types in BALF were determined by modified Wright–Giemsa staining. (B) Single cells from lung were collected through enzymatic procedure and analyzed by flow cytometry for differential cell counts. (C) Chemokine concentrations in BALF and lung homogenates were determined by ELISA. (D) C. pneumoniae IFU in the lungs was quantified as described in Materials and Methods. For (A)–(D), the experiment was performed twice (n = 5 [Cpn] and n = 6 [Cpn + Crom]). Data shown are a representative experiment. Data are mean ± SD. *p < 0.05, ***p < 0.001, Mann–Whitney test.
Because our data suggested that MC degranulation is required for proper immune infiltration into the airways, we hypothesized that inducing excess degranulation pharmacologically would have the opposite effect, with greater numbers of cells in the BALF and an increased bacterial burden. We used compound 48/80, an MC degranulation inducer, to investigate the role of MC degranulation in C. pneumoniae lung infection (40, 54). WT mice were treated with compound 48/80 (2 mg/kg body weight) intratracheally 2 d after C. pneumoniae inoculation, followed by sacrifice on day 5. As predicted, compound 48/80–treated mice displayed increased total cell numbers in BALF (Fig. 5A), whereas the total cell number in the lung was not changed (Fig. 5B). Compound 48/80–treated mice also had increased KC in BALF and MIP-2 in the lung homogenates compared with nontreated mice (Fig. 5C). Finally, C. pneumoniae IFU were significantly increased in compound 48/80–treated mice compared with control mice (Fig. 5D). These data suggest that MC degranulation plays a role in immune cell infiltration of the airway and is beneficial for C. pneumoniae replication.
Compound 48/80 increased inflammatory cell infiltration into the airspace and bacterial number in the lung. WT mice were treated with compound 48/80 (2 mg/kg body weight) 2 d post–C. pneumoniae infection and sacrificed on day 5. (A) Total and differential cell counts in BALF. Cell types in BALF were determined by modified Wright–Giemsa staining. (B) Single cells from lung were collected through enzymatic procedure and analyzed by flow cytometry. (C) Chemokine concentrations in BALF and lung homogenates were determined by ELISA. (D) C. pneumoniae IFU in the lung from C. pneumoniae–infected WT and Wsh mice was quantified as described in Materials and Methods. For (A)–(D), the experiment was performed twice (n = 5 [Cpn] and n = 7 [Cpn + Comp]). Data shown are a representative experiment. Data are mean ± SD. *p < 0.05, **p < 0.01, Mann–Whitney test.
MC-deficient mice have impaired immune cell distribution in the lung during C. pneumoniae infection
Next, we observed the histology of lung tissue from C. pneumoniae–infected WT and Wsh mice. At day 3 postinfection, there were no obvious differences between WT and Wsh mice (data not shown). However, at both days 5 and 14 we found that infiltrating cells had accumulated in the perivascular regions of Wsh lungs, whereas in WT mice the immune cells were more evenly distributed (Fig. 6A). Also at days 5 and 14, Wsh lungs displayed patchy inflammatory lesions in lung parenchyma, whereas WT lung inflammation was much more diffuse (Fig. 6A). The perivascular accumulations in Wsh mice contained large numbers of B and T cells (Fig. 6A). Although these structures looked superficially like inducible BALT, their appearance by 5 d postinfection precludes them from being normal inducible BALT, because they generally are a product of a long-term immune response. Additionally, these accumulations did not provide any advantage of acquired immunity later, such as C. pneumoniae–specific IgG production or effector memory T cell numbers in draining lymph nodes and IFN-γ production in spleen (data not shown).
Wsh mice display patchy inflammation, accumulated T and B cells perivascularly, and accumulated neutrophils in the lung during C. pneumoniae–induced lung inflammation. WT and Wsh mice were infected with C. pneumoniae intratracheally. Lungs were harvested for H&E staining and immunohistochemistry for B cells and T cells on days 5 and 14 (A) and for neutrophils on day 5 (B). Arrows denote neutrophil staining. This experiment was performed three times (total n = 7 [WT and Wsh] day 5, n = 7 [WT], and n = 10 [Wsh] day 14). Scale bar, 0.5 mm.
Neutrophil staining revealed a patchy distribution in Wsh lungs compared with WT lungs, which showed a very diffuse staining pattern (Fig. 6B), although the neutrophils were not quite as clustered as were the T and B cells. These data suggest that MCs play a role in regulating inflammatory cell distribution in the lung and infiltration into airways.
MC-deficient mice are defective in claudin degradation during C. pneumoniae lung infection
Next, we investigated the role that claudins might play in the defective immune cell infiltration into airways of Wsh mice. Claudins are important components of tight junctions, and their degradation is required for paracellular permeability of fluid, as well as immune cells (40). We analyzed claudin-5 and -18 protein levels in the lungs of WT and Wsh mice by Western blotting. When infected with C. pneumoniae, the lungs of Wsh mice displayed significantly reduced claudin degradation (both claudin-5 and -18) in comparison with WT mice (Fig. 7A), although there was no difference in the amounts of claudin proteins in the lungs of uninfected WT and Wsh animals (Fig. 7A). Because claudins are degraded by MMP-9 in vivo (40, 55, 56), we assayed the presence of MMP-9 in BALF of infected WT and Wsh mice and found that secreted MMP-9 was indeed reduced in Wsh BALF compared with WT BALF (Fig. 7B). MCs are known to secrete MMP-9 (32, 55, 56). During degranulation, MCs also release chymase, a major MMP-9 activator in vivo (30, 32). Thus, it is possible that, during a C. pneumoniae infection in mice, MCs release and activate MMP-9 (most likely via chymase), which then degrades claudin-5 and -18 in epithelial tight junctions and allows infiltrating immune cells into the airspaces.
Cell–cell tight junction molecule degradation was reduced in Wsh lung compared with WT lung during C. pneumoniae lung infection. WT and Wsh mice were infected with 2 × 106 IFU C. pneumoniae intratracheally and sacrificed on day 5. (A) Lung cell lysates were separated by SDS-PAGE, and immunoblotting was performed with the indicated Abs. The representative Western blots show relative amounts of claudin-5 and -18 in the lung from naive and C. pneumoniae–infected WT and Wsh mice. (B) BALF was separated by SDS-PAGE, and immunoblotting was performed with anti-MMP-9 Ab to determine secreted MMP-9 in BALF. In (A), the experiment was performed three times (n = 5 [WT] and n = 6 [Wsh]). In (B), the experiment was performed twice (n = 4 [WT] and n = 4 [Wsh]). Data shown are a representative experiment. Data are mean ± SD. *p < 0.05, Mann–Whitney test.
In addition to claudin degradation, TREM-1 was reported to be required for neutrophil transepithelial migration in the lung (25, 29, 30). However, we did not find any significant difference in TREM-1 cell surface expression between WT and Wsh neutrophils in BALF or lung during infection (data not shown).
MC reconstitution restores WT phenotype to Wsh mice
Wsh mice have an inverted Kit gene upstream of the normal gene that leads to a selective reduction in Kit expression and resulting MC deficiency. However, these mice also exhibit other abnormalities, including splenic myeloid and megakaryocytic hyperplasia (57). Therefore, to assess the MC deficiency phenotype in Wsh mice, we adoptively transferred WT MCs into Wsh mice and infected them with C. pneumoniae. Reconstitution with WT MCs resulted in an increased bacterial burden, similar to WT mice (Fig. 8A). Additionally, histopathology revealed a diffuse immune cell staining in MC-reconstituted mice that was similar to that seen in WT mice (Fig. 8B). These data demonstrated that the difference in bacterial burden between Wsh and WT mice is solely due to the lack of MCs and is not related to any other underlying features of Wsh mice. MC reconstitution also restored claudin-5 degradation and MMP-9 secretion and activation (Fig. 8C), highlighting the critical role of MCs in these processes during C. pneumoniae infection.
MC reconstitution in Wsh mice restores normal responses to C. pneumoniae infection. (A–C) Wsh mice were given 5 × 106 of MCs i.v. and allowed to reconstitute for 12 wk. Then the mice were infected with C. pneumoniae and sacrificed 5 d later. (A) Lung C. pneumoniae IFU was quantified at day 5 postinfection. (B) H&E staining. (C) Western blot analysis of claudin-5 and MMP-9 in the lung. Data are mean ± SD. *p < 0.05, one-way ANOVA and Tukey post hoc test.
In this study, we investigated the role that MCs play during C. pneumoniae infection of the lung in mice. Using MC-deficient mice, we were surprised to discover that Wsh mice were protected from C. pneumoniae infection, with decreased mortality, reduced pulmonary inflammation, and lower bacterial titers. These observations were juxtaposed with a decrease in immune cell infiltrates into the airspaces, as well as an increase in immune cells into the lungs. Interestingly, although there was no difference in vascular permeability between Wsh and WT mice in the whole lung, there was less Evans blue dye detected in BALF of Wsh mice compared with WT mice. Histopathology of the lungs revealed Wsh mice to have patchy inflammation with large immune cell clusters in a perivascular distribution, whereas WT mice had a much more diffuse staining pattern. Pharmacological manipulation of MCs with cromolyn, a MC stabilizer, phenocopied MC deficiency with decreased inflammation and bacterial burden, whereas compound 48/80, which induces MC degranulation, exacerbated infection with increased inflammation and bacterial burden. The results of these pharmacological interventions suggest that MC degranulation is important in the response to C. pneumoniae infection and, possibly, following bacterial persistence. MC deficiency also coincided with a lack of claudin-5 and -18 degradation in the lungs during C. pneumoniae infection, with reduced amounts of MMP-9 in BALF, suggesting that MCs may be required for the opening of tight cell junctions to allow infiltrating immune cells access to the airspaces. Critically, MC reconstitution in Wsh mice led to normal bacterial growth, claudin-5 degradation, and increased MMP secretion.
Although it is clear that MCs play a critical role in immune cell recruitment to the airspaces during C. pneumoniae infection, further investigation is needed to identify how these MCs are activated. In our model, it is likely that MCs are not directly activated by C. pneumoniae, but rather by cytokines produced by immune cells in response to C. pneumoniae infection. Peritoneal MCs exposed to C. pneumoniae directly were not activated (data not shown), suggesting that a secondary mechanism is at play. Indeed, MCs were shown to have far-reaching effects, with systemic MC activation playing a critical role in local inflammatory responses (58). However, a previous publication found that human MCs could produce cytokines in response to C. pneumoniae directly (in vitro) and that, in a pulmonary C. pneumoniae infection in mice, MCs were degranulated at a greater rate (18). Thus, it may be possible that C. pneumoniae infection can induce cytokine production directly, as well as indirectly induce degranulation. Indeed, we found evidence for MC degranulation 4 d after C. pneumoniae infection based upon increased LAMP1 detection on the surface of MCs in the lung.
In recent studies, MCs have emerged as an important cell type in both viral and bacterial infections. Francisella tularensis can activate MCs via TLR2, which results in enhanced macrophage killing of Francisella by secreting IL-4 (25, 29, 59). TNF-α produced by MCs plays a critical role in neutrophil recruitment to the lungs during Klebsiella pneumoniae infection (28, 59). MC-deficient mice are also more susceptible to Mycoplasma pneumoniae infection, with greater bacterial burden and mortality (28). Dietrich et al. found that MCs are primed to respond to both Gram-positive and Gram-negative bacteria by the secretion of proinflammatory cytokines (12). However, unlike these studies, we found that MC-deficient mice were protected from mortality and had a reduced C. pneumoniae burden in the lungs. This is somewhat similar to the role that MCs play during influenza A virus infection in mice. MC-deficient mice were protected from body weight loss and inflammation when infected with H1N1 influenza A virus (14, 15). However, despite the lack of inflammation, there was no difference in viral titers in that model. It might come from the difference in target cells between C. pneumoniae and H1N1 influenza A, which infects epithelial cells more than macrophages (60). Thus, although there seems to be a general consensus that MCs induce a potent inflammatory response to infection, depending on the organism, this inflammation can be beneficial, damaging, or, as in our study, lead to unproductive inflammation and greater bacterial growth. Indeed, in a recent study, MC-deficient mice were protected from Streptococcus pneumoniae pneumonia, although this was independent of MC degranulation (61). C. pneumoniae requires host cells for productive replication. Initial pulmonary infection occurs mainly in alveolar macrophages and, to a lesser degree, in epithelial cells. In our study, MCs facilitated the influx of neutrophils and other macrophage/monocytes into the airspaces where the bacterial progeny of the first round of replication could infect them, leading to a longer and more damaging infection. Indeed, it was reported previously that neutrophil depletion promotes bacterial clearance during C. pneumoniae lung infection (10, 16, 17) and that neutrophils are a critical site of C. pneumoniae replication (62, 63), again suggesting that neutrophils play an important role as an infectious target of C. pneumoniae.
The key to the protection found against C. pneumoniae infection in MC-deficient mice was primarily the lack of immune cell recruitment to the airspaces, despite increased chemokine production. In other models, MCs were found to be important in immune cell recruitment. In the experimental autoimmune encephalomyelitis model of multiple sclerosis, MCs in the dura mater and pia mater exacerbate the inflammation by facilitating neutrophil recruitment and promoting blood brain barrier and cerebral spinal fluid–blood barrier breakdown to allow immune cells access to the CNS (19–21). Additionally, in a Pseudomonas LPS model of pulmonary inflammation, MCs mediated the influx of neutrophils into the lung (12, 24–29). One possible mechanism for the control of neutrophil infiltration into the lungs was by controlling TREM-1 expression on neutrophils. TREM-1 was found to be required for transepithelial migration of neutrophils (30). However, we did not find any differences in TREM-1 expression in neutrophils in MC-deficient mice, indicating that the regulation of immune cell infiltration by MCs in C. pneumoniae infection is independent of TREM-1. Additionally, MC-deficient mice displayed abnormal T and B cell distribution in the lung later in the course of C. pneumoniae infection, suggesting that MCs regulate neutrophil transmigration, as well as the migration of other cells during infection.
MCs can make and secrete chymase, a critical protease that can activate many other cellular products by their cleavage (32). One such downstream product is MMP-9, an important metalloproteinase that is responsible for cleaving many surface exposed cellular proteins (35). In our study, we found that MMP-9 was present in BALF of WT infected mice, and it was drastically reduced in MC-deficient mice. One function of MMP-9 is to break down tight junctions, allowing cellular infiltration (35). Claudins are a group of extracellular adhesion proteins responsible for tight junction formation (38), and MMP-9 was shown to degrade claudins in epithelial tight junctions to allow transepithelial migration of immune cells (39, 40). Our data clearly showed that, during C. pneumoniae infection in mice, both claudin-5 and -18 were degraded in the lungs and that Wsh mice were protected from this degradation. Reconstitution of MCs led to the restoration of claudin-5 degradation and MMP-9 secretion concomitantly with an increased bacterial burden, similar to WT mice. Therefore, we propose that the lack of immune cell infiltrates into the airways in MC-deficient mice during C. pneumoniae infection is due to the inability of the host to break down the epithelial cell tight junctions as a consequence of the lack of MC-derived MMP-9. Although a disrupted chemokine gradient could also explain the reduction in immune cell infiltrates into the airspaces, there was actually increased KC in BALF and lungs, suggesting that this is not the case. Although migration of cells into the airspaces was inhibited in MC-deficient mice, the clusters of B and T cells were more pronounced than those of neutrophils, as seen by histology. It is possible that the neutrophils achieved a slightly more diffuse staining pattern compared with that of B and T cells because they can secrete their own MMP-9 (37, 64, 65), which may allow them to migrate slightly more interstitially than the B and T cells. It is also possible that neutrophilic MMP-9 is necessary for claudin degradation. However, because there are actually more neutrophils in the lungs of infected Wsh mice, this seems less likely. Alternatively, it could be that the neutrophils are not activated, but this would still place them downstream of MCs in the immune response.
One caveat to our experiments is the use of the Wsh mouse model. Several recent studies found that, in some cases, MC deficiency was not the reason for the observed phenotype; the mutation itself affected other developmental pathways (43). Of potential concern was the finding that these mice may have differential Treg numbers and activity. In one study investigating oral tolerance, both Wsh and KitW/W-v mice had increased numbers of Tregs that were not affected by MC reconstitution (44). Another study found an opposite result, with greatly reduced Treg numbers, leading to greater inflammation in Wsh mice (45). However, in our experiments we did not find any difference in Tregs in the lungs of our infected WT and MC-deficient mice (Supplemental Fig. 2E). It also was reported that Wsh mice frequently develop myeloid and megakaryocytic proliferation in the spleen and bone marrow, resulting in neutrophilia, basophilia, increased MC progenitor numbers in the spleen, and elevated platelet numbers in the blood (47). Nevertheless, our study demonstrated that Wsh mice had less neutrophil migration in alveoli. Furthermore, pharmacological intervention of WT mice during C. pneumoniae infection phenocopied MC deficiency (cromolyn) or led to greater inflammation and greater bacterial burden (compound 48/80) (Figs. 4, 5). Finally, MC reconstitution restored phenotypic features of C. pneumoniae infection, indicating that it is unlikely that our observations regarding the use of Wsh mice are due to any other potential inherent defect in these mice and are due, in fact, to the lack of MCs and their responses during C. pneumoniae infection. Finally, although certain MC-deficient mice (i.e., WBB6F1-KitW/W-v) were reported to have associated neutropenia, the mice that we used (WT and Wsh) do not; instead, they have increased numbers of neutrophils, which we found in the lungs (66). Thus, although we do not believe that intrinsic PMN alterations affected our observations on the role of MCs, we cannot completely rule out this possibility.
C. pneumoniae remains an enigmatic infectious organism with associations to many important human diseases. Understanding this unique organism's complex lifecycle and the immune system's response to infection is a high priority. In this study, we found that the normal immune response is actually beneficial to C. pneumoniae infection by providing it with host cells to infect and propagate. Without the presence of MCs, inflammation was mild, mortality was reduced, and C. pneumoniae was cleared rapidly from the hosts. Not only do these studies provide important new paradigms for how we understand infections, they may provide new avenues of treatments and protective strategies. Additionally, although MCs have long been considered to primarily play a role in Th2-mediated diseases, they were revealed to be major players in both inflammatory disorders and infections. Our findings place MCs as critical gatekeepers during a pulmonary bacterial infection, facilitating immune cell transepithelial migration in the lungs into the airways and, thus, provide a new target for intervention strategies.
The authors have no financial conflicts of interest.
We thank Ganghua Huang, Wenxuan Zhang, and Polly Sun for experimental assistance. We also thank Drs. Barry Stripp and William Parks (Department of Pulmonary Medicine, Cedars-Sinai Medical Center) for critical reading of this manuscript.
This work was supported by National Institutes of Health Grants AI-067995 and 2R56AI067995-06 (to M.A.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
BALF
BMMC
bone marrow–derived MC
IFU
infectious unit
MCPT
MC protease
matrix metalloprotease
TREM-1
triggering receptor expressed on myeloid cells 1
regulatory T cell
KitW-sh/W-sh
wild-type.
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You are going to email the following Mast Cells Play an Important Role in Chlamydia pneumoniae Lung Infection by Facilitating Immune Cell Recruitment into the Airway
Norika Chiba, Kenichi Shimada, Shuang Chen, Heather D. Jones, Randa Alsabeh, Anatoly V. Slepenkin, Ellena Peterson, Timothy R. Crother, Moshe Arditi
The Journal of Immunology April 15, 2015, 194 (8) 3840-3851; DOI: 10.4049/jimmunol.1402685
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Show more INFECTIOUS DISEASE AND HOST RESPONSE


Jacqueline (Forde) Straker, known to everyone who loved her as "Jackie," passed away on June 7, 2018 at the age of 65. Jackie was born in St. Michael, Barbados, W.I. on December 19, 1952 to the late Grafton Wharton and the late Irene Morris. Jackie emigrated to Boston, MA in 1971.
Before retiring in 2009, Jackie was employed at Boston Gas Company for 37 years. Jackie was an active member of St. Bartholomew's Church in Cambridge and was a founding member of the Caribbean Heritage Association. Jackie often gave her time and any resources she had to those in need. She will be remembered for her extreme generosity and her beautiful, welcoming smile.
Jackie will be lovingly remembered by her devoted husband of 41 years, Darnell Straker of Medford, MA, her children, Carolyn Gill (Rashaan Brown) of Boston, MA, Carolyn Daley (Justin) of Brockton, MA, Andre Straker (Allison) of Lowell, MA, Derek Straker of Marlborough, MA and Kayla Straker (Troy Cox) of Brockton, MA, her six grandchildren Preston, Jaxon and Amaya Daley and Cameron, Vivian and Miles Straker, her brothers Ancil Forde of Barbados, Frederick A. Forde and his wife Angela of Barbados and Frederick W. Forde of Atlanta, GA, her sister Veldene Bradshaw of Barbados, as well as a host of other family members and friends. She was predeceased by her brother, Wade Morris and her sister Pancilla Smith.
A celebration of Jackie's life will take place on Friday, June 22, 2018 at St. Bartholomew's Church, 239 Harvard Street, Cambridge, MA 02139. Visitation will be held from 9:00 – 10:00 AM, directly followed by the funeral service at 10:00 AM. The burial will immediately follow the service at Oak Grove Cemetery in Medford, MA.


Minna is full of romance and natural inspiration. Her 3-leaf design is meticulously paved with white diamonds, while her beautifully contoured silhouette is feminine and light. Each ring is custom tailored to fit the exact dimensions of its unique center diamond, and no two pieces will ever be exactly alike. Please allow 3-4 weeks for fabrication, as this piece is custom made to order.


Jim Bohannon is a Talk Show Host for the Jim Bohannon Show of the Westwood One Radio Networks with 47 videos in the C-SPAN Video Library; the first appearance was a 1989 Journalists' Roundtable as a Correspondent for the Mutual Broadcasting System. The year with the most videos was 2006 with six videos. The year with the highest average number of views per program was 2008 with an average of 957 views per program. Most appearances with Michael Harrison (11), Blanquita Cullum (9), Ellen Ratner (7). Most common tags: Radio, Political Coverage, Washington Politics.


Regulation of diet and sleep is recommended herein for the yogis. Too much eating means eating more than is required to keep the body and soul together. There is no need for men to eat animals, because there is an ample supply of grains, vegetables, fruits and milk. Such simple foodstuff is considered to be in the mode of goodness according to the Bhagavad-gita. Animal food is for those in the mode of ignorance. Therefore, those who indulge in animal food, drinking, smoking and eating food which is not first offered to Krishna will suffer sinful reactions because of eating only polluted things. Bhunjate te tv agham papa ye pacanty atma-karanat. Anyone who eats for sense pleasure, or cooks for himself, not offering his food to Krishna, eats only sin. One who eats sin and eats more than is allotted to him cannot execute perfect yoga. It is best that one eat only the remnants of foodstuff offered to Krishna. A person in Krishna consciousness does not eat anything which is not first offered to Krishna. Therefore, only the Krishna conscious person can attain perfection in yoga practice. Nor can one who artificially abstains from eating, manufacturing his own personal process of fasting, practice yoga. The Krishna conscious person observes fasting as it is recommended in the scriptures. He does not fast or eat more than is required, and he is thus competent to perform yoga practice. One who eats more than required will dream very much while sleeping, and he must consequently sleep more than is required. One should not sleep more than six hours daily. One who sleeps more than six hours out of twenty-four is certainly influenced by the mode of ignorance. A person in the mode of ignorance is lazy and prone to sleep a great deal. Such a person cannot perform yoga.
One should fill the stomach half with food, one quarter with water, and one quarter remaining for the movement of air.
6.16 Over-eating and excessive fasting are opposed to Yoga. So also are excessive recreation and non-recreation, too much of sleep and too much of vigil. So too, are overwork and idleness.
The regulations regarding the eating of food and the regimen of sleep that are to be followed by those practising yoga or the science of the individual consciousness attaining communion with the ultimate consciousness are now being given by Lord Krishna. By not following these injunctions it is not possible to even commence practising any form of yoga.
Abstention from performing regulatory fasting such as Ekadasi which is the 11th day of the waxing or waning moon is sanctioned only for the weak and invalid. In the Narada Purana it states: That one who is strong having given up sleep, food, fear, lethargy and actions, meditates within on the Supreme Lord Krishna or His authorised incarnations with eyes half-closed focused on the tip of the nose or the space between the eyebrows attains equanimity of mind.
After describing the results of meditation Lord Krishna gives the regulations regarding the eating of food and the regimen of sleep habits. One who eats like a glutton or sleeps like a sloth as well as one who fasts too much and sleeps to little are never qualified to practise yoga or the science of the individual consciousness attaining communion with the ultimate consciousness. In regard to food it is described by Pantajali that the stomach should be filled one-half with food, one-quarter with liquid and one-quarter should be kept free for the movement of air.
6.16-17 Yogosti etc. Yuktahara etc. For foods : for sense-objects that are being brought [by sense-organs]. Effort : activity for enjoying [them]. Its appropriateness is neither to have unlimited indulgence, nor to have unlimited abstention. The same is in all cases. The rest [of the text] is clear. On the authority of the Sage [Vyasa], the form jagaratah etc. [may be viewed correct] as those in the Vedic literature. The same is in other similar instances also.
na — never; ati — too much; aśnataḥ — of one who eats; tu — but; yogaḥ — linking with the Supreme; asti — there is; na — nor; ca — also; ekāntam — overly; anaśnataḥ — abstaining from eating; na — nor; ca — also; ati — too much; svapna-śīlasya — of one who sleeps; jāgrataḥ — or one who keeps night watch too much; na — not; eva — ever; ca — and; arjuna — O Arjuna.


Keep up to date with all current and emerging Environmental Legislation and ensure compliance.
Adhere to the conditions set out in our Discharge Licence by ( ) and aim to have zero non compliance's.
Monitor the implementation of the policy by carrying out periodic audits of compliance and where necessary introduce remedial measures.
Develop a monitoring program to assess its environmental performance and communicate its environmental management performance through Annual Environmental Reports.
Ensure all employees, in the course of their duties, act in accordance with our environmental standards.
In addition areas of particular attention within the business will be the selections of non-polluting technology, waste minimization, re-use/recycling and the reduction in energy and water usage.


text:
IAEA To Vote On Demand Iran Freeze New Nuclear Site
IAEA Director-General Muhammad el-Baradei (right) told reporters at a joint press conference with German Foreign Minister Guido Westerwelle on November 25 that a nuclear-fuel swap within Iran is "not an option."
VIENNA (Reuters) -- World powers are demanding that Iran immediately mothball a uranium-enrichment site it hid for years, heightening fears it is secretly planning to build atom bombs, in a resolution drafted by the UN nuclear watchdog.
Diplomats forecast majority approval for the resolution in a vote by the International Atomic Energy Agency's (IAEA) 35-nation governing board today or on November 27 in what would be its first action against Iran in almost four years.
The move reflects dismay over Iran's September disclosure of a second enrichment site it had been building clandestinely for two years, and frustration at Iran's holdup of an IAEA-brokered plan to give it fuel for its nuclear medical program if it parts with enriched uranium that could be used in weapons.
IAEA Director-General Muhammad el-Baradei today rejected Iran's assertions the deal lacks guarantees it will get the fuel in the end, a stance Western powers regard as stalling and a tacit admission its LEU will not be used for electricity generation, as Tehran insists.
"In view of the degree of mutual mistrust, it has extensive built-in guarantees, consisting of the IAEA taking custody of the (LEU) until it returned to Iran in the form of fuel," he told a meeting of the agency's 35-nation Board of Governors, his last before he retires on November 30 after 12 years in office.
El-Baradei noted that as an alternative, he had proposed transferring the LEU to a neutral third country such as Turkey, "which has the confidence of all parties," and remain there under IAEA supervision until Iran gets the reactor fuel.
Mounting Pressure
"I am disappointed that Iran so far has not agreed to the original proposal or alternative modalities, both of which I believe are balanced and fair and would greatly alleviate the concerns relating to Iran's nuclear programme." el-Baradei told Reuters in a rare interview on November 25 that Iran's demand to dilute the fuel pact was unacceptable because it could mean Tehran retaining enough enriched uranium for use in a nuclear weapon.
"They are ready to put material under IAEA control on an [Iranian] island in the Persian Gulf. But the whole idea as I explained to them, to defuse this crisis, is to take the material out of Iran," said el-Baradei.
"I do not think this will work as far as the West is concerned."
The last IAEA board resolution passed against Iran was in February 2006, when governors referred Tehran's case to the UN Security Council over its refusal to suspend enrichment and open up completely to IAEA inspections and investigations.
The new measure's sponsors were the United States, Britain, France, Germany, Russia, and China, the sextet locked in a long standoff with Iran over its shadowy enrichment activity, alleged nuclear bomb research and restrictions on IAEA inspections.
...Including From Russia
Russian and Chinese support is significant, and expected to secure rare developing nation votes against Iran at the IAEA, since the two have often blocked a united stance against Iran in international security bodies.
It was unclear whether Moscow and Beijing's expression of disenchantment with Iran, an important trade partner for both, would translate into readiness for harsher UN sanctions Western powers will push for if the fuel deal falls through.
But Russia's Foreign Ministry today urged Iran to adhere to the deal.
"The Russia side especially underlined the necessity of observing the principles of the agreement, reached in negotiations on this theme in Geneva on October 1 between representatives of Iran and the six countries," a statement on the Russian Foreign Ministry website said.
The statement was released after Deputy Russian Foreign Minister Sergei Ryabkov met Iran's ambassador to Moscow.
The statement said both sides exchanged views about Iran's nuclear program following the November 20 meeting in Brussels by political directors of Russia and the other five countries involved in the talks with Tehran.
A November 16 IAEA report said Iran violated a transparency statute by admitting the existence of the Fordow enrichment site only two months ago, at least two years after building began, and raised concern it could be harboring more secret sites.
Iran had previously assured the IAEA it was not concealing nuclear activity with potential weapons applications.
Resolution Could Backfire
The draft resolution, a restricted copy of which was obtained by Reuters, urged Iran to immediately halt construction of the Fordow plant, clarify its original purpose, and confirm it has no more hidden atomic activity or covert plans for any.
It voiced "serious concern" -- a diplomatic euphemism for alarm -- over its cover-up of the Fordow project and said it was in blatant breach of UN demands for an enrichment suspension.
It also called on Iran to shelve all enrichment-related activity as demanded by Security Council resolutions since 2006, grant unfettered IAEA inspections and open up to an IAEA probe into suspicions it conducted illicit nuclear weapons research.
But el-Baradei suggested to Reuters in the November 25 interview that the new resolution could backfire by aggravating Iran's siege mentality, boosting nuclear hard-liners.
Iran's envoy to the IAEA, Ali Asghar Soltanieh, told Germany's "Sueddeutsche Zeitung" newspaper today that the resolution would "endanger the prevailing constructive atmosphere" and have "long-term consequences."
He was alluding to IAEA efforts to broker a compromise to salvage the nuclear-fuel supply plan and Iran's stated readiness to resolve IAEA questions about the Fordow enrichment site.
"The P5+1 [six powers] will win the battle in the Board of Governors, but it will be only a simple majority, but lose the war to get Iran to be more cooperative," a senior diplomat from the bloc of developing nations said.
Tehran says the bunkered Fordow site, which is to start operations in 2011, is a backup for its much larger Natanz enrichment center in case it is bombed by foes such as Israel.
Western nuclear analysts say Fordow's low capacity makes it unsuitable for any purpose but to enrich smaller quantities of uranium suitable for a bomb. Enrichment plants generally need tens of thousands of centrifuges to feed a nuclear power plant.
compiled from multiple Reuters reports

redpajama_set_name: RedPajamaCommonCrawl


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Another solid week in video games in the bag and damn this one was filled with a lot of exciting things. We were able to get out our reviews for Adr1ft and Batman from Telltale and we also were able to see THQ come back into form in a small way. Also a whole lot of updates to other video games like Gears Of War 4 and Resident Evil 7 for us all to chew on. We are also so very close to another huge gaming convention here too that will most likely come with a whole lot of other news and tidbits all their own. It is a great time to be a gamer now isn't it?


You can always use more skulls to deaden up the place. This set of three should do just fine. They are perfect for mounting on walls or incorporating into furniture because of their unique three dimensional face only design. Made from a hard foam. Each is 9" tall.


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E1: GRID: On TR 9.2.0.4 Grid Focus Is Not Set to Summary Total in P4210 When Using Go To End.
On Tools Release 9.2.0.4, Grid Focus is not being set to the Summary Total when Go To End is used on a Find. The Grid Focus is set to the top record.
The Focus may be observed to be set to the bottom of the Grid for a brief moment before relocating Grid Focus to the top of the Grid.
3. Observe that Grid Focus is returned to the top of the Grid.
4. Using Vertical Scroll in Grid to go to bottom, reveals the Summary Total/Open Amount.


text:
Wikipedia, early Internet culture, and conspiracy theory jokes
There is a 2008 comic book, Umbrella Academy, which contains a joke about the assassination of United States President John F. Kennedy (JFK).In 2019 the comic became a video series and presented the Kennedy assassination joke again. I liked the comic and I recently watched the video series. The joke is incidental, but since the joke is part of my native Texas culture, I understand it and can explain it. Here is the joke and how it changed the course of Wikipedia history.
In summary, from the 1960s to present, many discussions make humor with conspiracy theories of assassination of JFK. The form of the humor is in tall tales, where anyone who wants to participate creates an outlandish and complicated explanations of who wanted JFK dead and why. In a typical story, some minor detail changes the course of history, like a butterfly effect in "A sound of Thunder". Dozens of these stories are in popular circulation, and people from Texas like me have heard many of these stories throughout our lives and can easily recognize them as attempted humor. In 2005 some random person with an Internet connection posted a Kennedy conspiracy theory into the Wikipedia biography of journalist John Seigenthaler. Wikipedia had an excellent quality control process in place, and in less than a day, a reviewer removed the conspiracy theory and even drafted original biographical text of good quality in its place. Then and even now this is a commendable show of the highest quality web moderation as compared to any other online community. Still, somehow Seigenthaler became enraged and directed his anger at Wikipedia. His anger was ridiculous then, because he failed to recognize he cultural context of years of Kennedy assassination jokes, and also because this was an era when young people understood the concept of publishing text online but older people like Seigenthaler did not, and also because the Wikipedia community was confident in its quality control process because this was an occurrence where Wikipedia's review was operating exceptionally well as compared to any competing media outlet. Seigenthaler had unusual access to media as a conventional journalist and drew as much mass media attention to his story as he could. While his complaint was silly, and while Wikipedia was in the more fortunate and favorable position in this conflict, the Wikipedia community treated his protest seriously. Everyone in wiki recognized that in general, we all want to have confidence in our moderation and to be able to give a clear explanation of our publishing commitment. The Wikipedia community's own outcome was the creation of the Biographies of Living Persons Policy, in which the Wikipedia community committed to prioritize the review of content added to biographies of living people. The general rule in Wikipedia is that if someone adds content to a Wikipedia article, then that content should be a summary of an existing published reliable source which they cite. The biographies of living persons policy directs the Wikipedia community to quickly review all incoming edits to that class of articles, with the first and easiest quality check being to simply confirm that editors cite sources. The Wikipedia community has come to celebrate this policy. 15 years on, I wanted to tell the story around this policy again. The story at present is that American media still makes jokes about Kennedy's assassination, as evidenced by the joke's recurrence in this popular bigger budget superhero video series. I would not want anyone to forget that so much of the discussion about this policy came from the dramatic irony of a large number of people in Wikipedia witnessing Seigenthaler's lack of awareness of the culture of tall tales around the Kennedy assassination, and that the Internet now permits anyone to post anything online, and that his complaint was showcasing the unparalleled strength of Wikipedia's quality control rather than attacking any shortcoming.
Kennedy's assassination and tall tales
In 1963 United States President John F. Kennedy got a bullet in his head while in a public motorcade in Dallas, Texas. He was in a moving car and a sniper shot him from far away while crowds watched. Of course everyone talked about the incident. Kennedy was a socially and politically disruptive president for many reasons, and in the conversation after his death, many people speculated on who might have wanted him dead and who would benefit from him being gone. The government captured the assassin, the mysterious Lee Harvey Oswald, who neglected to communicate any particular reason for killing the president. Within two days of Oswald's capture, a mafia affiliate named Jack Ruby assassinated him, then Ruby proceeded to publicly and prominently tease claims that he had something important to say about some weird web of conspiracies. Jack Ruby died before being able to tell his story. The facts of this story are strange enough. Complicating all of this, in Texas there is a culture to promote conspiracy theories, which people take seriously, and to tell outlandish tall tales, which is a hobby many people take as part of the local culture. Sometimes the two mix, such as when someone communicates a conspiracy theory as a tall tale so that they can spread a rumor in a socially acceptable way. By mixing truth, fiction, and speculation, information spreads easily in Texas, and individual listeners can believe what they wish. The way that people play the game and tell the joke is to create an outlandish explanation for who killed Kennedy and what complicated chain of events utterly disrupts all of society to eventually trickle into some small benefit for some criminal mastermind who planned everything while being far removed from any direct action. When I was 18 years old in my first year of college I had a paper copy of the Warren Commission and saw pictures of "Bob" as graffiti in my community, and was enthusiastic to discuss JFK's assassination and "Bob"'s comparable assassination in the Dallas area where I lived, where Kennedy died, and which "Bob" designated as holy in the Church of the Subgenius.
Kennedy's assassination in fiction
In the Umbrella Academy one of the heroes has an exciting life as a time traveling assassin for hire. Comic book characters somehow often are simultaneously heroes and commit mass murders, and in the universe of adventure fiction, somehow teams including the Marvel heroes, the DC heroes, or any number of other individuals or groups kill tens or hundreds or hundreds of thousands of people casually and with little consequence. Mass murders in comics are often background details for advancing the plot, which is typically interpersonal relationships. In the Umbrella Academy, the plot is that a group of siblings have difficult relationships with each other because of an unusual childhood. To express these family relationships, the comic book incidentally mentions that one of the children joined the interdimensional galaxy police and had to kill president Kennedy and bomb the Hindenburg.
Enough audiences find this funny that many media sources make light of this. I will not critique the humor of any this. I can see a future when new media has data about all past media, and when humanity will be able to count the number of times when anyone has told tall tales or made jokes about the Kennedy assassination. We are not there yet, but for the sake of this story, I confirm as a Texan that lots of people in Texas tell stories about Kennedy's assassination, making a joke of it. The TV Tropes wiki has a compilation of media which presents JFK jokes, or if not jokes, whatever JFK's assassination means now as a frame for storytelling.
Wikipedia's Biographies of Living Persons Policy
Wikipedia's Biographies of Living Person's Policy is a masterwork of Wikipedia culture and ideology. To understand this policy is to understand multiple new and amazing social changes brought by the advent of Internet. The Wikipedia community recognized these changes and articulated their significance in this policy before anyone else knew what was happening.
The social context of this policy is that the world became different as the public gained access to options for publishing user generated content. Here are some of facts which people in the Wikipedia community knew in 2005 at the introduction of this policy, and which power brokers in media and the world in general did not know:
People read content on the Internet
Anyone can publish information online
When someone publishes information online, other people can find and read that
It is possible for an individual who has no particular access or money or power to be able to publish whatever they like, even without permission from a wealthy person or power broker
There is not a boss of the Internet, or an obvious particular organization which has responsibility for everything published on in Internet accessible media
Internet media is not a fad, and online media is growing in popularity
I am belaboring details about the Internet which are obvious now, and will be obvious for as long as humanity has the Internet as now as we believe will exist with humanity forever, but if Siegenthaler was like his contemporaries he would doubt, disbelieve, or dismiss all these ideas. These concepts were mostly unknown in 2005 and were still amazing revelations then. Back in 2005 most people would have either not believed or not understood the significance of these statements.
More specifically to Wikipedia and that BLP policy, here are some other facts which the Wikipedia community knew which others did not know:
The Wikipedia community has superior insights into online media as compared to leaders in journalism, broadcasting, or traditional academic communication theory
The Wikipedia community has a new media moderation system in place which works natively in a digital media environment, and which is superior in a digital environment as compared to any attempt to adapt non-digital moderation to virtual spaces
Despite not having designated leadership or a public relations head, the Wikipedia community is able to coordinate communication effectively, and can, for example, respond effectively to mass-media criticism with the crowdsourced creation of a policy response
In observing complaints from traditional media against Wikipedia, the Wikipedia community operates as a hive mind to develop a robust critical discourse. The Wikipedia community has high consciousness of its strengths and weaknesses, and for any given complaint, Wikipedia users can tap into published knowledge of exactly how what to say to answer criticism.
The Wikipedia community already was feeling its power then at the time when other traditional powerholders in media did not even imagine that a decline was in process.
From Seigenthaler's perspective, and from the mass media coverage at the time, the old guard felt like they were criticizing Wikipedia, youth culture, the Internet in general, and encroachment of digital media talent upon their non-digital publishing professional skill sets. This was not the case then, and the Wikipedia community never felt like Seigenthaler had a reasonable complaint. When the Wikipedia community drafted this policy it was not even to change behavior, but rather, it was to put into words what everyone was already doing and which the new media generation understood without being told and due to their experience from the nature of the system.
One of the most encouraging aspects of the Seigenthaler complaint was that he was complaining about content which Wikipedia's moderation system had detected and removed within 24 hours, which was better than what Google, Facebook, Microsoft, Apple, or any of the other big players could do at that time. Part of the social context of this complaint about Wikipedia was that Wikipedia was a channel for community empowerment and nonprofit media, when actually all of the big players were experimenting with user generated content and all of them permitted similar user activity. The media conditions the public to tolerate bad behavior from the corporate sector, because they are supposed to be self-serving and profit seeking and problematic, but critical of nonprofit community projects with less resources, because those projects have a mission to seek goodness. The way things ought to be is that the public should set the ethics of public space, and when corporate entities seek to enter the public commons, then they have to comply with community norms. Too often the reality is that corporations do what they want and send blame downward to whomever is less privileged in society. I am not dismissing the Seigenthaler complaint, but I will note that lots of websites publish lots of nonsense, and that Wikipedia moderates that nonsense in a better, more quickly, more ethically, and with more criticism than other platforms with more billions of dollars.
About lies in Texas
I grew up in Texas which is the state where the assassination happened, and since part of Texas culture is sharing fake folklore, people there enjoy telling and listening to outlandish explanations for why things happened. Kennedy assassination stories can be a mix of somber regret, concerned citizens against government oppression, wild fiction for fun, or provocation by people in on the joke at the expense of people not in on the joke. In the future people will forget the historical context, but obviously the intent of saying that someone killed JFK is a joke. In the Seigenthaler vandalism, the outlandish accusation was that he was an assassin mastermind who killed both president Kennedy and also his brother Robert Kennedy in another conspiracy Other context which the future will forget is that in the early days of the Internet people were bewildered by online publishing in general, and understanding new media like user generated content or Wikipedia required multiple levels of abstraction when a person was unaware of foundational premises. Seigenthaler was confused, but came out very publicly complaining about Wikipedia. To me, blaming him for killing Kennedy would be comparable to telling a story of him abducting Harold Holt, or having Jimmy Hoffa's dead body, or being intimately involved in any other story in the list of conspiracy theories.
The Wikipedia community responded seriously to Seigenthaler's complaint but he never seemed to be conscious of how ridiculous it was for him to be offended because someone said something weird about him on the Internet. He was still imagining that Wikipedia had an editorial board like a conventional publishing house or newspaper or television news studio. Everything turned out fine for Wikipedia because we needed an excuse to have a policy for biographies of living persons, and having an overblown incident of cultural misunderstanding as an origin story is a great beginning. The media record about all of this is strange throughout, because internationally, many people did not realize the joke in the Kennedy murder accusation, and even in the United States somehow the media was not following that the vandal intended to be funny. Anyway, I am saying all this to share that this contemporary television show made this same lame decades-old joke, and with yet more time passed, and with Wikipedia being more international, I know that many people will miss the context. I grew up in an environment where people discussed the possibility that Kennedy shot first and I am not able to see anyone have this discussion without thinking of the jokes.
Posted in Texas, Wikipedia | Tagged conspiracy, death, policy
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