IL-21R Signaling Suppresses IL-17+ Gamma Delta T Cell Responses and Production of IL-17 Related Cytokines in the Lung at Steady State and After Influenza A Virus Infection

Influenza A virus (IAV) infection of the respiratory tract elicits a robust immune response, which is required for efficient virus clearance but at the same time can contribute to lung damage and enhanced morbidity. IL-21 is a member of the type I cytokine family and has many different immune-modulatory functions during acute and chronic virus infections, although its role in IAV infection has not been fully evaluated. In this report we evaluated the contributions of IL-21/IL-21 receptor (IL-21R) signaling to host defense in a mouse model of primary IAV infection using IL-21R knock out (KO) mice. We found that lack of IL-21R signaling had no significant impact on virus clearance, adaptive T cell responses, or myeloid cell accumulations in the respiratory tract. However, a subset of inflammatory cytokines were elevated in the bronchoalveolar lavage fluid of IL-21R KO mice, including IL-17. Although there was only a small increase in Th17 cells in the lungs of IL-21R KO mice, we observed a dramatic increase in gamma delta (γδ) T cells capable of producing IL-17 both after IAV infection and at steady state in the respiratory tract. Finally, we found that IL-21R signaling suppressed the accumulation of IL-17⁺ γδ T cells in the respiratory tract intrinsically. Thus, our study reveals a previously unrecognized role of IL-21R signaling in regulating IL-17 production by γδ T cells.     

Role of IL-1β in Experimental Cystic Fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftr ^tm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1^−/−), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1^−/− double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1^−/− and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients.     

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ^−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ^−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A⁺ CD45⁺ cells in the lungs and these effects were abolished in TCRδ^−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ^−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A⁺ CD45⁺ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A⁺ γδ T cells. Il17a mRNA and IL-17A⁺ γδ T cells were also lower in obese Cpe^fat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A⁺ γδ T cells to the lung.     

A Balanced IL-1β Activity Is Required for Host Response to Citrobacter rodentium Infection

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3^−/− and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3^−/− mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3^−/− mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3^−/− mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3^−/− compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3^−/− mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3^−/− macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3^−/− mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3^−/− mice but increased tissue damage when given to WT mice.     

Granuloma Formation and Host Defense in Chronic Mycobacterium tuberculosis Infection Requires PYCARD/ASC but Not NLRP3 or Caspase-1

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1β secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3^−/−, Casp-1^−/−, and WT mice showed no differences in pulmonary IL-1β production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard^−/− mice. Decreased survival of Pycard^−/− animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.     

In Pulmonary Paracoccidioidomycosis IL-10 Deficiency Leads to Increased Immunity and Regressive Infection without Enhancing Tissue Pathology

Background

Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM.

Methodology/Principal Findings

Wild type (WT) and IL-10^−/− C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10^−/− mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10^−/− and WT mice were i.t. infected with 1×10⁶ Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10^−/− mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10^−/− mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4⁺ and CD8⁺ T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10^−/− mice.

Conclusions/Significance

Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.     

Type I Interferon Induction during Influenza Virus Infection Increases Susceptibility to Secondary Streptococcus pneumoniae Infection by Negative Regulation of γδ T Cells

The majority of deaths following influenza virus infection result from secondary bacterial superinfection, most commonly caused by Streptococcus pneumoniae. Several models have been proposed to explain how primary respiratory viral infections exacerbate secondary bacterial disease, but the mechanistic explanations have been contradictory. In this study, mice were infected with S. pneumoniae at different days after primary influenza A (X31) virus infection. Our findings show that the induction of type I interferons (IFNs) during a primary nonlethal influenza virus infection is sufficient to promote a deadly S. pneumoniae secondary infection. Moreover, mice deficient in type I interferon receptor (IFNAR knockout [KO] mice) effectively cleared the secondary bacterial infection from their lungs, increased the recruitment of neutrophils, and demonstrated an enhanced innate expression of interleukin-17 (IL-17) relative to wild-type (WT) mice. Lung γδ T cells were responsible for almost all IL-17 production, and their function is compromised during secondary S. pneumoniae infection of WT but not IFNAR KO mice. Adoptive transfer of γδ T cells from IFNAR KO mice reduced the susceptibility to secondary S. pneumoniae infection in the lung of WT mice. Altogether, our study highlights the importance of type I interferon as a key master regulator that is exploited by opportunistic pathogens such as S. pneumoniae. Our findings may be utilized to design effective preventive and therapeutic strategies that may be beneficial for coinfected patients during influenza epidemics.     

Limited Anti-Inflammatory Role for Interleukin-1 Receptor Like 1 (ST2) in the Host Response to Murine Postinfluenza Pneumococcal Pneumonia

Interleukin-1 receptor like 1 (ST2) is a negative regulator of Toll-like receptor (TLR) signaling. TLRs are important for host defense during respiratory tract infections by both influenza and Streptococcus (S.) pneumoniae. Enhanced susceptibility to pneumococcal pneumonia is an important complication following influenza virus infection. We here sought to determine the role of ST2 in primary influenza A infection and secondary pneumococcal pneumonia. ST2 knockout (st2 ^−/−) and wild-type (WT) mice were intranasally infected with influenza A virus; in some experiments mice were infected 2 weeks later with S. pneumoniae. Both mouse strains cleared the virus similarly during the first 14 days of influenza infection and had recovered their weights equally at day 14. Overall st2^−/− mice tended to have a stronger pulmonary inflammatory response upon infection with influenza; especially 14 days after infection modest but statistically significant elevations were seen in lung IL-6, IL-1β, KC, IL-10, and IL-33 concentrations and myeloperoxidase levels, indicative of enhanced neutrophil activity. Interestingly, bacterial lung loads were higher in st2^−/− mice during the later stages of secondary pneumococcal pneumonia, which was associated with relatively increased lung IFN-γ levels. ST2 deficiency did not impact on gross lung pathology in either influenza or secondary S. pneumoniae pneumonia. These data show that ST2 plays a limited anti-inflammatory role during both primary influenza and postinfluenza pneumococcal pneumonia.     

IL-4 Receptor-Alpha-Dependent Control of Cryptococcus neoformans in the Early Phase of Pulmonary Infection

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lung inflammation and meningoencephalitis in immunocompromised people. Previously we showed that mice succumb to intranasal infection by induction of pulmonary interleukin (IL)-4Rα–dependent type 2 immune responses, whereas IL-12-dependent type 1 responses confer resistance. In the experiments presented here, IL-4Rα^−/− mice unexpectedly show decreased fungal control early upon infection with C. neoformans, whereas wild-type mice are able to control fungal growth accompanied by enhanced macrophage and dendritic cell recruitment to the site of infection. Lower pulmonary recruitment of macrophages and dendritic cells in IL-4Rα^−/− mice is associated with reduced pulmonary expression of CCL2 and CCL20 chemokines. Moreover, IFN-γ and nitric oxide production are diminished in IL-4Rα^−/− mice compared to wild-type mice. To directly study the potential mechanism(s) responsible for reduced production of IFN-γ, conventional dendritic cells were stimulated with C. neoformans in the presence of IL-4 which results in increased IL-12 production and reduced IL-10 production. Together, a beneficial role of early IL-4Rα signaling is demonstrated in pulmonary cryptococcosis, which contrasts with the well-known IL-4Rα-mediated detrimental effects in the late phase.     

Interleukin-22 (IL-22) Production by Pulmonary Natural Killer Cells and the Potential Role of IL-22 during Primary Influenza Virus Infection

We set out to test the hypothesis that interleukin-22 (IL-22), a cytokine crucial for epithelial cell homeostasis and recovery from tissue injury, would be protective during influenza virus infection. Recent studies have identified phenotypically and functionally unique intestinal NK cells capable of producing the cytokine IL-22. Unlike gut NK cells that produce IL-22, the surface phenotypes of lung NK cells were similar to those of spleen NK cells and were characteristically mature. With mitogen stimulation, both single and double IL-22- and gamma interferon (IFN-γ)-producing lung NK cells were detected. However, only the IL-22⁺ IFN-γ⁻ lung NK subset was observed after stimulation with IL-23. IL-23 receptor (IL-23R) blocking dramatically inhibited IL-22 production, but not IFN-γ production. Furthermore, we found that NK1.1⁺ or CD27⁻ lung NK cells were the primary sources of IL-22. After influenza virus infection, lung NK cells were quickly activated to produce both IFN-γ and IL-22 and had increased cytotoxic potential. The level of IL-22 in the lung tissue declined shortly after infection, gradually returning to the baseline after virus clearance, although the IL-22 gene expression was maintained. Furthermore, depletion of NK cells with or without influenza virus infection reduced the protein level of IL-22 in the lung. Anti-IL-22 neutralization in vivo did not dramatically affect weight loss and survival after virus clearance. Unexpectedly, anti-IL-22-treated mice had reduced virus titers. Our data suggest that during primary respiratory viral infection, IL-22 seems to a play a marginal role for protection, indicating a differential requirement of this cytokine for bacterial and viral infections.NK cells are important innate immune effectors that patrol the body for invading pathogens and tumors. Primary biological functions of NK cells include natural cytotoxicity and cytokine generation, through which NK cells directly or indirectly control infections and tumors and regulate the immune system (8). Accumulating evidence has unveiled other novel functions of NK cells that are associated with their anatomic locations. For example, in the uterus, NK cells support reproductive tissue development by providing a variety of cytokines, growth factors, and angiogenic factors (18, 26). The uterine NK cells also demonstrate a unique receptor repertoire, the Ly49 phenotype of which is strikingly different from that of spleen NK cells (39).Very recently, an NK1.1 low or negative subset of NK cells (CD3⁻ NKp46⁺) has been identified in the intestinal mucosa and found to be capable of making interleukin-22 (IL-22) (7, 24, 31, 32). IL-22 is one of the IL-10 cytokine family members that have been shown to be important in regulating mucosal epithelial cell function, maintaining barrier integrity, and protection from bacterial infections in the gut and lung (4, 43). Interestingly, gut NK cells are distinguished by an immature phenotype, as evidenced by the lack of multiple traditional NK cell markers, such as Ly49A, Ly49D, Ly49C/I, and Ly49G2, and by altered expression of several markers, such as CD122, NK1.1, CD49b (DX5), CD11b, CD27, and CD127, in comparison with spleen NK cells (24, 31, 32). Functionally, gut NK cells lack the capability of gamma interferon (IFN-γ) production and cytotoxicity (24, 31, 32). Taken together, the unique nontraditional features of gut NK cells indicate a distinct developmental process (11, 36) in which they acquire the ability to produce IL-22 and thus are crucial components against intestinal bacterial infections.In addition to the gut, the respiratory tract is an important mucosal system that can be easily invaded by microorganisms. In the lung, NK cells constitute about 10% of the total resident lymphocytes, a relatively higher percentage than that distributed in most other lymphoid tissues and nonlymphoid tissues (17), indicating potential crucial involvement of NK cells in lung infections. Indeed, lung NK cells are known to be vital for containing numerous pulmonary infections, including those caused by Mycobacterium tuberculosis, Cryptococcus neoformans, Bordetella pertussis, respiratory syncytial virus, and influenza virus (12, 16). The potential mechanism of NK cell defense in lung infections has been attributed to NK cell IFN-γ production and their cytolytic functions. However, IL-22 has been implicated in protection against respiratory infection with Gram-negative bacteria, such as Klebsiella pneumoniae, where IL-22 levels increase after infection (4). Whether lung NK cell production of IL-22 in the context of respiratory virus infection or IL-22 itself is important for viral protection has not been investigated.In this study, we investigated the phenotypes and IL-22 production potential of lung NK cells in the context of influenza virus infection. The data show that lung NK cells are phenotypically similar to spleen NK cells yet capable of producing IL-22 upon in vitro stimulation and after influenza virus infection in vivo. Unlike gut NK cells, IL-22-producing lung NK cells are capable of making IFN-γ and display cytolytic potential. After influenza virus infection, in spite of the detection of IL-22-producing NK cells in the lung, IL-22 levels actually went down, and mice treated with anti-IL-22 antibodies had reduced virus titers, with little change in disease severity. These observations show that IL-22 serves different roles in bacterial versus virus infections of the lung and suggest that it may be actively regulated to limit proliferation of cells targeted by the influenza virus.     

Mouse Macrophage Galactose-type Lectin (mMGL) is Critical for Host Resistance against Trypanosoma cruzi Infection

The C-type lectin receptor mMGL is expressed exclusively by myeloid antigen presenting cells (APC) such as dendritic cells (DC) and macrophages (Mφ), and it mediates binding to glycoproteins carrying terminal galactose and α- or β-N-acetylgalactosamine (Gal/GalNAc) residues. Trypanosoma cruzi (T. cruzi) expresses large amounts of mucin (TcMUC)-like glycoproteins. Here, we show by lectin-blot that galactose moieties are also expressed on the surface of T. cruzi. Male mMGL knockout (-/-) and wild-type (WT) C57BL/6 mice were infected intraperitoneally with 10⁴ T. cruzi trypomastigotes (Queretaro strain). Following T. cruzi infection, mMGL-/- mice developed higher parasitemia and higher mortality rates compared with WT mice. Although hearts from T. cruzi-infected WT mice presented few amastigote nests, mMGL-/- mice displayed higher numbers of amastigote nests. Compared with WT, Mφ from mMGL-/- mice had low production of nitric oxide (NO), interleukin (IL)-12 and tumor necrosis factor (TNF)-α in response to soluble T. cruzi antigens (TcAg). Interestingly, upon in vitro T. cruzi infection, mMGL-/- Mφ expressed lower levels of MHC-II and TLR-4 and harbored higher numbers of parasites, even when mMGL-/- Mφ were previously primed with IFN-γ or LPS/IFN-γ. These data suggest that mMGL plays an important role during T. cruzi infection, is required for optimal Mφ activation, and may synergize with TLR-4-induced pathways to produce TNF-α, IL-1β and NO during the early phase of infection.     

IL-4Rα-Dependent Alternative Activation of Macrophages Is Not Decisive for Mycobacterium tuberculosis Pathology and Bacterial Burden in Mice

Classical activation of macrophages (caMph or M1) is crucial for host protection against Mycobacterium tuberculosis (Mtb) infection. Evidence suggests that IL-4/IL-13 alternatively activated macrophages (aaMph or M2) are exploited by Mtb to divert microbicidal functions of caMph. To define the functions of M2 macrophages during tuberculosis (TB), we infected mice deficient for IL-4 receptor α on macrophages (LysM^creIL-4Rα^-/lox) with Mtb. We show that absence of IL-4Rα on macrophages does not play a major role during infection with Mtb H37Rv, or the clinical Beijing strain HN878. This was demonstrated by similar mortality, bacterial burden, histopathology and T cell proliferation between infected wild-type (WT) and LysM^creIL-4Rα^-/lox mice. Interestingly, we observed no differences in the lung expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg1), well-established markers for M1/M2 macrophages among the Mtb-infected groups. Kinetic expression studies of IL-4/IL-13 activated bone marrow-derived macrophages (BMDM) infected with HN878, followed by gene set enrichment analysis, revealed that the MyD88 and IL-6, IL-10, G-CSF pathways are significantly enriched, but not the IL-4Rα driven pathway. Together, these results suggest that IL-4Rα-macrophages do not play a central role in TB disease progression.     

Defining the Roles of IFN-γ and IL-17A in Inflammation and Protection against Helicobacter pylori Infection

CD4⁺ T cells have been shown to be essential for vaccine-induced protection against Helicobacter pylori infection. However, the effector mechanisms leading to reductions in the gastric bacterial loads of vaccinated mice remain unclear. We have investigated the function of IFN-γ and IL-17A for vaccine-induced protection and inflammation (gastritis) using IFN-γ-gene-knockout (IFN-γ^-/-) mice, after sublingual or intragastric immunization with H. pylori lysate antigens and cholera toxin. Bacteria were enumerated in the stomachs of mice and related to the gastritis score and cellular immune responses. We report that sublingually and intragastrically immunized IFN-γ^-/- mice had significantly reduced bacterial loads similar to immunized wild-type mice compared to respective unimmunized infection controls. The reduction in bacterial loads in sublingually and intragastrically immunized IFN-γ^-/- mice was associated with significantly higher levels of IL-17A in stomach extracts and lower gastritis scores compared with immunized wild-type mice. To study the role of IL-17A for vaccine-induced protection in sublingually immunized IFN-γ^-/- mice, IL-17A was neutralized in vivo at the time of infection. Remarkably, the neutralization of IL-17A in sublingually immunized IFN-γ^-/- mice completely abolished protection against H. pylori infection and the mild gastritis. In summary, our results suggest that IFN-γ responses in the stomach of sublingually immunized mice promote vaccine-induced gastritis, after infection with H. pylori but that IL-17A primarily functions to reduce the bacterial load.     

Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3?/? Macrophages

In this study, we have utilized wild-type (WT), ASC^−/−, and NLRP3^−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC^−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3^−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3^−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.     

Inflammasome-dependent pyroptosis and IL-18 protect against Burkholderia pseudomallei lung infection while IL-1β is deleterious

Burkholderia pseudomallei is a Gram-negative bacterium that infects macrophages and other cell types and causes melioidosis. The interaction of B. pseudomallei with the inflammasome and the role of pyroptosis, IL-1β, and IL-18 during melioidosis have not been investigated in detail. Here we show that the Nod-like receptors (NLR) NLRP3 and NLRC4 differentially regulate pyroptosis and production of IL-1β and IL-18 and are critical for inflammasome-mediated resistance to melioidosis. In vitro production of IL-1β by macrophages or dendritic cells infected with B. pseudomallei was dependent on NLRC4 and NLRP3 while pyroptosis required only NLRC4. Mice deficient in the inflammasome components ASC, caspase-1, NLRC4, and NLRP3, were dramatically more susceptible to lung infection with B. pseudomallei than WT mice. The heightened susceptibility of Nlrp3^-/- mice was due to decreased production of IL-18 and IL-1β. In contrast, Nlrc4^-/- mice produced IL-1β and IL-18 in higher amount than WT mice and their high susceptibility was due to decreased pyroptosis and consequently higher bacterial burdens. Analyses of IL-18-deficient mice revealed that IL-18 is essential for survival primarily because of its ability to induce IFNγ production. In contrast, studies using IL-1RI-deficient mice or WT mice treated with either IL-1β or IL-1 receptor agonist revealed that IL-1β has deleterious effects during melioidosis. The detrimental role of IL-1β appeared to be due, in part, to excessive recruitment of neutrophils to the lung. Because neutrophils do not express NLRC4 and therefore fail to undergo pyroptosis, they may be permissive to B. pseudomallei intracellular growth. Administration of neutrophil-recruitment inhibitors IL-1ra or the CXCR2 neutrophil chemokine receptor antagonist antileukinate protected Nlrc4^-/- mice from lethal doses of B. pseudomallei and decreased systemic dissemination of bacteria. Thus, the NLRP3 and NLRC4 inflammasomes have non-redundant protective roles in melioidosis: NLRC4 regulates pyroptosis while NLRP3 regulates production of protective IL-18 and deleterious IL-1β.     

Progesterone-Based Therapy Protects Against Influenza by Promoting Lung Repair and Recovery in Females

Over 100 million women use progesterone therapies worldwide. Despite having immunomodulatory and repair properties, their effects on the outcome of viral diseases outside of the reproductive tract have not been evaluated. Administration of exogenous progesterone (at concentrations that mimic the luteal phase) to progesterone-depleted adult female mice conferred protection from both lethal and sublethal influenza A virus (IAV) infection. Progesterone treatment altered the inflammatory environment of the lungs, but had no effects on viral load. Progesterone treatment promoted faster recovery by increasing TGF-β, IL-6, IL-22, numbers of regulatory Th17 cells expressing CD39, and cellular proliferation, reducing protein leakage into the airway, improving pulmonary function, and upregulating the epidermal growth factor amphiregulin (AREG) in the lungs. Administration of rAREG to progesterone-depleted females promoted pulmonary repair and improved the outcome of IAV infection. Progesterone-treatment of AREG-deficient females could not restore protection, indicating that progesterone-mediated induction of AREG caused repair in the lungs and accelerated recovery from IAV infection. Repair and production of AREG by damaged respiratory epithelial cell cultures in vitro was increased by progesterone. Our results illustrate that progesterone is a critical host factor mediating production of AREG by epithelial cells and pulmonary tissue repair following infection, which has important implications for women’s health.     

Expression of Suppressor of Cytokine Signaling 1 (SOCS1) Impairs Viral Clearance and Exacerbates Lung Injury during Influenza Infection

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1^−/− mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1^−/−IFN-γ^−/− mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1^−/−IFN-γ^−/− mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ^−/− or WT animals. Enhanced viral clearance in SOCS1^−/−IFN-γ^−/− mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1^−/−IFN-γ^−/− mice had no significant effect on their enhanced resistance to influenza infection, while CD8⁺ splenocytes from SOCS1^−/−IFN-γ^−/− mice were sufficient to rescue RAG1^−/− animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1^−/− mice reconstituted with SOCS1^−/−IFN-γ^−/− adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.