Macrophages (MΦ) play an essential role in innate immune responses and can either display a pro-inflammatory, classically activated phenotype (M1) or undergo an alternative activation program (M2) promoting immune regulation. M-CSF is used to differentiate monocytes into MΦ and IFN-γ or IL-4+IL-13 to further polarize these cells towards M1 or M2, respectively. Recently, differentiation using only GM-CSF or M-CSF has been described to induce a M1- or M2-like phenotype, respectively. In this study, we combined both approaches by differentiating human MΦ in GM-CSF or M-CSF followed by polarization with either IFN-γ or IL-4+IL-13. We describe the phenotypic differences between CD14hi CD163hi CD206int FOLR2-expressing M-CSF MΦ and CD14lo CD163lo CD206hi GM-CSF MΦ but show that both macrophage populations reacted similarly to further polarization with IFN-γ or IL-4+IL-13 with up- and down-regulation of common M1 and M2 marker genes. We also show that high expression of the mannose receptor (CD206), a marker of alternative activation, is a distinct feature of GM-CSF MΦ. Changes of the chromatin structure carried out by chromatin modification enzymes (CME) have been shown to regulate myeloid differentiation. We analyzed the expression patterns of CME during MΦ polarization and show that M1 up-regulate the histone methyltransferase MLL and demethylase KDM6B, while resting and M2 MΦ were characterized by DNA methyltransferases and histone deacetylases. We demonstrate that MLL regulates CXCL10 expression and that this effect could be abrogated using a MLL-Menin inhibitor. Taken together we describe the distinct phenotypic differences of GM-CSF or M-CSF MΦ and demonstrate that MΦ polarization is regulated by specific epigenetic mechanisms. In addition, we describe a novel role for MLL as marker for classical activation. Our findings provide new insights into MΦ polarization that could be helpful to distinguish MΦ activation states.
Control of inflammation is critical for therapy of infectious diseases. Pathogen-associated and/or danger-associated molecular patterns (PAMPs and DAMPs, respectively) are the two major inducers of inflammation. Because the CD24-Siglec G/10 interactions selectively repress inflammatory response to DAMPs, microbial disruption of the negative regulation would provide a general mechanism to exacerbate inflammation. Here we show that the sialic acid-based pattern recognitions of CD24 by Siglec G/10 are targeted by sialidases in polybacterial sepsis. Sialidase inhibitors protect mice against sepsis by a CD24-Siglecg-dependent mechanism, whereas a targeted mutation of either CD24 or Siglecg exacerbates sepsis. Bacterial sialidase and host CD24 and Siglecg genes interact to determine pathogen virulence. Our data demonstrate a critical role for disrupting sialic acid-based pattern recognitions in microbial virulence and suggest a therapeutic approach to dampen harmful inflammatory response during infection.
Theiler's murine encephalomyelitis virus induces a demyelinating disease (TMEV-IDD) of the central nervous system (CNS) in susceptible mouse strains with accompanying histopathology characterized by mononuclear cell infiltrates. In susceptible strains of mice such as SJL, virus establishes a persistent infection in macrophages, induces a CNS infiltration by macrophages, T cells, and B cells, which results in chronic-progressive paralysis. In the present report the authors have investigated the functional role of CCL2 (monocyte chemotactic protein-1) in the induction and progression of demyelinating disease. Treatment of infected mice at day 0, 14, or 28 with anti-CCL2 resulted in a significant decrease in the clinical disease progression. Further analysis of anti-CCL2–treated mice revealed decreased CNS inflammation and mononuclear cell infiltration with an accompanying change in inflammatory cytokine responses. There was an overall decrease in the absolute numbers of CNS-infiltrating CD4+ T cells, macrophages, and B cells. Finally, anti-CCL2 treatment resulted in decreased viral load in the CNS. These data directly demonstrate a role for CCL2 in the pathogenesis of TMEV-IDD.
cell trafficking; chemokines; demyelinating disease; multiple sclerosis; neuroimmunology; Theiler's virus
Chemokines are key mediators of leukocyte recruitment during pathogenic insult and also play a prominent role in homeostasis. While most chemokine receptors bind to multiple chemokines, CCR6 is unique in that this receptor is one of only a few that can bind only a single chemokine ligand, CCL20. CCR6 is an important receptor that is involved in regulating several aspects of mucosal immunity, including the ability to mediate the recruitment of immature dendritic cells (DCs) and mature DCs, and professional antigen presenting cells (APCs) to the sites of epithelial inflammation. Further, CCR6 mediates the homing of both CD4+ T (T-helper; Th) cells and DCs to the gut mucosal lymphoid tissue. DCs, which are known to be essential immune cells in innate immunity and in the initiation of adaptive immunity, play a central role in initiating a primary immune response Herein, we summarize the role of CCR6 in immune responses at epithelial and mucosal sites in both the lung and gut based on a review of the current literature.
CCR6; innate immunity; mucosal immunity; dendritic cell
The cytosolic RNA helicases melanoma differentiation-associated gene 5 (MdA5) and retinoic acid-inducible gene-I (RIG-I) and their adaptor IFNβ promotor stimulator (IPS-1) have been implicated in the recognition of viral RNA and the production of type I interferon (IFN). Complementing the endosomal Toll-like receptors (TLR), Mda5 and RIG-I provide alternative mechanisms for viral detection in cells with reduced phagocytosis or autophagy. The infection route of Respiratory Syncytial Virus (RSV) - via fusion of virus particles with the cell membrane - points to IPS-1 signaling as the pathway of choice for downstream antiviral responses. In the present study, viral clearance and inflammation resolution were indeed strongly affected by the absence of an initial IPS-1-mediated IFNβ response. Despite the blunted inflammatory response in IPS-1 deficient alveolar epithelial cells, pulmonary macrophages and CD11b+ dendritic cells (DC), lungs of RSV-infected IPS-1 knockout (KO) mice showed augmented recruitment of inflammatory neutrophils, monocytes and DC. Interestingly, pulmonary CD103+ DC could functionally compensate for IPS-1 deficiency with the up-regulation of certain inflammatory cytokines and chemokines, possibly via TLR3 and TLR7 signaling. The increased inflammation and reduced viral clearance in IPS-1 KO mice was accompanied by increased T-cell activation and IFNγ production. Experiments with bone marrow chimeras indicated that RSV-induced lung pathology was most severe when IPS-1 expression was lacking in both immune and non-immune cell populations. Similarly, viral clearance was rescued upon restored IPS-1 signaling in either the non-immune or the immune compartment. These data support a non-redundant function for IPS-1 in controlling RSV-induced inflammation and viral replication.
Hypersensitivity pneumonitis (HP) is an inflammatory lung disease that develops following repeated exposure to inhaled particulate antigen. Stachybotrys chartarum (SC) is a dimorphic fungus that has been implicated in a number of respiratory illnesses, including HP (1). In this study we have developed a murine model of SC- induced HP that reproduces pathology observed in human HP and hypothesized that TLR9–mediated IL-23/IL-17 responses are required for the generation of granulomatous inflammation induced by inhaled SC. Mice that undergo i.p. sensitization and i.t. challenge with 106 SC spores developed granulomatous inflammation with multinucleate giant cells, accompanied by increased accumulation of T cells. SC sensitization and challenge resulted in robust pulmonary expression of IL-17 and IL-23. SC-mediated granulomatous inflammation required intact IL-23/IL-17 responses and required TLR9, as TLR9−/− mice displayed reduced IL-17 and IL-23 expression in whole lung associated with decreased accumulation of IL-17 expressing CD4+ and γδ T cells. As compared to SC-sensitized dendritic cells (DC) isolated from WT mice, DC isolated from TLR9−/− mice had a reduced ability to produce IL-23 in responses to SC. Moreover, shRNA knockdown of IL-23 in DC abolished IL-17 production from splenocytes in response to antigen challenge. Finally, the i.t. reconstitution of IL-23 in TLR9−/− mice recapitulated the immunopathology observed in WT mice. In conclusion, our studies suggest that TLR9 is critical for development of Th17-mediated granulomatous inflammation in the lung in response to SC.
Fungus; toll receptors; lung; cytokines
Rationale: Accumulating evidence supports the hypothesis that the continuous host response to a persistent challenge can polarize the cytokine environment toward a Th2 cytokine phenotype, but the mechanisms responsible for this skewing are not clear.
Objectives: We investigated the role of Toll-like receptor 9 (TLR9) in a Th2-driven pulmonary granulomatous response initiated via the embolization of Schistosoma mansoni eggs to the lungs of mice.
Methods: Mice were intravenously injected with S. mansoni eggs. Histological and flow cytometric analysis, cytokine measurement, adoptive transfer of bone marrow (BM)-derived dendritic cells (DCs), and in vitro T-cell treatments with antigen-presenting cells were examined.
Measurements and Main Results: In comparison to wild-type mice, TLR9−/− mice showed increased pulmonary granuloma size, augmented collagen deposition, increased Th2 cytokine phenotype, and impaired accumulation of DCs. BM-derived DCs, but not macrophages, recovered from animals with developed Th2-type lung granulomas promoted the production of type 2 cytokines from CD4+ T cells. BM-derived DCs from TLR9−/− mice induced impaired Th1 cytokine and enhanced Th2 cytokine production by T cells, compared with DCs from WT mice. Macrophages from TLR9−/− mice expressed a significantly higher alternatively activated (M2) phenotype characterized by increased “found in inflammatory zone-1” (FIZZ1) and arginase-1 expression. The adoptive transfer of BM-derived DCs from syngeneic WT mice into TLR9−/− mice restored the granuloma phenotype seen in WT mice.
Conclusions: These studies suggest that TLR9 plays an important mechanistic role in the maintenance of the pulmonary granulomatous response.
granuloma; pulmonary fibrosis; innate immunity; dendritic cell; macrophage
Survivors of severe sepsis exhibit increased morbidity and mortality in response to secondary infections. Although bacterial secondary infections have been widely studied, there remains a paucity of data concerning viral infections post-sepsis. In an experimental mouse model of severe sepsis (cecal ligation and puncture, CLP) followed by respiratory syncytial virus (RSV) infection, exacerbated immunopathology was observed in the lungs of CLP mice compared to RSV-infected sham surgery mice. This virus-associated immunopathology was evidenced by increased mucus production in the lungs of RSV-infected CLP mice and correlated with increased IL-17 production in the lungs. RSV infected CLP mice exhibited increased levels of Th2 cytokines and reduced IFNγ in the lungs and lymph nodes compared to RSV-infected sham mice. In addition, CD4 T cells from CLP mice produced increased IL-17 in vitro irrespective of the presence of exogenous cytokines or blocking antibodies. This increased IL-17 production correlated wth increased STAT3 transcription factor binding to the IL-17 promoter in CD4 T cells from CLP mice. Further, in vivo neutralization of IL-17 prior to RSV infection led to a significant reduction in virus induced mucus production and Th2 cytokines. Taken together, these data provide evidence that post septic CD4+T cells are primed toward IL-17 production via increased STAT3-mediated gene transcription, which may contribute to the immunopathology of a secondary viral infection.
Inflammation; infection; mucus; cytokines; lymphocytes
The present study addressed the modulatory role of CCR4 in TLR9-mediated innate immunity and explored the underlying molecular mechanisms. Our results demonstrated that CCR4-deficient mice were resistant to both septic peritonitis induced by cecal ligation and puncture (CLP) and CpG DNA/D-galactosamine-induced shock. In bone marrow-derived macrophages (BMMΦs) from CLP-treated CCR4-deficient mice, TLR9-mediated pathways of MAPK/AP-1, PI3K/Akt, and IκB kinase (IKK) /NF-κB were impaired compared to WT cells. While TLR9 expression was not altered, the intensity of internalized CpG DNA was increased in CCR4-deficient macrophages when compared to WT macrophages. Pharmacological inhibitor studies revealed that impaired activation of JNK, PI3K/Akt, and/or IKK/NF-κB could be responsible for decreased proinflammatory cytokine expression in CCR4-deficient macrophages. Interestingly, the CCR4-deficient BMMΦs exhibited an alternatively activated (M2) phenotype and the impaired TLR9-mediated signal transduction responses in CCR4-deficient cells were similar to the signaling responses observed in WT BMMΦs skewed to an alternatively activated phenotype. These results indicated that macrophages deficient in CCR4 impart a regulatory influence on TLR9-mediated innate immunity.
Chemokines; Toll-like receptors; Macrophages
Ligands from dying cells are a source of Toll-like receptor (TLR) activating agents. Although TLR3 is known to respond to RNA from necrotic cells, the relative importance of this response in vivo during acute inflammatory processes has not been fully explored. We observed the involvement of TLR3 activation during experimental polymicrobial septic peritonitis and ischemic gut injury in the absence of an exogenous viral stimulus. In TLR3-deficient mice, increased chemokine/cytokine levels and neutrophil recruitment characterized the initial inflammatory responses in both injury models. However, the levels of inflammatory chemokines and tumor necrosis factor α quickly returned to baseline in tlr3−/− mice, and these mice were protected from the lethal effects of sustained inflammation. Macrophages from tlr3−/− mice responded normally to other TLR ligands but did not respond to RNA from necrotic neutrophils. Importantly, an immunoneutralizing antibody directed against TLR3 attenuated the generation of inflammatory chemokines evoked by byproducts from necrotic neutrophils cultured with wild-type macrophages. In vivo, anti-TLR3 antibody attenuated the tissue injury associated with gut ischemia and significantly decreased sepsis-induced mortality. Collectively, these data show that TLR3 is a regulator of the amplification of immune response and serves an endogenous sensor of necrosis, independent of viral activation.
TLR9 activation is important for the maintenance of mycobacteria-elicited pulmonary granulomatous responses, hallmarks of protective immune responses following mycobacterial infection. However, the mechanism or mechanisms underlying this effect of TLR9 are not clear. Here, we show that Tlr9-deficient mice challenged with a Mycobacterium antigen display an altered Th17 cytokine profile, decreased accumulation of granuloma-associated myeloid DCs, and profoundly impaired delta-like 4 (dll4) Notch ligand expression. Mechanistic analysis revealed that WT bone marrow–derived DCs but not macrophages promoted the differentiation of Th17 cells from bacillus Calmette-Guérin–challenged (BCG-challenged) lung CD4+ T cells. Both lung and bone marrow DCs isolated from Tlr9-deficient mice inoculated with Mycobacterium antigen expressed lower levels of dll4 Notch ligand than the same cells isolated from WT mice. Passively immunizing WT mice with neutralizing antibodies specific for dll4 during granuloma formation resulted in larger granulomas and lower levels of Th17-related cytokines. In addition, dll4 specifically regulated Th17 activation in vitro. Together, these results suggest dll4 plays an important role in promoting Th17 effector activity during a mycobacterial challenge. Furthermore, TLR9 seems to be required for optimal dll4 expression and the regulation of Mycobacterium antigen–elicited granuloma formation in mice.
Murine gammaherpesvirus 68 (MHV68) is a natural rodent pathogen that has been used as a model to study the pathogenesis of human gammaherpesviruses. Like other herpesviruses, MHV68 causes acute infection and establishes life-long latency in the host. Recently, it has been shown that mice latently infected with MHV68 have resistance to unrelated pathogens in secondary infection models. We therefore hypothesized that latent MHV68 infection could modulate the host response to influenza A virus. To test this hypothesis, mice were infected intranasally with influenza virus following the establishment of MHV68 latency. Mice latently infected with MHV68 showed significantly higher survival to influenza A virus infection than did PBS mock-infected mice. Latent MHV68 infection led to lower influenza viral loads and decreased inflammatory pathology in the lungs. Alveolar macrophages of mice latently infected with MHV68 showed activated status, and adoptive transfer of those activated macrophages into mice followed the infection with influenza A virus had significantly greater survival rates than control mice, suggesting that activated alveolar macrophages are a key mechanistic component in protection from secondary infections.
gammaherpesvirus; influenza A virus; alveolar macrophages; neutrophils
Current dogma supports the concept that the expression of a disease-inducing signature cytokine phenotype is important to the maintenance stage of chronic lung disorders. This cytokine phenotype has been characterized as a polarization toward type 2 cytokines, which are profibrotic and immunoregulatory. The biology of this latter activity could mechanistically explain pathogen-induced exacerbation of chronic lung inflammation, as a skewed cytokine profile in the lung alters dendritic cell function, activates fibroblasts, and facilitates a subsequent “second hit” by an infectious pathogen. In this setting, cytokine biology is also linked to Toll-like receptors (TLRs) in the maintenance of lung immunity, as the activity of this receptor–ligand system by both leukocytes and stromal cells is likely an important component of disease chronicity. The participation of dendritic cells via TLRs in chronic lung disease could facilitate communication circuits established between antigen-presenting cells and lymphocytes. Data suggest that TLR activation via myeloid differentiation factor 88 adaptor protein leads to the induction of a Notch ligand known as Delta-like-4 on dendritic cells that activate the Notch receptor on T cells, promoting a helper T-cell type 1 cytokine response. It is likely that the evolution of host defense signals designed to recognize patterns emitted from a hostile microbial environment may now be superimposed on adaptive immunity and provide the underpinning to support the maintenance of chronic lung disease.
virus; dendritic cell; innate immunity
Type-1 and type-2 lung granulomas, respectively, elicited by bead immobilized Mycobacteria bovis and Schistosoma mansoni egg antigens (Ags) display different patterns of chemokine expression. This study tested the hypothesis that chemokine expression patterns were related to upstream cytokine signaling. Using quantitative transcript analysis, we defined expression profiles for 16 chemokines and then examined the in vivo effects of neutralizing antibodies against interferon-γ (IFN-γ), interleukin (IL)-4, IL-10, IL-12, and IL-13. Transcripts for CXCL2, −5, −9, −10, and −11 and the CCL chemokine, CCL3, and lymphotactin (XCL1), were largely enhanced by Th1-related cytokines, IFN-γ or IL-12. Transcripts for CCL11, CCL22, CCL17, and CCL1 were enhanced largely by Th2-related cytokines, IL-4, IL-10, or IL-13. Transcripts for CCL4, CCL2, CCL8, CCL7, and CCL12 were potentially induced by either Th1- or Th2-related cytokines, although some of these showed biased expression. IFN-γ and IL-4 enhanced the greatest complement of transcripts, and their neutralization had the greatest anti-inflammatory effect on type-1 and type-2 granulomas, respectively. Th1/Th2 cross-regulation was evident because endogenous Th2 cytokines inhibited type-1, whereas Th1 cytokines inhibited type-2 biased chemokines. These findings reveal a complex cytokine–chemokine regulatory network that dictates profiles of local chemokine expression during T cell–mediated granuloma formation.
Toll-like receptor (TLR) activation has been implicated in acetaminophen (APAP)-induced hepatotoxicity. Herein, we hypothesize that TLR3 activation significantly contributed to APAP-induced liver injury. In fasted wildtype (WT) mice, APAP caused significant cellular necrosis, edema, and inflammation in the liver, and the de novo expression and activation of TLR3 was found to be necessary for APAP-induced liver failure. Specifically, liver tissues from similarly fasted TLR3-deficient (tlr3−/−) mice exhibited significantly less histological and biochemical evidence of injury after APAP challenge. Similar protective effects were observed in WT mice in which TLR3 was targeted through immunoneutralization at 3 h post-APAP challenge. Among three important death ligands (i.e. TNFα, TRAIL, and FASL) known to promote hepatocyte death after APAP challenge, TNFα was the only ligand that was significantly reduced in APAP-challenged tlr3−/− mice compared with APAP-challenged WT controls. In vivo studies demonstrated that TLR3 activation contributed to TNFα production in the liver presumably via F4/80+ and CD11c+ immune cells. In vitro studies indicated that there was cooperation between TNFα and TLR3 in the activation of JNK signaling in isolated and cultured liver epithelial cells (i.e. nMuLi). Moreover, TLR3 activation enhanced the expression of phosphorylated JNK in APAP injured livers. Thus, the current study demonstrates that TLR3 activation contributes to APAP-induced hepatotoxicity.
C57BL/6 mice were maintained for up to 18-months on high-fat and low-fat diets with or without a multi-mineral-supplement derived from the skeletal remains of the red marine algae Lithothamnion calcareum. Numerous grossly observable liver masses were visible in animals on the “western-style” high-fat diet sacrificed at 12 and 18 months. The majority of the masses were in male mice (20 out of 100 males versus 3 out of 100 females; p=0.0002). There were more liver masses in animals on the high-fat diet than on the low-fat diet (15 out of 50 on high-fat versus 5 out of 50 on low-fat; p=0.0254). The multi-mineral supplement reduced the number of liver masses in mice on both diets (3 out of 25 male mice in the low-fat diet group without the supplement versus 1 out of 25 mice with supplement; 12 of 25 male mice in the high-fat diet group without the supplement versus 3 of 25 mice with supplement [p=0.0129]). Histological evaluation revealed a total of 17 neoplastic lesions (9 adenomas and 8 hepatocellular carcinomas), and 18 pre-neoplastic lesions. Out of 8 hepatocellular carcinomas, 7 were found in unsupplemented diet groups. Steatosis was widely observed in livers with and without grossly observable masses, but the multi-mineral supplement had no effect on the incidence of steatosis or its severity. Taken together, these findings suggest that a multi-mineral-rich natural product can protect mice against neoplastic and pre-neoplastic proliferative liver lesions that may develop in the face of steatosis.
Calcium; hepatocellular carcinoma; liver disease; minerals; trace elements
Although arachidonic acid cascade has been shown to be involved in sepsis, little is known about the role of prostaglandin D2 and its newly found receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), on the septic response. Severe sepsis is associated with the failure of neutrophil migration. To investigate whether CRTH2 influences neutrophil recruitment and the lethality during sepsis, sepsis was induced by cecal ligation and puncture (CLP) surgery in mice. CRTH2 knockout (−/−) mice were highly resistant to CLP-induced sepsis, which was associated with lower bacterial load and lower production of TNF-α, IL-6, and CCL3. IL-10, an anti-inflammatory cytokine, was higher in CRTH2−/− mice, blunting CLP-induced lethality in CRTH2−/− mice. Neutrophil accumulation in the peritoneum was more pronounced after CLP in CRTH2−/− mice, which was associated with higher CXCR2 level in circulating neutrophils. Furthermore, sepsis caused a decrease in the level of acetylation of histone H3, an activation mark, at the CXCR2 promoter in WT neutrophils, suggesting that CXCR2 expression levels are epigenetically regulated. Finally, both pharmacological depletion of neutrophils and inhibition of CXCR2 abrogated the survival benefit in CRTH2−/− mice. These results demonstrate that genetic ablation of CRTH2 improved impaired neutrophil migration and survival during severe sepsis, which was mechanistically associated with epigenetic mediated CXCR2 expression. Thus, CRTH2 is a potential therapeutic target for polymicrobial sepsis.
Granulomas represent a spectrum of inflammatory sequestration responses that may be initiated by a variety of agents, including non-infectious environmental factors and infectious microbial pathogens. Although this reaction is designed to be protective, the associated tissue injury is often responsible for a profound degree of pathology. While many of the mechanisms that sustain the development of the granuloma are enigmatic, it is accepted that the maintenance of this inflammatory process is dependent upon dynamic interactions between an inciting agent, inflammatory mediators, various immune and inflammatory cells, and structural cells of the involved tissue. The best studied of the host-dependent processes during granuloma development is the innate and adaptive immune response. The innate immune response by antigen-presenting cells [APCs; dendritic cells (DCs) and macrophages] is initiated quickly to protect from overwhelming pathogens, but with time, can also activate the adaptive immune response. APCs, essential regulators of the innate immune response, can respond to microbial ligands through Toll-like receptors (TLRs), which function in the recognition of microbial components and play an important role to link the innate and adaptive immune responses. CD4+ T helper (Th) cells are essential regulators of adaptive immune responses and inflammatory diseases. Recently, the Notch system has been shown to be an important bridge between APCs and T cell communication circuits. In the present review, we discuss recent findings that explore the mechanisms in the linkage of innate and adaptive immunity, including granulomatous formation though TLRs and Notch activation.
Notch signaling; Toll-like receptor; dendritic cell; T helper cell; innate immunity; acquired immunity
Toll like receptors (TLR) bridge innate immunity and host responses, including inflammation. Sterile inflammation such as a venous thrombus (VT) may involve TLR signaling, including TLR9.
Methods and Results
TLR9 signaling on thrombus resolution was investigated using a mouse model of stasis VT. VT were significantly larger in TLR9 −/− mice as compared with WT at 2 and 8 days, despite a 2 fold increase in thrombus PMN at 2d, and monocytes at 8d, while thrombus collagen and neovascularization was 55% and 37% less at 8d. Coincidently, decreased fibrinogen and increased thrombin-antithrombin complex were observed in TLR9 −/− mice thrombi. Vein wall IFNα, IL1α, and IL2 was significantly reduced in TLR9 −/− mice as compared to WT. Thrombus cell death pathway markers were not significantly altered at 2d, but caspase 1 was reduced in TLR9 −/− thrombi at 8d. MyD88 confers TLR9 intracellular signaling, but MyD88−/− mice had similar VT resolution as WT. However, inhibition of the NOTCH ligand delta-like 4 was associated with larger VT. Finally, stimulation with a TLR9 agonist was associated with smaller VT.
TLR9 signaling is integral for early and mid VT resolution through modulation of sterile inflammation, maintaining a TH1 milieu, and effects on the thrombosis pathway.
Previous studies have suggested that neonates rely heavily on innate immunity for their antimicrobial response to bacterial infections. However, the innate immune response by neonates to bacterial infection remains poorly characterized. Here, we show that in a murine model of neonatal polymicrobial sepsis, CXC ligand 10 (CXCL10) concentrations increase in the blood and peritoneum concordant with the peritoneal recruitment of granulocytes and macrophages. Additionally, CXC receptor 3 (CXCR3) expression on elicited peritoneal macrophages and granulocytes increases following sepsis. Blockade of CXCL10 worsens not only recruitment and phagocytic function of peritoneal granulocytes and macrophages but also survival. Deletion of CXCR3 also significantly increases mortality to a septic challenge. Finally, we demonstrate that the protective adjuvant effect of pretreatment with a Toll-like receptor 4 agonist to neonatal sepsis is dependent on an endogenous CXCL10 response and that pretreatment of neonates with CXCL10 can also significantly improve macrophage and granulocyte function and modestly improve outcome to polymicrobial sepsis. Together, these data suggest a critical role for CXCL10 signaling during neonatal sepsis.
Influenza A viral infections have been identified as the etiologic agents for historic pandemics, and contribute to the annual mortality associated with acute viral pneumonia. While both innate and acquired immunity are important in combating influenza virus infection, the mechanism connecting these arms of the immune system remains unknown. Recent data have indicated that the Notch system is an important bridge between antigen-presenting cells (APCs) and T cell communication circuits and plays a central role in driving the immune system to overcome disease. In the present study, we examine the role of Notch signaling during influenza H1N1 virus infection, focusing on APCs. We demonstrate here that macrophages, but not dendritic cells (DCs), increased Notch ligand Delta-like 1 (Dll1) expression following influenza virus challenge. Dll1 expression on macrophages was dependent on retinoic acid-inducible gene-I (RIG-I) induced type-I IFN pathway, and not on the TLR3-TRIF pathway. We also found that IFNα-Receptor knockout mice failed to induce Dll1 expression on lung macrophages and had enhanced mortality during influenza virus infection. Our results further showed that specific neutralization of Dll1 during influenza virus challenge induced higher mortality, impaired viral clearance, and decreased levels of IFN-γ. In addition, we blocked Notch signaling by using γ-secretase inhibitor (GSI), a Notch signaling inhibitor. Intranasal administration of GSI during influenza infection also led to higher mortality, and higher virus load with excessive inflammation and an impaired production of IFN-γ in lungs. Moreover, Dll1 expression on macrophages specifically regulates IFN-γ levels from CD4+and CD8+T cells, which are important for anti-viral immunity. Together, the results of this study show that Dll1 positively influences the development of anti-viral immunity, and may provide mechanistic approaches for modifying and controlling the immune response against influenza H1N1 virus infection.
Influenza viruses cause annual epidemics and occasional pandemics that have claimed the lives of millions. Both innate and acquired immunity are essential for protection against influenza virus, and Notch and Notch ligands provide a key bridge between innate and acquired immunity. However, the role of Notch system during influenza virus infection is unknown. Here, we show that Notch ligand Delta-like 1 (Dll1) expression was up-regulated in influenza virus H1N1 challenged macrophages, and was dependent on both retinoic-acid–inducible protein I (RIG-I) and IFNα receptor (IFNαR)-mediated pathways. IFNαR-deficient mice challenged with influenza virus in vivo also display a profoundly impaired Dll1 expression with increased mortality and abrogated IFN-γ production. Treatment of WT mice during influenza infection, with either neutralizing antibodies specific for Dll1 or a γ-secretase inhibitor (GSI), which blocks Notch signaling, resulted in increased mortality, impaired viral clearance, and lower IFN-γ production. In addition, Dll1 specifically regulated IFN-γ production from both CD4+and CD8+T cells in vitro. Together, these results suggest that Notch signaling through macrophage-dependent Dll1 is critical in providing an anti-viral response during influenza infection by linking innate and acquired immunity.
TLRs are a family of receptors that mediate immune system pathogen recognition. In the respiratory system, TLR activation has both beneficial and deleterious effects in asthma. For example, clinical data indicate that TLR6 activation exerts protective effects in asthma. Here, we explored the mechanism or mechanisms through which TLR6 mediates this effect using mouse models of Aspergillus fumigatus–induced and house dust mite antigen–induced (HDM antigen–induced) chronic asthma. Tlr6–/– mice with fungal- or HDM antigen–induced asthma exhibited substantially increased airway hyperresponsiveness, inflammation, and remodeling compared with WT asthmatic groups. Surprisingly, whole-lung levels of IL-23 and IL-17 were markedly lower in Tlr6–/– versus WT asthmatic mice. Tlr6–/– DCs generated less IL-23 upon activation with lipopolysaccharide, zymosan, or curdlan. Impaired IL-23 generation in Tlr6–/– mice also corresponded with lower levels of expression of the pathogen-recognition receptor dectin-1 and expansion of Th17 cells both in vivo and in vitro. Exogenous IL-23 treatment of asthmatic Tlr6–/– mice restored IL-17A production and substantially reduced airway hyperresponsiveness, inflammation, and lung fungal burden compared with that in untreated asthmatic Tlr6–/– mice. Together, our data demonstrate that TLR6 activation is critical for IL-23 production and Th17 responses, which both regulate the allergic inflammatory response in chronic fungal-induced asthma. Thus, therapeutics targeting TLR6 activity might prove efficacious in the treatment of clinical asthma.
Previous epidemiological studies in humans and experimental studies in animals indicate that survivors of severe sepsis exhibit deficiencies in the activation and effector function of immune cells. In particular, CD4+ T lymphocytes can exhibit reduced proliferative capacity and improper cytokine responses following sepsis. To further investigate the cell-intrinsic defects of CD4+ T cells following sepsis, splenic CD4+ T cells from sham surgery and post-septic mice were transferred into lymphopenic mice. These recipient mice were then subjected to both TH1-(purified protein derivative) and TH2-(Schistosoma mansoni egg antigen) driven models of granulomatous lung inflammation. Post-septic CD4+ T cells mediated smaller TH1 and larger TH2 lung granulomas as compared to mice receiving CD4+ T cells from sham surgery donors. However, cytokine production by lymph node cells in antigen restimulation assays indicated increased pan-specific cytokine expression by post-septic CD4+ T cell recipient mice in both TH1 and TH2 granuloma models. These include increased production of TH2 cytokines in TH1 inflammation, and increased production of TH1 cytokines in TH2 inflammation. These results suggest that cell-intrinsic defects in CD4+ T cell effector function can have deleterious effects on inflammatory processes post-sepsis, due to a defect in the proper regulation of TH-specific cytokine expression.
Immunosuppression following severe sepsis remains a significant human health concern, as long-term morbidity and mortality rates of patients who have recovered from life-threatening septic shock remain poor. Mouse models of severe sepsis indicate this immunosuppression may be partly due to alterations in myeloid cell function; however, the effect of severe sepsis on subsequent CD4+ T cell responses remains unclear. In the present study, CD4+ T cells from mice subjected to an experimental model of severe sepsis (cecal ligation and puncture, CLP) were analyzed in vitro. CD4+ CD62L+ T cells from CLP mice exhibited reduced proliferative capacity and altered gene expression. Additionally, CD4+ CD62L+ T cells from CLP mice exhibit dysregulated cytokine production after in vitro skewing with exogenous cytokines, indicating a decreased capability of these cells to commit to either the TH1 or TH2 lineage. Repressive histone methylation marks were also evident at promoter regions for the TH1 cytokine interferon-γ (IFN-γ) and the TH2 transcription factor GATA-3 in naïve CD4+ T cells from CLP mice. These results provide evidence that CD4+ T cell subsets from postseptic mice exhibit defects in activation and effector function, possibly due to chromatin remodeling proximal to genes involved in cytokine production or gene transcription.
Sepsis; CD4+ T cell; Inflammation; Epigenetics; Mouse