Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia

Fungal infections have skyrocketed in immune-compromised patients lacking CD4⁺ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A⁺ CD8⁺ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4⁺ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ⁺ CD8⁺ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8⁺ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.     

Impaired Innate Immunity in Tlr4 ?/? Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8⁺ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8⁺ T cells specific for H-2K^b-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2^−/−, Tlr4^−/−, Tlr9^{−/ −} or Myd88^−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8⁺ T cells at levels comparable to WT mice, although the frequency of IFN-γ⁺CD4⁺ cells was diminished in infected Myd88^−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4^−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi.     

Natural killer cell intrinsic toll-like receptor MyD88 signaling contributes to IL-12-dependent IFN-γ production by mice during infection with Toxoplasma gondii

Myeloid differentiation factor 88 (MyD88)-dependent IL-12 secretion by dendritic cells is critical for natural killer cell-mediated IFN-γ production and innate resistance to Toxoplasma gondii. Although MyD88^−/− mice challenged with T. gondii have defective IL-12 responses and succumb to infection, administration of IL-12 to MyD88^−/− mice fails to prevent acute mortality, suggesting that MyD88 may mediate signals within natural killer cells important for IL-12-dependent IFN-γ production and innate resistance to this parasite. In this study, we found that T. gondii antigens and IL-12 could synergistically trigger IFN-γ secretion by natural killer cells, which was dependent on toll-like receptor-MyD88 signaling. Further analysis showed that p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, c-Jun N-terminal kinase and NF-κB multiple pathways downstream of MyD88 contributed to IFN-γ production by natural killer cells. Moreover, the well-established toll-like receptor agonists, T. gondii profilin (Tgprofilin) and T. gondii heat shock protein 70 (TgHSP70) could evoke a similar IFN-γ secretory response in natural killer cells to that evoked by T. gondii antigens. In vivo adoptive transfer experiments showed that, upon challenge with T. gondii, NOD/SCID-β2 microglobulin null (NOD/SCID-β2m^−/−) mice injected i.v. with MyD88^−/− natural killer cells had reduced serum IFN-γ levels and increased splenic tachyzoite burdens compared with those injected i.v. with wild-type natural killer cells. Taken together, these findings demonstrate a critical role for natural killer cell intrinsic toll-like receptor-MyD88 signaling in IL-12-dependent early IFN-γ production and innate resistance to T. gondii.     

CXCR3-Dependent CD4+ T Cells Are Required to Activate Inflammatory Monocytes for Defense against Intestinal Infection

Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4⁺ and CD8⁺ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3^−/− mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4⁺ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b⁺Ly6C/G⁺ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3^−/− mice. Strikingly, adoptive transfer of wild-type but not Ifnγ^−/− CD4⁺ T lymphocytes into Cxcr3^−/− animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes.     

Differential Regulation of Effector- and Central-Memory Responses to Toxoplasma gondii Infection by IL-12 Revealed by Tracking of Tgd057-Specific CD8+ T Cells

Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-γ-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2K^b-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-γ production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-γ recall responses. Importantly, isolated CD62L^hi KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-γ-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.     

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.     

Deficiency of Leishmania Phosphoglycans Influences the Magnitude but Does Not Affect the Quality of Secondary (Memory) Anti-Leishmania Immunity

Despite inducing very low IFN-γ response and highly attenuated in vivo, infection of mice with phosphoglycan (PG) deficient Leishmania major (lpg2-) induces protection against virulent L. major challenge. Here, we show that mice infected with lpg2- L. major generate Leishmania-specific memory T cells. However, in vitro and in vivo proliferation, IL-10 and IFN-γ production by lpg2- induced memory cells were impaired in comparison to those induced by wild type (WT) parasites. Interestingly, TNF recall response was comparable to WT infected mice. Despite the impaired proliferation and IFN-γ response, lpg2- infected mice were protected against virulent L. major challenge and their T cells mediated efficient infection-induced immunity. In vivo depletion and neutralization studies with mAbs demonstrated that lpg2- L. major-induced resistance was strongly dependent on IFN-γ, but independent of TNF and CD8⁺ T cells. Collectively, these data show that the effectiveness of secondary anti-Leishmania immunity depends on the quality (and not the magnitude) of IFN-γ response. These observations provide further support for consideration of lpg2- L. major as a live-attenuated candidate for leishmanization in humans since it protects strongly against virulent challenge, without inducing pathology in infected animals.     

P47phox?/? Mice Are Compromised in Expansion and Activation of CD8+ T Cells and Susceptible to Trypanosoma cruzi Infection

Macrophage activation of NAD(P)H oxidase (NOX2) and reactive oxygen species (ROS) is suggested to kill Trypanosoma cruzi that causes Chagas disease. However, the role of NOX2 in generation of protective immunity and whether these mechanisms are deregulated in the event of NOX2 deficiency are not known, and examined in this study. Our data showed that C57BL/6 p47^phox−/− mice (lack NOX2 activity), as compared to wild-type (WT) mice, succumbed within 30 days post-infection (pi) to low doses of T. cruzi and exhibited inability to control tissue parasites. P47^phox−/− bone-marrow and splenic monocytes were not compromised in maturation, phagocytosis and parasite uptake capacity. The deficiency of NOX2 mediated ROS was compensated by higher level of inducible nitric oxide synthase (iNOS) expression, and nitric oxide and inflammatory cytokine (TNF-α, IFN-γ, IL-1β) release by p47^phox−/− macrophages as compared to that noted in WT controls infected by T. cruzi. Splenic activation of Th1 CD4⁺T cells and tissue infiltration of immune cells in T. cruzi infected p47^phox−/− mice were comparable to that noted in infected control mice. However, generation and activation of type 1 CD8⁺T cells was severely compromised in p47^phox−/− mice. In comparison, WT mice exhibited a robust T. cruzi-specific CD8⁺T cell response with type 1 (IFN-γ+TNF-α>IL-4+IL-10), cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺) phenotype. We conclude that NOX2/ROS activity in macrophages signals the development of antigen-specific CD8⁺T cell response. In the event of NOX2 deficiency, a compromised CD8⁺T cell response is generated, leading to increased parasite burden, tissue pathogenesis and mortality in chagasic mice.     

The Protein Kinase Double-Stranded RNA-Dependent (PKR) Enhances Protection against Disease Cause by a Non-Viral Pathogen

PKR is well characterized for its function in antiviral immunity. Using Toxoplasma gondii, we examined if PKR promotes resistance to disease caused by a non-viral pathogen. PKR^−/− mice infected with T. gondii exhibited higher parasite load and worsened histopathology in the eye and brain compared to wild-type controls. Susceptibility to toxoplasmosis was not due to defective expression of IFN-γ, TNF-α, NOS2 or IL-6 in the retina and brain, differences in IL-10 expression in these organs or to impaired induction of T. gondii-reactive T cells. While macrophages/microglia with defective PKR signaling exhibited unimpaired anti-T. gondii activity in response to IFN-γ/TNF-α, these cells were unable to kill the parasite in response to CD40 stimulation. The TRAF6 binding site of CD40, but not the TRAF2,3 binding sites, was required for PKR phosphorylation in response to CD40 ligation in macrophages. TRAF6 co-immunoprecipitated with PKR upon CD40 ligation. TRAF6-PKR interaction appeared to be indirect, since TRAF6 co-immunoprecipitated with TRAF2 and TRAF2 co-immunoprecipitated with PKR, and deficiency of TRAF2 inhibited TRAF6-PKR co-immunoprecipitation as well as PKR phosphorylation induced by CD40 ligation. PKR was required for stimulation of autophagy, accumulation the autophagy molecule LC3 around the parasite, vacuole-lysosomal fusion and killing of T. gondii in CD40-activated macrophages and microglia. Thus, our findings identified PKR as a mediator of anti-microbial activity and promoter of protection against disease caused by a non-viral pathogen, revealed that PKR is activated by CD40 via TRAF6 and TRAF2, and positioned PKR as a link between CD40-TRAF signaling and stimulation of the autophagy pathway.     

Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Methodology/Principal Findings

Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.

Conclusion/Significance

Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.     

Lymphocytoid choriomeningitis virus activates plasmacytoid dendritic cells and induces a cytotoxic T-cell response via MyD88

Toll-like receptors (TLRs) and retinoic acid-inducible gene I-like helicases (RLHs) are two major machineries recognizing RNA virus infection of innate immune cells. Intracellular signaling for TLRs and RLHs is mediated by their cytoplasmic adaptors, i.e., MyD88 or TRIF and IPS-1, respectively. In the present study, we investigated the contributions of TLRs and RLHs to the cytotoxic T-lymphocyte (CTL) response by using lymphocytoid choriomeningitis virus (LCMV) as a model virus. The generation of virus-specific cytotoxic T lymphocytes was critically dependent on MyD88 but not on IPS-1. Type I interferons (IFNs) are known to be important for the development of the CTL response to LCMV infection. Serum levels of type I IFNs and proinflammatory cytokines were mainly dependent on the presence of MyD88, although IPS-1^−/− mice showed a decrease in IFN-α levels but not in IFN-β and proinflammatory cytokine levels. Analysis of Ifna6^+/GFP reporter mice revealed that plasmacytoid dendritic cells (DCs) are the major source of IFN-α in LCMV infection. MyD88^−/− mice were highly susceptible to LCMV infection in vivo. These results suggest that recognition of LCMV by plasmacytoid DCs via TLRs is responsible for the production of type I IFNs in vivo. Furthermore, the activation of a MyD88-dependent innate mechanism induces a CTL response, which eventually leads to virus elimination.     

Interferon Gamma Suppresses Collagen-Induced Arthritis by Regulation of Th17 through the Induction of Indoleamine-2,3-Deoxygenase

C57BL/6 mice are known to be resistant to the development of collagen-induced arthritis (CIA). However, they show a severe arthritic phenotype when the Ifng gene is deleted. Although it has been proposed that IFN-γ suppresses inflammation in CIA via suppressing Th17 which is involved in the pathogenesis of CIA, the exact molecular mechanism of the Th17 regulation by IFN-γ is poorly understood. This study was conducted to 1) clarify that arthritogenic condition of IFN-γ knockout (KO) mice is dependent on the disinhibition of Th17 and 2) demonstrate that IFN-γ-induced indoleamine2,3dioxgenase (IDO) is engaged in the regulation of Th17. The results showed that the IFN-γ KO mice displayed increased levels of IL-17 producing T cells and the exacerbation of arthritis. Also, production of IL-17 by the splenocytes of the IFN-γ KO mice was increased when cultured with type II collagen. When Il17 was deleted from the IFN-γ KO mice, only mild arthritis developed without any progression of the arthritis score. The proportion of CD44^highCD62L^low memory-like T cells were elevated in the spleen, draining lymph node and mesenteric lymph node of IFN-γ KO CIA mice. Meanwhile, CD44^lowCD62L^high naïve T cells were increased in IFN-γ and IL-17 double KO CIA mice. When Th17 polarized CD4+ T cells of IFN-γ KO mice were co-cultured with their own antigen presenting cells (APCs), a greater increase in IL-17 production was observed than in co-culture of the cells from wild type mice. In contrast, when APCs from IFN-γ KO mice were pretreated with IFN-γ, there was a significant reduction in IL-17 in the co-culture system. Of note, pretreatment of 1-methyl-DL- tryptophan, a specific inhibitor of IDO, abolished the inhibitory effects of IFN-γ. Given that IFN-γ is a potent inducer of IDO in APCs, these results suggest that IDO is involved in the regulation of IL-17 by IFN-γ.     

Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis

Background

Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far.

Methodology/Principal Findings

Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2^-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2^-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2^-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra-intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2^-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2^-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals.

Conclusion/Significance

NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner.     

Activation of Toll-Like Receptor 2 Prevents Suppression of T-Cell Interferon γ Production by Modulating p38/Extracellular Signal-Regulated Kinase Pathways following Alcohol and Burn Injury

Recent studies indicate that toll-like receptors (TLRs) are expressed on T cells and that these receptors directly or indirectly activate the adaptive immune system. We have shown previously that acute alcohol/ethanol (EtOH) intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell interleukin-2 (IL-2) and interferon γ (IFN-γ) production. We examined whether direct stimulation of T cells with TLR2, 4, 5 and 7 agonists modulates CD3-mediated T-cell IL-2/IFN-γ release following EtOH and burn injury. Male mice were gavaged with EtOH (2.9 gm/kg) 4 h prior to receiving an ~12.5% total body surface area sham or full-thickness burn injury. Animals were killed on d 1 after injury and T cells were purified from MLN and spleens. T cells were cultured with plate-bound anti-CD3 in the presence or absence of various TLR ligands. Although TLR2, 4 and 5 agonists potentiate anti-CD3–dependent IFN-γ by T cells, the TLR2 agonist alone induced IFN-γ production independent of CD3 stimulation. Furthermore, T cells were treated with inhibitors of myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), p38 and/or extracellular signal-regulated kinase (ERK) to determine the mechanism by which TLR2 mediates IL-2/IFN-γ production. IL-2 was not influenced by TLR agonists. MyD88 and TIRAP inhibitory peptides dose-dependently diminished the ability of T cells to release IFN-γ. p38 and ERK inhibitors also abolished TLR2-mediated T-cell IFN-γ. Together, our findings suggest that TLR2 directly modulates T-cell IFN-γ production following EtOH and burn injury, independent of antigen-presenting cells. Furthermore, we demonstrated that MyD88/TIRAP-dependent p38/ERK activation is critical to TLR2-mediated T-cell IFN-γ release following EtOH and burn injury.     

FimH Adhesin of Type 1 Fimbriae Is a Potent Inducer of Innate Antimicrobial Responses Which Requires TLR4 and Type 1 Interferon Signalling

Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs). We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-β production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88^−/−, Trif^−/−, IFN−α/βR^−/− or IRF3^−/− mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88^−/−, IFN-α/βR^−/−, IRF3^−/− or TLR4^−/− mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-β, but not IFN-α or IFN-γ. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC) and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6) mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4^−/− mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens.     

CD8+ T Cells Mediate Female-Dominant IL-4 Production and Airway Inflammation in Allergic Asthma

The prevalence and severity of bronchial asthma are higher in females than in males after puberty. Although antigen-specific CD8⁺ T cells play an important role in the development of asthma through their suppressive effect on cytokine production, the contribution of CD8⁺ T cells to sex differences in asthmatic responses remains unclear. In the present study, we investigated the sex-specific effect of CD8⁺ T cells in the suppression of asthma using an ovalbumin mouse model of asthma. The number of inflammatory cells in bronchoalveolar lavage (BAL) fluid, lung type 2 T-helper cytokine levels, and interleukin-4 (IL-4) production by bronchial lymph node cells were significantly higher in female wild-type (WT) mice compared with male mice, whereas no such sex differences were observed between male and female cd8α-disrupted mice. The adaptive transfer of male, but not female, CD8⁺ T cells reduced the number of inflammatory cells in the recovered BAL fluid of male recipient mice, while no such sex difference in the suppressive activity of CD8⁺ T cells was observed in female recipient mice. Male CD8⁺ T cells produced higher levels of IFN-γ than female CD8⁺ T cells did, and this trend was associated with reduced IL-4 production by male, but not female, CD4⁺ T cells. Interestingly, IFN-γ receptor expression on CD4⁺ T cells was significantly lower in female mice than in male mice. These results suggest that female-dominant asthmatic responses are orchestrated by the reduced production of IFN-γ by CD8⁺ T cells and the lower expression of IFN-γ receptor on CD4⁺ T cells in females compared with males.     

Insights into the Mechanisms of Protective Immunity against Cryptococcus neoformans Infection Using a Mouse Model of Pulmonary Cryptococcosis

Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening pneumonia and meningoencephalitis in immune compromised individuals. Previous studies have shown that immunization of BALB/c mice with an IFN-γ-producing C. neoformans strain, H99γ, results in complete protection against a second pulmonary challenge with an otherwise lethal cryptococcal strain. The current study evaluated local anamnestic cell-mediated immune responses against pulmonary cryptococcosis in mice immunized with C. neoformans strain H99γ compared to mice immunized with heat-killed C. neoformans (HKC.n.). Mice immunized with C. neoformans strain H99γ had significantly reduced pulmonary fungal burden post-secondary challenge compared to mice immunized with HKC.n. Protection against pulmonary cryptococcosis was associated with increased pulmonary granulomatous formation and leukocyte infiltration followed by a rapid resolution of pulmonary inflammation, which protected the lungs from severe allergic bronchopulmonary mycosis (ABPM)-pathology that developed in the lungs of mice immunized with HKC.n. Pulmonary challenge of interleukin (IL)-4 receptor, IL-12p40, IL-12p35, IFN-γ, T cell and B cell deficient mice with C. neoformans strain H99γ demonstrated a requirement for Th1-type T cell-mediated immunity, but not B cell-mediated immunity, for the induction of H99γ-mediated protective immune responses against pulmonary C. neoformans infection. CD4⁺ T cells, CD11c⁺ cells, and Gr-1⁺ cells were increased in both proportion and absolute number in protected mice. In addition, significantly increased production of Th1-type/pro-inflammatory cytokines and chemokines, and conversely, reduced Th2-type cytokine production was observed in the lungs of protected mice. Interestingly, protection was not associated with increased production of cytokines IFN-γ or TNF-α in lungs of protected mice. In conclusion, immunization with C. neoformans strain H99γ results in the development of protective anti-cryptococcal immune responses that may be measured and subsequently used in the development of immune-based therapies to combat pulmonary cryptococcosis.     

Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection

Cyclic paroxysm and high fever are hallmarks of malaria and are associated with high levels of pyrogenic cytokines, including IL-1β. In this report, we describe a signature for the expression of inflammasome-related genes and caspase-1 activation in malaria. Indeed, when we infected mice, Plasmodium infection was sufficient to promote MyD88-mediated caspase-1 activation, dependent on IFN-γ-priming and the expression of inflammasome components ASC, P2X7R, NLRP3 and/or NLRP12. Pro-IL-1β expression required a second stimulation with LPS and was also dependent on IFN-γ-priming and functional TNFR1. As a consequence of Plasmodium-induced caspase-1 activation, mice produced extremely high levels of IL-1β upon a second microbial stimulus, and became hypersensitive to septic shock. Therapeutic intervention with IL-1 receptor antagonist prevented bacterial-induced lethality in rodents. Similar to mice, we observed a significantly increased frequency of circulating CD14⁺CD16⁻Caspase-1⁺ and CD14^dimCD16⁺Caspase-1⁺ monocytes in peripheral blood mononuclear cells from febrile malaria patients. These cells readily produced large amounts of IL-1β after stimulation with LPS. Furthermore, we observed the presence of inflammasome complexes in monocytes from malaria patients containing either NLRP3 or NLRP12 pyroptosomes. We conclude that NLRP12/NLRP3-dependent activation of caspase-1 is likely to be a key event in mediating systemic production of IL-1β and hypersensitivity to secondary bacterial infection during malaria.