The Polysaccharide Capsule of Streptococcus pneumonia Partially Impedes MyD88-Mediated Immunity during Pneumonia in Mice

Toll-like receptors (TLR) and the downstream adaptor protein MyD88 are considered crucial for protective immunity during bacterial infections. Streptococcus (S.) pneumoniae is a human respiratory pathogen and a large majority of clinical pneumococcal isolates expresses an external polysaccharide capsule. We here sought to determine the role of pneumococcal capsule in MyD88-mediated antibacterial defense during S. pneumonia pneumonia. Wild type (WT) and Myd88^-/- mice were inoculated intranasally with serotype 2 S. pneumoniae D39 or with an isogenic capsule locus deletion mutant (D39∆cps), and analysed for bacterial outgrowth and inflammatory responses in the lung. As compared to WT mice, Myd88^-/- mice infected with D39 demonstrated a modestly impaired bacterial clearance accompanied by decreased inflammatory responses in the lung. Strikingly, while WT mice rapidly cleared D39∆cps, Myd88^-/- mice showed 10⁵-fold higher bacterial burdens in their lungs and dissemination to blood 24 hours after infection. These data suggest that the pneumococcal capsule impairs recognition of TLR ligands expressed by S. pneumoniae and thereby partially impedes MyD88-mediated antibacterial defense.     

Bacterial Infections in Myd88-Deficient Mice

Three breeding colonies of Myd88^−/− mice had a history of significant morbidity and mortality. Although strain-specific poor reproductive performance might explain neonatal death and dystocia, mice were found dead or required euthanasia because of moribundity, distended abdomen, head tilt, and seizures. Histopathology results included bacteremia, placentitis, metritis, peritonitis with abscess formation, and suppurative meningoencephalitis. Intralesional gram-negative coccobacilli were present, often in extremely high number. Cultures of samples of the cardiac blood of a mouse and from water-bottle sipper tubes provided to some affected mice grew Pseudomonas aeruginosa. In addition, affected tissues from 2 mice and feces from a third tested PCR-positive for P. aeruginosa. Although the mice had received autoclaved reverse-osmosis–purified drinking water, we suspect that the mice were inoculated with P. aeruginosa through contaminated sipper tubes. Because of the deficiency in most of the Toll-like receptor signaling pathways, these Myd88^−/− mice were unlikely to have developed competitive innate and adaptive immune responses, resulting in bacterial infections. These clinical cases underscore the importance of understanding how genotype, phenotype and environment affect animal health. Sound husbandry and experimental practices are needed to prevent the exposure of immunodeficient mice to pathogens.Abbreviations: TLR, Toll-like receptorRodent colonies typically are managed on a ‘herd-health’ basis, with prevention, treatment, and control measures at the colony level rather than the individual level. Although sporadic cases of sick and dead mice are not unusual in large breeding colonies, unexpectedly high morbidity and mortality rates typically warrant clinical investigation and analysis of the disease triangle: the conceptual model of the interactions among the host, environment, and pathogen. Of these 3 factors, the host usually is well-characterized pertaining to its genotype, phenotype, and experimental manipulations. Similarly, the environmental conditions in a laboratory animal setting typically are controlled. However, infections with adventitious pathogens can occur and cause disease outbreak in susceptible animals. For example, poorly sanitized cages can act as fomites for pathogens that may cause clinical infections in susceptible mice.Immunodeficient mice are prone to exhibit clinical signs when infected with a pathogen. One such strain is Myd88^−/−, which lack the myeloid differentiation factor 88 (MyD88) protein, a cytoplasmic adaptor molecule essential for the signaling of IL1 and Toll-like receptor (TLR) family.^14,27 MyD88 plays a central role in innate and adaptive immune response, because it is essential for cellular responses to IL1, IL18, and many bacterial cell wall components including lipopolysaccharide, peptidoglycan, and lipopeptide.^1,10,27 Because of their phenotype, Myd88^−/− mice are commonly used for infectious disease and immunology studies.Here, we present a case series of Myd88^−/− mice that had a history of morbidity and mortality attributed to bacterial infections caused by gram-negative coccobacilli; the organism was identified to be Pseudomonas aeruginosa in one case. We underscore the importance of understanding host responses to pathogens and the provision of sound husbandry procedures to prevent infections in immunodeficient mice, such as the Myd88^−/− strain.     

Ischemia-Reperfusion Lung Injury Is Attenuated in MyD88-Deficient Mice

Ischemia-reperfusion lung injury is a common cause of acute morbidity and mortality in lung transplant recipients and has been associated with subsequent development of bronchiolitis obliterans syndrome. Recognition of endogenous ligands released during cellular injury (damage-associated molecular patterns; DAMPs) by Toll-like receptors (TLRs), especially TLR4, has increasingly been recognized as a mechanism for inflammation resulting from tissue damage. TLR4 is implicated in the pathogenesis of ischemia-reperfusion injury of multiple organs including heart, liver, kidney and lung. Additionally, activation of TLRs other than TLR4 by DAMPs has been identified in tissues other than the lung. Because all known TLRs, with the exception of TLR3, signal via the MyD88 adapter protein, we hypothesized that lung ischemia-reperfusion injury was mediated by MyD88-dependent signaling. To test this hypothesis, we subjected C57BL/6 wildtype, Myd88 ^-/-, and Tlr4 ^-/- mice to 1 hr of left lung warm ischemia followed by 4 hr of reperfusion. We found that Myd88 ^-/- mice had significantly less MCP-1/CCL2 in the left lung following ischemia-reperfusion as compared with wildtype mice. This difference was associated with dramatically reduced lung permeability. Interestingly, Tlr4 ^-/- mice had only partial protection from ischemia-reperfusion as compared to Myd88 ^-/- mice, implicating other MyD88-dependent pathways in lung injury following ischemia-reperfusion. We also found that left lung ischemia-reperfusion caused remote inflammation in the right lung. Finally, using chimeric mice with MyD88 expression restricted to either myeloid or non-myeloid cells, we found that MyD88-dependent signaling in myeloid cells was necessary for ischemia-reperfusion induced lung permeability. We conclude that MyD88-dependent signaling through multiple receptors is important in the pathogenesis of acute lung inflammation and injury following ischemia and reperfusion.     

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.     

MyD88 Is a Critical Regulator of Hematopoietic Cell-Mediated Neuroprotection Seen after Stroke

Neuroinflammation is critical in the neural cell death seen in stroke. It has been shown that CNS and peripheral responses drive this neuroinflammatory response in the brain. The Toll-like receptors (TLRs) are important regulators of inflammation in response to both exogenous and endogenous stressors. Taking advantage of a downstream adapter molecule that controls the majority of TLR signalling, this study investigated the role of the TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the control of CNS and peripheral inflammation. Reversible middle-cerebral artery occlusion was used as the model of stroke in vivo; in vitro primary cultured neurons and glia were subject to four hours of oxygen and glucose deprivation (OGD). Both in vitro and in vivo Myd88^−/− animals or cells were compared with wild type (WT). We found that after stroke Myd88^−/− animals have a larger infarct volume compared to WT animals. Interestingly, in vitro there was no difference between the survival of Myd88^−/− and WT cells following OGD, suggesting that peripheral responses were influencing stroke outcome. We therefore generated bone marrow chimeras and found that Myd88^−/− animals have a smaller stroke infarct than their radiation naive counterparts if their hematopoietic cells are WT. Furthermore, WT animals have a larger stroke than their radiation naive counterparts if the hematopoietic cells are Myd88^−/−. We have demonstrated that MyD88-dependent signalling in the hematopoietic cell lineage reduces infarct size following stroke and that infiltrating cells to the site of neuroinflammation are neuroprotective following stroke.     

Interleukin-1 Receptor–Associated Kinase M–Deficient Mice Demonstrate an Improved Host Defense during Gram-negative Pneumonia

Pneumonia is a common cause of morbidity and mortality and the most frequent source of sepsis. Bacteria that try to invade normally sterile body sites are recognized by innate immune cells through pattern recognition receptors, among which toll-like receptors (TLRs) feature prominently. Interleukin-1 receptor (IL-1R)–associated kinase (IRAK)-M is a proximal inhibitor of TLR signaling expressed by epithelial cells and macrophages in the lung. To determine the role of IRAK-M in host defense against bacterial pneumonia, IRAK-M-deficient (IRAK-M^−/−) and normal wild-type (WT) mice were infected intranasally with Klebsiella pneumoniae. IRAK-M mRNA was upregulated in lungs of WT mice with Klebsiella pneumonia, and the absence of IRAK-M resulted in a strongly improved host defense as reflected by reduced bacterial growth in the lungs, diminished dissemination to distant body sites, less peripheral tissue injury and better survival rates. Although IRAK-M^−/− alveolar macrophages displayed enhanced responsiveness toward intact K. pneumoniae and Klebsiella lipopolysaccharide (LPS) in vitro, IRAK-M^−/− mice did not show increased cytokine or chemokine levels in their lungs after infection in vivo. The extent of lung inflammation was increased in IRAK-M^−/− mice shortly after K. pneumoniae infection, as determined by semiquantitative scoring of specific components of the inflammatory response in lung tissue slides. These data indicate that IRAK-M impairs host defense during pneumonia caused by a common gram-negative respiratory pathogen.     

Receptor for Advanced Glycation End Products (RAGE) Serves a Protective Role during Klebsiella pneumoniae - Induced Pneumonia

Klebsiella species is the second most commonly isolated gram-negative organism in sepsis and a frequent causative pathogen in pneumonia. The receptor for advanced glycation end products (RAGE) is expressed on different cell types and plays a key role in diverse inflammatory responses. We here aimed to investigate the role of RAGE in the host response to Klebsiella (K.) pneumoniae pneumonia and intransally inoculated rage gene deficient (RAGE^-/-) and normal wild-type (Wt) mice with K. pneumoniae. Klebsiella pneumonia resulted in an increased pulmonary expression of RAGE. Furthermore, the high-affinity RAGE ligand high mobility group box-1 was upregulated during K. pneumoniae pneumonia. RAGE deficiency impaired host defense as reflected by a worsened survival, increased bacterial outgrowth and dissemination in RAGE^-/- mice. RAGE^-/- neutrophils showed a diminished phagocytosing capacity of live K. pneumoniae in vitro. Relative to Wt mice, RAGE^-/- mice demonstrated similar lung inflammation, and slightly elevated—if any—cytokine and chemokine levels and unchanged hepatocellular injury. In addition, RAGE^-/- mice displayed an unaltered response to intranasally instilled Klebsiella lipopolysaccharide (LPS) with respect to pulmonary cell recruitment and local release of cytokines and chemokines. These data suggest that (endogenous) RAGE protects against K. pneumoniae pneumonia. Also, they demonstrate that RAGE contributes to an effective antibacterial defense during K. pneumoniae pneumonia, at least partly via its participation in the phagocytic properties of professional granulocytes. Additionally, our results indicate that RAGE is not essential for the induction of a local and systemic inflammatory response to either intact Klebsiella or Klebsiella LPS.     

Overexpression of the Endothelial Protein C Receptor Is Detrimental during Pneumonia-Derived Gram-negative Sepsis (Melioidosis)

Background

The endothelial protein C receptor (EPCR) enhances anticoagulation by accelerating activation of protein C to activated protein C (APC) and mediates anti-inflammatory effects by facilitating APC-mediated signaling via protease activated receptor-1. We studied the role of EPCR in the host response during pneumonia-derived sepsis instigated by Burkholderia (B.) pseudomallei, the causative agent of melioidosis, a common form of community-acquired Gram-negative (pneumo)sepsis in South-East Asia.

Methodology/Principal Findings

Soluble EPCR was measured in plasma of patients with septic culture-proven melioidosis and healthy controls. Experimental melioidosis was induced by intranasal inoculation of B. pseudomallei in wild-type (WT) mice and mice with either EPCR-overexpression (Tie2-EPCR) or EPCR-deficiency (EPCR^−/−). Mice were sacrificed after 24, 48 or 72 hours. Organs and plasma were harvested to measure colony forming units, cellular influxes, cytokine levels and coagulation parameters. Plasma EPCR-levels were higher in melioidosis patients than in healthy controls and associated with an increased mortality. Tie2-EPCR mice demonstrated enhanced bacterial growth and dissemination to distant organs during experimental melioidosis, accompanied by increased lung damage, neutrophil influx and cytokine production, and attenuated coagulation activation. EPCR^−/− mice had an unremarkable response to B. pseudomallei infection as compared to WT mice, except for a difference in coagulation activation in plasma.

Conclusion/Significance

Increased EPCR-levels correlate with accelerated mortality in patients with melioidosis. In mice, transgenic overexpression of EPCR aggravates outcome during Gram-negative pneumonia-derived sepsis caused by B. pseudomallei, while endogenous EPCR does not impact on the host response. These results add to a better understanding of the regulation of coagulation during severe (pneumo)sepsis.     

MyD88-deficient bone marrow cells accelerate onset and reduce survival in a mouse model of amyotrophic lateral sclerosis

Increasing evidence suggests that neurotoxicity of secreted superoxide dismutase 1 (SOD1) mutants is associated with amyotrophic lateral sclerosis (ALS). We show here that mutant SOD1 protein activates microglia via a myeloid differentiation factor 88 (MyD88)–dependent pathway. This inflammatory response is also associated with a marked recruitment of bone marrow–derived microglia (BMDM) in the central nervous system. We then generated chimeric SOD1^G37R and SOD1^G93A mice by transplantation of bone marrow (BM) cells from MyD88-deficient or green fluorescent protein (GFP)–expressing mice. SOD1^G37R mice receiving MyD88^−/− BM cells exhibit a significantly earlier disease onset and shorter lifespan compared with mice transplanted with control GFP cells. This compelling beneficial effect of MyD88-competent BMDM is a previously unrecognized natural innate immune mechanism of neuroprotection in a mouse model of late-onset motor neuron disease.     

NFATc1 Mediates Toll-Like Receptor-Independent Innate Immune Responses during Trypanosoma cruzi Infection

Host defense against the intracellular protozoan parasite Trypanosoma cruzi depends on Toll-like receptor (TLR)-dependent innate immune responses. Recent studies also suggest the presence of TLR-independent responses to several microorganisms, such as viruses, bacteria, and fungi. However, the TLR-independent responses to protozoa remain unclear. Here, we demonstrate a novel TLR-independent innate response pathway to T. cruzi. Myd88 ^−/− Trif ^−/− mice lacking TLR signaling showed normal T. cruzi-induced Th1 responses and maturation of dendritic cells (DCs), despite high sensitivity to the infection. IFN-γ was normally induced in T. cruzi-infected Myd88 ^−/− Trif ^−/− innate immune cells, and further was responsible for the TLR-independent Th1 responses and DC maturation after T. cruzi infection. T. cruzi infection induced elevation of the intracellular Ca²⁺ level. Furthermore, T. cruzi-induced IFN-γ expression was blocked by inhibition of Ca²⁺ signaling. NFATc1, which plays a pivotal role in Ca²⁺ signaling in lymphocytes, was activated in T. cruzi-infected Myd88^−/−Trif^−/− innate immune cells. T. cruzi-infected Nfatc1 ^−/− fetal liver DCs were impaired in IFN-γ production and DC maturation. These results demonstrate that NFATc1 mediates TLR-independent innate immune responses in T. cruzi infection.     

Modulation of the Intestinal Microbiota Alters Colitis-Associated Colorectal Cancer Susceptibility

It is well established that the intestinal microbiota plays a key role in the pathogenesis of Crohn''s disease (CD) and ulcerative colitis (UC) collectively referred to as inflammatory bowel disease (IBD). Epidemiological studies have provided strong evidence that IBD patients bear increased risk for the development of colorectal cancer (CRC). However, the impact of the microbiota on the development of colitis-associated cancer (CAC) remains largely unknown. In this study, we established a new model of CAC using azoxymethane (AOM)-exposed, conventionalized-Il10^−/− mice and have explored the contribution of the host intestinal microbiota and MyD88 signaling to the development of CAC. We show that 8/13 (62%) of AOM-Il10^−/− mice developed colon tumors compared to only 3/15 (20%) of AOM- wild-type (WT) mice. Conventionalized AOM-Il10^−/− mice developed spontaneous colitis and colorectal carcinomas while AOM-WT mice were colitis-free and developed only rare adenomas. Importantly, tumor multiplicity directly correlated with the presence of colitis. Il10^−/− mice mono-associated with the mildly colitogenic bacterium Bacteroides vulgatus displayed significantly reduced colitis and colorectal tumor multiplicity compared to Il10^−/− mice. Germ-free AOM-treated Il10^−/− mice showed normal colon histology and were devoid of tumors. Il10^−/−; Myd88^−/− mice treated with AOM displayed reduced expression of Il12p40 and Tnfα mRNA and showed no signs of tumor development. We present the first direct demonstration that manipulation of the intestinal microbiota alters the development of CAC. The TLR/MyD88 pathway is essential for microbiota-induced development of CAC. Unlike findings obtained using the AOM/DSS model, we demonstrate that the severity of chronic colitis directly correlates to colorectal tumor development and that bacterial-induced inflammation drives progression from adenoma to invasive carcinoma.     

TLR–Dependent Control of Francisella tularensis Infection and Host Inflammatory Responses

Background

Francisella tularensis is the causative agent of tularemia and is classified as a Category A select agent. Recent studies have implicated TLR2 as a critical element in the host protective response to F. tularensis infection, but questions remain about whether TLR2 signaling dominates the response in all circumstances and with all species of Francisella and whether F. tularensis PAMPs are predominantly recognized by TLR2/TLR1 or TLR2/TLR6. To address these questions, we have explored the role of Toll-like receptors (TLRs) in the host response to infections with F. tularensis Live Vaccine Strain (LVS) and F. tularensis subspecies (subsp.) novicida in vivo.

Methodology/Principal Findings

C57BL/6 (B6) control mice and TLR– or MyD88-deficient mice were infected intranasally (i.n.) or intradermally (i.d.) with F. tularensis LVS or with F. tularensis subsp. novicida. B6 mice survived >21 days following infection with LVS by both routes and survival of TLR1^−/−, TLR4^−/−, and TLR6^−/− mice infected i.n. with LVS was equivalent to controls. Survival of TLR2^−/− and MyD88^−/− mice, however, was significantly reduced compared to B6 mice, regardless of the route of infection or the subspecies of F. tularensis. TLR2^−/− and MyD88^−/− mice also showed increased bacterial burdens in lungs, liver, and spleen compared to controls following i.n. infection. Primary macrophages from MyD88^−/− and TLR2^−/− mice were significantly impaired in the ability to secrete TNF and other pro-inflammatory cytokines upon ex vivo infection with LVS. TNF expression was also impaired in vivo as demonstrated by analysis of bronchoalveolar lavage fluid and by in situ immunofluorescent staining.

Conclusions/Significance

We conclude from these studies that TLR2 and MyD88, but not TLR4, play critical roles in the innate immune response to F. tularensis infection regardless of the route of infection or the subspecies. Moreover, signaling through TLR2 does not depend exclusively on TLR1 or TLR6 during F. tularensis LVS infection.     

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.     

TLR-2/TLR-4 TREM-1 Signaling Pathway Is Dispensable in Inflammatory Myeloid Cells during Sterile Kidney Injury

Inflammatory macrophages are abundant in kidney disease, stimulating repair, or driving chronic inflammation and fibrosis. Damage associated molecules (DAMPs), released from injured cells engage pattern recognition receptors (PRRs) on macrophages, contributing to activation. Understanding mechanisms of macrophage activation during kidney injury may lead to strategies to alleviate chronic disease. We identified Triggering-Receptor-in-Myeloid-cells (TREM)-1, a regulator of TLR signaling, as highly upregulated in kidney inflammatory macrophages and tested the roles of these receptors in macrophage activation and kidney disease. Kidney DAMPs activated macrophages in vitro, independently of TREM-1, but partially dependent on TLR-2/−4, MyD88. In two models of progressive interstitial kidney disease, TREM-1 blockade had no impact on disease or macrophage activation in vivo, but TLR-2/−4, or MyD88 deficiency was anti-inflammatory and anti-fibrotic. When MyD88 was mutated only in the myeloid lineage, however, there was no bearing on macrophage activation or disease progression. Instead, TLR-2/−4 or MyD88 deficiency reduced activation of mesenchyme lineage cells resulting in reduced inflammation and fibrosis, indicating that these pathways play dominant roles in activation of myofibroblasts but not macrophages. To conclude, TREM-1, TLR2/4 and MyD88 signaling pathways are redundant in myeloid cell activation in kidney injury, but the latter appear to regulate activation of mesenchymal cells.     

MyD88 Is Required for Protection from Lethal Infection with a Mouse-Adapted SARS-CoV

A novel human coronavirus, SARS-CoV, emerged suddenly in 2003, causing approximately 8000 human cases and more than 700 deaths worldwide. Since most animal models fail to faithfully recapitulate the clinical course of SARS-CoV in humans, the virus and host factors that mediate disease pathogenesis remain unclear. Recently, our laboratory and others developed a recombinant mouse-adapted SARS-CoV (rMA15) that was lethal in BALB/c mice. In contrast, intranasal infection of young 10-week-old C57BL/6 mice with rMA15 results in a nonlethal infection characterized by high titer replication within the lungs, lung inflammation, destruction of lung tissue, and loss of body weight, thus providing a useful model to identify host mediators of protection. Here, we report that mice deficient in MyD88 (MyD88^−/−), an adapter protein that mediates Toll-like receptor (TLR), IL-1R, and IL-18R signaling, are far more susceptible to rMA15 infection. The genetic absence of MyD88 resulted in enhanced pulmonary pathology and greater than 90% mortality by day 6 post-infection. MyD88^−/− mice had significantly higher viral loads in lung tissue throughout the course of infection. Despite increased viral loads, the expression of multiple proinflammatory cytokines and chemokines within lung tissue and recruitment of inflammatory monocytes/macrophages to the lung was severely impaired in MyD88^−/− mice compared to wild-type mice. Furthermore, mice deficient in chemokine receptors that contribute to monocyte recruitment to the lung were more susceptible to rMA15-induced disease and exhibited severe lung pathology similar to that seen in MyD88^−/−mice. These data suggest that MyD88-mediated innate immune signaling and inflammatory cell recruitment to the lung are required for protection from lethal rMA15 infection.     

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent,TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb ^−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm^avir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb ^−/− mice are efficiently infected by S.Tm^avir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb ^−/− Myd88 ^−/− mice indicated that the S.Tm^avir-inflicted disease in Cybb ^−/− mice hinges on CD11c⁺CX₃CR1⁺ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm^avir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.     

Probiotic Bifidobacterium breve Induces IL-10-Producing Tr1 Cells in the Colon

Specific intestinal microbiota has been shown to induce Foxp3⁺ regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103⁺ dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103⁺ DCs from Il10 ^−/−, Tlr2 ^−/−, and Myd88 ^−/− mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103⁺ DCs failed to induce IL-10 production from co-cultured Il27ra ^−/− T cells. B. breve treatment of Tlr2 ^−/− mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103⁺ DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4⁺ T cells from wild-type mice, but not Il10 ^−/− mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.     

Non-Essential Role for TLR2 and Its Signaling Adaptor Mal/TIRAP in Preserving Normal Lung Architecture in Mice

Myeloid differentiation factor 88 (MyD88) and MyD88-adaptor like (Mal)/Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) play a critical role in transducing signals downstream of the Toll-like receptor (TLR) family. While genetic ablation of the TLR4/MyD88 signaling axis in mice leads to pulmonary cell death and oxidative stress culminating in emphysema, the involvement of Mal, as well as TLR2 which like TLR4 also signals via MyD88 and Mal, in the pathogenesis of emphysema has not been studied. By employing an in vivo genetic approach, we reveal here that unlike the spontaneous pulmonary emphysema which developed in Tlr4^−/− mice by 6 months of age, the lungs of Tlr2^−/− mice showed no physiological or morphological signs of emphysema. A more detailed comparative analysis of the lungs from these mice confirmed that elevated oxidative protein carbonylation levels and increased numbers of alveolar cell apoptosis were only detected in Tlr4^−/− mice, along with up-regulation of NADPH oxidase 3 (Nox3) mRNA expression. With respect to Mal, the architecture of the lungs of Mal^−/− mice was normal. However, despite normal oxidative protein carbonylation levels in the lungs of emphysema-free Mal^−/− mice, these mice displayed increased levels of apoptosis comparable to those observed in emphysematous Tlr4^−/− mice. In conclusion, our data provide in vivo evidence for the non-essential role for TLR2, unlike the related TLR4, in maintaining the normal architecture of the lung. In addition, we reveal that Mal differentially facilitates the anti-apoptotic, but not oxidant suppressive, activities of TLR4 in the lung, both of which appear to be essential for TLR4 to prevent the onset of emphysema.