Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis

Chronic periodontitis is a local inflammatory disease induced by a dysbiotic microbiota and leading to destruction of the tooth-supporting structures. Microbial nucleic acids are abundantly present in the periodontium, derived through release after phagocytic uptake of microbes and/or from biofilm-associated extracellular DNA. Binding of microbial DNA to its cognate receptors, such as Toll-like receptor 9 (TLR9), can trigger inflammation. In this study, we utilized TLR9 knockout (TLR9^−/−) mice and wild-type (WT) controls in a murine model of Porphyromonas gingivalis-induced periodontitis and report the first in vivo evidence that TLR9 signaling mediates the induction of periodontal bone loss. P. gingivalis-infected WT mice exhibited significantly increased bone loss compared to that in sham-infected WT mice or P. gingivalis-infected TLR9^−/− mice, which were resistant to bone loss. Consistent with this, the expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNF), and receptor-activator of nuclear factor kappa B ligand (RANKL) were significantly elevated in the gingival tissues of the infected WT mice but not in infected TLR9^−/− mice compared to their levels in controls. Ex vivo studies using splenocytes and bone marrow-derived macrophages revealed significantly diminished cytokine production in TLR9^−/− cells relative to the cytokine production in WT cells in response to P. gingivalis, thereby implicating TLR9 in inflammatory responses to this organism. Intriguingly, compared to the cytokine production in WT cells, TLR9^−/− cells exhibited significantly decreased proinflammatory cytokine production upon challenge with lipopolysaccharide (LPS) (TLR4 agonist) or Pam3Cys (TLR2 agonist), suggesting possible cross talk between TLR9, TLR4, and TLR2. Collectively, our results provide the first proof-of-concept evidence implicating TLR9-triggered inflammation in periodontal disease pathogenesis, thereby identifying a new potential therapeutic target to control periodontal inflammation.     

p21WAF1/Cip1 limits senescence and acinar‐to‐ductal metaplasia formation during pancreatitis

Trans‐differentiation of pancreatic acinar cells into ductal‐like lesions, a process defined as acinar‐to‐ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre‐malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down‐regulation of p21^WAF1/Cip1, a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down‐regulation is implicated in controlling the early events of acinar cell trans‐differentiation and ADM formation. p21‐mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein‐induced pancreatitis, using wild‐type (WT) and p21‐deficient (p21^?/?) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up‐regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down‐regulation is associated with ADM formation. In support of this hypothesis, p21^?/? mice showed a significant increase in number and size of metaplasia. In addition, p21 over‐expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell‐autonomous manner. p21^?/? mice displayed increased expression and relocalization of β‐catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate‐keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Regulation of airway inflammation and remodeling in asthmatic mice by TLR3/TRIF signal pathway

This paper aims to investigate the effect of Toll-like receptors 3 (TLR3)/TIR-domain-containing adapter-inducing interferon-β (TRIF) signal pathway on the airway inflammation and remodeling in asthmatic mice. C57BL/6 and TLR3^−/− mice were randomly divided into three groups (10 mice per group), including Control group (mice inhaled phosphate buffer saline (PBS)), Asthma group (mice inhaled ovalbumin (OVA)) and polyriboinosinic-ribocytidylic acid (poly (I: C)) group (asthmatic mice were injected intraperitoneally with TLR3 agonist poly (I: C)). Hematoxylin-eosin (HE) staining, Wright-Giemsa staining, Enzyme-linked immunosorbent assay (ELISA), Immunohistochemistry, Hydroxyproline assay, quantitative real time polymerase chain reaction (qRT-PCR) and Western blot were used to assess for the indices of airway inflammation and remodeling. In terms of WT mice, all asthma groups with or without the addition of poly (I: C) showed exaggerated inflammation and remodeling in the airways as compared to Control group, which were more seriously in poly (I: C) group than Asthma group. Furthermore, we observed the significant inhibition of airway inflammation and remodeling in the TLR3^−/− mice in both Asthma no matter with or without addition of poly (I: C) than the WT mice. TLR3 knockout could obviously relieve the airway inflammation and remodeling in asthma through inhibiting TLR3/TRIF signaling pathway.     

Toll-Like Receptor 2 (TLR2) and TLR9 Play Opposing Roles in Host Innate Immunity against Salmonella enterica Serovar Typhimurium Infection

Toll-like receptors (TLRs) are evolutionarily conserved host proteins that are essential for effective host defense against pathogens. However, recent studies suggest that some TLRs can negatively regulate immune responses. We observed here that TLR2 and TLR9 played opposite roles in regulating innate immunity against oral infection of Salmonella enterica serovar Typhimurium in mice. While TLR9^−/− mice exhibited shortened survival, an increased cytokine storm, and more severe Salmonella hepatitis than wild-type (WT) mice, TLR2^−/− mice exhibited the opposite phenomenon. Further studies demonstrated that TLR2 deficiency and TLR9 deficiency in macrophages both disrupted NK cell cytotoxicity against S. Typhimurium-infected macrophages by downregulating NK cell degranulation and gamma interferon (IFN-γ) production through decreased macrophage expression of the RAE-1 NKG2D ligand. But more importantly, we found that S. Typhimurium-infected TLR2^−/− macrophages upregulated inducible nitric oxide synthase (iNOS) expression, resulting in a lower bacterial load than that in WT macrophages in vitro and livers in vivo as well as low proinflammatory cytokine levels. In contrast, TLR9^−/− macrophages showed decreased reactive oxygen species (ROS) expression concomitant with a high bacterial load in the macrophages and in livers of TLR9^−/− mice. TLR9^−/− macrophages were also more susceptible than WT macrophages to S. Typhimurium-induced necroptosis in vitro, likely contributing to bacterial spread and transmission in vivo. Collectively, these findings indicate that TLR2 negatively regulates anti-S. Typhimurium immunity, whereas TLR9 is vital to host defense and survival against S. Typhimurium invasion. TLR2 antagonists or TLR9 agonists may thus serve as potential anti-S. Typhimurium therapeutic agents.     

Toll-like receptor 4 mediates tolerance in macrophages stimulated with Toxoplasma gondii-derived heat shock protein 70

Peritoneal macrophages (PMs) from toll-like receptor 4 (TLR4)-deficient and wild-type (WT) mice were responsive to recombinant Toxoplasma gondii-derived heat shock protein 70 (rTgHSP70) and natural TgHSP70 (nTgHSP70) in NO release, but those from TLR2-, myeloid differentiation factor 88 (MyD88)-, and interleukin-1R-associated kinase 4 (IRAK4)-deficient mice were not. Polymyxin B did not inhibit PM activation by TgHSP70 and nTgHSP70 from WT and TLR4-deficient mice, while it inhibited PM activation by lipopolysaccharide. Pretreatment of PMs from WT but not from TLR4-deficient mice with rTgHSP70 resulted in suppression of NO release on restimulation with rTgHSP70. Similarly, pretreatment of PMs from WT but not TLR4-deficient mice with nTgHSP70 resulted in suppression of NO release on restimulation with nTgHSP70. Polymyxin B did not inhibit rTgHSP70- and nTgHSP70-induced tolerance of PMs from TLR4-deficient mice. Furthermore, PMs from WT mice increased suppressor of cytokine-signaling-1 (SOCS-1) expression after restimulation with rTgHSP70, while those from TLR4-deficient mice did not. Phosphorylation of JNK and I-kappaBalpha occurred in rTgHSP70-induced tolerance of PMs from TLR4-deficient mice, but not in that from WT mice. These data indicated that TgHSP70 signaling mechanisms were mediated by TLR2, MyD88, and IRAK4, but not by TLR4. On the other hand, signaling of TgHSP70-induced tolerance was mediated by TLR4, and the expression of SOCS-1 suppressed the TLR2 signaling pathway.     

B and CD4+ T‐cell expression of TLR2 is critical for optimal induction of a T‐cell‐dependent humoral immune response to intact Streptococcus pneumoniae

TLR2^?/? mice immunized with Streptococcus pneumoniae (Pn) elicit normal IgM, but defective CD4⁺ T‐cell‐dependent type 1 IgG isotype production, associated with a largely intact innate immune response. We studied the T‐cell‐dependent phosphorylcholine (PC)‐specific IgG3 versus the T‐cell‐independent IgM response to Pn to determine whether TLR2 signals directly via the adaptive immune system. Pn‐activated TLR2^?/? BMDC have only a modest defect in cytokine secretion, undergo normal maturation, and when transferred into naïve WT mice elicit a normal IgM and IgG3 anti‐PC response, relative to WT BMDC. Pn synergizes with BCR and TCR signaling for DNA synthesis in purified WT B and CD4⁺T cells, respectively, but is defective in cells lacking TLR2. Pn primes TLR2^?/? mice for a normal CD4⁺ T‐cell IFN‐γ recall response. Notably, TLR2^?/? B cells transferred into RAG‐2^?/? mice with WT CD4⁺T cells, or TLR2^?/? CD4⁺T cells transferred into athymic nude mice, each elicit a defective IgG3, in contrast to normal IgM, anti‐PC response relative to WT cells. These data are the first to demonstrate a major role for B‐cell and CD4⁺ T‐cell expression of TLR2 for eliciting an anti‐bacterial humoral immune response.     

TLR9 mediated regulatory B10 cell amplification following sub-total body irradiation: Implications in attenuating EAE

Autoimmunity and inflammation are controlled in part by regulatory B (Breg) cells, including the recently identified IL-10-competent B10 cell subset that represents 1%–3% of mouse spleen B cells. In this study, the influence of irradiation on Breg/B10 cell generation and IL-10 production mediated by TLR9 signaling pathways was investigated. Spleen and peritoneal cavity Breg/B10 cell frequencies were significantly expanded three weeks after sub-total body irradiation (sub-TBI, 5 Gy or 10 Gy) in adult male wild type (WT) C57BL/6(B6) mice but not in TLR9^−/− mice. TLR9 agonist ODN1826 stimulation in vitro for 5 h induced more B10 cells to express cytoplasmic IL-10 in sub-TBI WT mice than in TLR9^−/− mice. Prolonged ODN1826 stimulation (48 h) induced additional spleen CD19^hiCD5⁺CD1d^hi B cells to express IL-10. TLR9-dependent signaling molecules, MyD88, TRAF6 and IRF8 are involved in sub-TBI induced Breg/B10 cells development and expansion. Furthermore, using a mouse model for multiple sclerosis, we show here that sub-TBI induced Breg/B10 cells dramatically inhibit disease onset and severity when transferred into mice with established experimental autoimmune encephalomyelitis (EAE). Adoptively transferred sub-TBI induced Breg cells significantly suppress inflammatory T cell responses of TH17 and TH1 types in EAE mice. In conclusion, sub-TBI can drive Breg/B10 cell development and expansion, which could be used as a novel tool for suppressing undesirable immunity. The ex vivo expansion and reinfusion of autologous Breg/B10 cells may provide a novel and effective in vivo treatment for severe autoimmune diseases that are resistant to current therapies.     

Myeloid differentiation antigen 88 deficiency impairs pathogen clearance but does not alter inflammation in Borrelia burgdorferi-infected mice

The spirochete Borrelia burgdorferi causes acute inflammation in mice that resolves with the development of pathogen-specific adaptive immunity. B. burgdorferi lipoproteins activate innate immune cells via Toll-like receptor 2 (TLR2), but TLR2-deficient mice are not resistant to B. burgdorferi-induced disease, suggesting the involvement of other TLRs or non-TLR mechanisms in the induction of acute inflammation. For this study, we used mice that were deficient in the intracellular adapter molecule myeloid differentiation antigen 88 (MyD88), which is required for all TLR-induced inflammatory responses, to determine whether the interruption of this pathway would alter B. burgdorferi-induced disease. Infected MyD88(-/-) mice developed carditis and arthritis, similar to the disease in wild-type (WT) mice analyzed at its peak (days 14 and 28) and during regression (day 45). MyD88(-/-) macrophages produced tumor necrosis factor alpha only when spirochetes were opsonized, suggesting a role for B. burgdorferi-specific antibody in disease expression. MyD88(-/-) mice produced stronger pathogen-specific Th2-dependent immunoglobulin G1 (IgG1) responses than did WT mice, and their IgM titers remained significantly elevated through 90 days of infection. Despite specific antibodies, the pathogen burden was 250-fold higher in MyD88(-/-) mice than in WT mice 45 days after infection; by 90 days of infection, the pathogen burden had diminished substantially in MyD88(-/-) mice, but it was still elevated compared to that in WT mice. The elevated pathogen burden may be explained in part by the finding that MyD88(-/-) peritoneal macrophages could ingest spirochetes but degraded them more slowly than WT macrophages. Our results show that MyD88-dependent signaling pathways are not required for B. burgdorferi-induced inflammation but are necessary for the efficient control of the pathogen burden by phagocytes.     

Requirement for TLR2 in the development of albuminuria,inflammation and fibrosis in experimental diabetic nephropathy

Inflammation and fibrosis are essential elements of diabetic nephropathy (DN). We tested the hypothesis that these elements are dependent upon Toll-like receptor 2 (TLR2) signalling by examining WT and TLR2^-/- mice in an experimental model of DN. Diabetes was induced in WT and TLR2^-/- mice by i.p. injection of streptozotocin. Kidney injury was assessed at 6, 12 and 24 weeks after induction of diabetes. Gene expression of TLR2, its endogenous ligands and downstream cytokines, chemokines and fibrogenic molecules were upregulated in kidneys from WT mice with streptozotocin diabetes. TLR2^-/- mice were protected against the development of DN, exhibiting less albuminuria, inflammation, glomerular hypertrophy and hypercellularity, podocyte and tubular injury as compared to diabetic WT controls. Marked reductions in interstitial collagen deposition, myofibroblast activation (α-SMA) and expression of fibrogenic genes (TGF-β and fibronectin) were also evident in TLR2 deficient mice. Consistent with our in vivo results, high glucose directly promoted TLR2 activation in podocytes and tubular epithelial cells (TECs) in vitro, resulting in NF-κB activation, inflammation and TGF-β production. We conclude that TLR2 was required for the full development of inflammation, kidney damage and fibrosis in this model of DN. As TLR2 is known to be expressed by intrinsic kidney cells and as high concentration glucose stimulated podocytes and TECs in vitro to express TLR2 and TLR2 ligands, pro-inflammatory and pro-fibrotic cytokines in a TLR2 dependent manner in the present study, it appears likely that TLR2 signalling in intrinsic kidney cells contributes to the pathogenesis of diabetic nephropathy.     

TLR2-deficiency of cKit+ bone marrow cells is associated with augmented potency to stimulate angiogenic processes

Objective: Toll-like receptor 2 (TLR2)-deficiency is associated with the preservation of vascular function and TLR2-deficient (TLR2^-/-) mice exhibit increased neovascularization following induction of hindlimb ischemia. Hematopoietic stem cells play an important role in ischemia-induced angiogenesis and we now investigated whether the effects observed in TLR2^-/- mice may be attributed to TLR2 deficiency on bone marrow-derived stem cells. Approach and Results: cKit-positive (cKit⁺) bone marrow cells (BMC) were isolated from wild type (WT) and TLR2^-/- mice employing MACS-bead technology. Co-incubation of TLR2^-/-cKit⁺ BMC with mature endothelial cells (ECs) resulted in increased tube formation of ECs on matrigel, augmented sprouting in a 3D-collagen matrix and increased migratory capacity compared to co-incubation with WT cKit⁺ BMC. In an in vivo matrigel plug assay, TLR2^-/-cKit⁺ BMC exhibited enhanced formation of capillary-like networks. In a murine model of hindlimb ischemia, administration of TLR2^-/- cKit⁺ BMC to WT mice augmented capillary density and reperfusion of ischemic M. gastrocnemius muscle tissue to the level of TLR2^-/- mice. Western Blot analysis revealed comparable expression of CXCR4 on TLR2^-/-cKit⁺ BMC but increased activation of the PI3K downstream signaling molecule protein kinase B (PKB/AKT) compared to WT cKit⁺ cells. Conclusions: The absence of TLR2 on cKit⁺ BMC is associated with augmented potency to support angiogenic processes in vitro and in vivo. Functional inhibition of TLR2 may therefore provide a novel tool to enhance stem cell function for the treatment of vascular diseases.     

MyD88 signaling contributes to early pulmonary responses to Aspergillus fumigatus

Toll-like receptors and the β-glucan receptor, dectin-1, mediate macrophage inflammatory responses to Aspergillus fumigatus through MyD88-dependent and -independent signaling mechanisms; however, pulmonary inflammatory responses in MyD88-deficient mice challenged with A. fumigatus are poorly defined. The role of MyD88 signaling in early pulmonary inflammation and fungal clearance was evaluated in C57BL/6J wild-type (WT) and MyD88-deficient (MyD88^−/−) mice. Early (<48 h) after infection, MyD88^−/− mice had higher fungal burdens than those of WT mice, although fungal burdens rapidly declined (>72 h) in both. MyD88^−/− mice had less consolidated inflammation, with fewer NK cells, in lung tissue early (24 h) after infection than did WT mice. At the latter time point, MyD88^−/− mouse lungs were characterized by a large amount of necrotic cellular debris and fibrin, while WT lungs had organized inflammation. Although there were equivalent numbers of macrophages in WT and MyD88^−/− mouse lung tissues, MyD88^−/− cells demonstrated delayed uptake of green fluorescent protein-expressing A. fumigatus (GFP-Af293); histologically, MyD88^−/− mouse lungs had more hyphal invasion of terminal airways and vessels, the appearance of bronchiolar epithelial cell necrosis, and necrotizing vasculitis. MyD88^−/− lung homogenates contained comparatively decreased amounts of interleukin-1β (IL-1β), IL-6, KC, and gamma interferon and paradoxically increased amounts of tumor necrosis factor alpha and macrophage inflammatory protein 1α. These data indicate that the MyD88-dependent pathway mediates acute pulmonary fungal clearance, inflammation, and tissue injury very early after infection. Resolution of abnormalities within a 3-day window demonstrates the importance of redundant signaling pathways in mediating pulmonary inflammatory responses to fungi.     

A disintegrin and metalloproteinase domain-15 deficiency leads to exaggerated cigarette smoke-induced chronic obstructive pulmonary disease (COPD)-like disease in mice

A disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by cells implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its contributions to COPD are unknown. To address this gap, ADAM15 levels were measured in samples from cigarette smoke (CS)-versus air-exposed wild-type (WT) mice. CS-induced COPD-like disease was compared in CS-exposed WT, Adam15^−/−, and Adam15 bone marrow chimeric mice. CS exposure increased Adam15 expression in lung macrophages and CD8⁺ T cells and to a lesser extent in airway epithelial cells in WT mice. CS-exposed Adam15^−/− mice had greater emphysema, small airway fibrosis, and lung inflammation (macrophages and CD8⁺ T cells) than WT mice. Adam15 bone marrow chimera studies revealed that Adam15 deficiency in leukocytes led to exaggerated pulmonary inflammation and COPD-like disease in mice. Adam15 deficiency in CD8⁺ T cells was required for the exaggerated pulmonary inflammation and COPD-like disease in CS-exposed Adam15^−/− mice (as assessed by genetically deleting CD8⁺ T cells in Adam15^−/− mice). Adam15 deficiency increased pulmonary inflammation by rendering CD8⁺ T cells and macrophages resistant to CS-induced activation of the mitochondrial apoptosis pathway by preserving mTOR signaling and intracellular Mcl-1 levels in these cells. These results strongly link ADAM15 deficiency to the pathogenesis of COPD.     

CXCR3 deficiency enhances tumor progression by promoting macrophage M2 polarization in a murine breast cancer model

Tumor associated macrophages play a vital role in determining the outcome of breast cancer. We investigated the contribution of the chemokine receptor CXCR3 to antitumor immune responses using a cxcr3 deficient mouse orthotopically injected with a PyMT breast cancer cell line. We observed that cxcr3 deficient mice displayed increased IL-4 production and M2 polarization in the tumors and spleens compared to WT mice injected with PyMT cells. This was accompanied by larger tumor development in cxcr3^−/− than in WT mice. Further, tumor-promoting myeloid derived immune cell populations accumulated in higher proportions in the spleens of cxcr3 deficient mice. Interestingly, cxcr3^−/− macrophages displayed a deficiency in up-regulating inducible nitric oxide synthase after stimulation by either IFN-γ or PyMT supernatants. Stimulation of bone marrow derived macrophages by PyMT supernatants also resulted in greater induction of arginase-1 in cxcr3^−/− than WT mice. Further, cxcr3^−/− T cells activated with CD3/CD28 in vitro produced greater amounts of IL-4 and IL-10 than T cells from WT mice. Our data suggests that a greater predisposition of cxcr3 deficient macrophages towards M2 polarization contributes to an enhanced tumor promoting environment in cxcr3 deficient mice. Although CXCR3 is known to be expressed on some macrophages, this is the first report that demonstrates a role for CXCR3 in macrophage polarization and subsequent breast tumor outcomes. Targeting CXCR3 could be a potential therapeutic approach in the management of breast cancer tumors.     

Role of the Toll-like receptor pathway in the recognition of orthopedic implant wear-debris particles

The inflammatory response to prosthetic implant-derived wear particles is the primary cause of bone loss and aseptic loosening of implants, but the mechanisms by which macrophages recognize and respond to particles remain unknown. Studies of innate immunity demonstrate that Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPS). All TLRs signal through myeloid differentiation factor 88 (MyD88), except TLR3 which signals through TIR domain containing adapter inducing interferon-beta (TRIF), and TLR4 which signals through both MyD88 and TRIF. We hypothesized that wear-debris particles may act as PAMPs/DAMPs and activate macrophages via TLRs. To test this hypothesis, we first demonstrated that inhibition of MyD88 decreases polymethylmethacrylate (PMMA) particle-induced production of TNF-α in RAW 264.7 macrophages. Next we compared particle-induced production of TNF-α among MyD88 knockout (MyD88(-/-)), TRIF knockout (TRIF(-/-)), and wild type (WT) murine macrophages. Relative to WT, disruption of MyD88 signaling diminished, and disruption of TRIF amplified the particle-induced production of TNF-α. Gene expression data indicated that this latter increase in TNF-α was due to a compensatory increase in expression of MyD88 associated components of the TLR pathway. Finally, using an in?vivo model, MyD88(-/-) mice developed less particle-induced osteolysis than WT mice. These results indicate that the response to PMMA particles is partly dependent on MyD88, presumably as part of TLR signaling; MyD88 may represent a therapeutic target for prevention of wear debris-induced periprosthetic osteolysis.     

Toll-like receptor 2 regulates organic dust-induced airway inflammation

Organic dust exposure in agricultural environments results in significant airway inflammatory diseases. Gram-positive cell wall components are present in high concentrations in animal farming dusts, but their role in mediating dust-induced airway inflammation is not clear. This study investigated the role of Toll-like receptor (TLR) 2, a pattern recognition receptor for gram-positive cell wall products, in regulating swine facility organic dust extract (DE)-induced airway inflammation in mice. Isolated lung macrophages from TLR2 knockout mice demonstrated reduced TNF-α, IL-6, keratinocyte chemoattractant/CXCL1, but not macrophage inflammatory protein-2/CXCL2 expression, after DE stimulation ex vivo. Next, using an established mouse model of intranasal inhalation challenge, we analyzed bronchoalveolar lavage fluid and lung tissue in TLR2-deficient and wild-type (WT) mice after single and repetitive DE challenge. Neutrophil influx and select cytokines/chemokines were significantly lower in TLR2-deficient mice at 5 and 24 hours after single DE challenge. After daily exposure to DE for 2 weeks, there were significant reductions in total cellularity, neutrophil influx, and TNF-α, IL-6, CXCL1, but not CXCL2 expression, in TLR2-deficient mice as compared with WT animals. Lung pathology revealed that bronchiolar inflammation, but not alveolar inflammation, was reduced in TLR2-deficient mice after repetitive exposure. Airway hyperresponsiveness to methacholine after dust exposure was similar in both groups. Finally, airway inflammatory responses in WT mice after challenge with a TLR2 agonist, peptidoglycan, resembled DE-induced responses. Collectively, these results demonstrate that the TLR2 pathway is important in regulating swine facility organic dust-induced airway inflammation, which suggests the importance of TLR2 agonists in mediating large animal farming-induced airway inflammatory responses.     

TLR2‐independent induction and regulation of chronic intestinal inflammation

Interactions between the intestinal microflora and host innate immune receptors play a critical role in intestinal homeostasis. Several studies have shown that TLR2 can modulate inflammatory responses in the gut. TLR2 signals enhance tight junction formation and fortify the epithelial barrier, and may play a crucial role in driving acute inflammatory responses towards intestinal bacterial pathogens. In addition, TLR2 agonists can have direct effects on both Th1 cells and Treg. To define the role of TLR2 in the induction and regulation of chronic intestinal inflammation we examined the effects of TLR2 deletion on several complementary models of inflammatory bowel disease. Our results show that TLR2 signals are not required for the induction of chronic intestinal inflammation by either innate or adaptive immune responses. We further show that TLR2^?/? mice harbor normal numbers of Foxp3⁺ Treg that are able to suppress intestinal inflammation as effectively as their WT counterparts. We also did not find any intrinsic role for TLR2 for pathogenic effector T‐cell responses in the gut. Thus, in contrast to their role in acute intestinal inflammation and repair, TLR2 signals may have a limited impact on the induction and regulation of chronic intestinal inflammation.     

Neutrophils negatively regulate induction of mucosal IgA responses after sublingual immunization

Induction of mucosal immunoglobulin-A (IgA) capable of providing a first line of defense against bacterial and viral pathogens remains a major goal of needle-free vaccines given via mucosal routes. Innate immune cells are known to play a central role in induction of IgA responses by mucosal vaccines, but the relative contribution of myeloid cell subsets to these responses has not firmly been established. Using an in vivo model of sublingual vaccination with Bacillus anthracis edema toxin (EdTx) as adjuvant, we examined the role of myeloid cell subsets for mucosal secretory IgA responses. Sublingual immunization of wild-type mice resulted in a transient increase of neutrophils in sublingual tissues and cervical lymph nodes. These mice later developed Ag-specific serum IgG responses, but not serum or mucosal IgA. Interestingly, EdTx failed to increase neutrophils in sublingual tissues and cervical lymph nodes of IKKβ^ΔMye mice, and these mice developed IgA responses. Partial depletion of neutrophils before immunization of wild-type mice allowed the development of both mucosal and serum IgA responses. Finally, co-culture of B cells with neutrophils from either wild-type or IKKβ^ΔMye mice suppressed secretion of IgA, but not IgM or IgG. These results identify a new role for neutrophils as negative regulators of IgA responses.