The Formylpeptide Receptor 2 (Fpr2) and Its Endogenous Ligand Cathelin-related Antimicrobial Peptide (CRAMP) Promote Dendritic Cell Maturation

Mouse formylpeptide receptor 2 (Fpr2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2, which interacts with pathogen and host-derived chemotactic agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-deficient (Fpr2^−/−) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the potential changes in the differentiation and maturation of DCs caused by Fpr2 deficiency. Bone marrow monocytes from Fpr2^−/− mouse mice incubated with GM-CSF and IL-4 in vitro showed normal expression of markers of immature DCs. However, upon stimulation with the TLR4 agonist LPS, Fpr2^−/− mouse DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2^−/− DCs also failed to induce allogeneic T-cell proliferation in vitro, and their recruitment into the T-cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, because neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We additionally observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation. The importance of CRAMP for DC maturation was further demonstrated by the observations that DCs from CRAMP^−/− mice expressed lower levels of costimulatory molecules and MHC II and exhibited poor chemotaxis in response to CCL21 after LPS stimulation. Our observations indicate a nonredundant role for Fpr2 and its agonist CRAMP in DC maturation in immune responses.     

A role for TLR4 in Clostridium difficile infection and the recognition of surface layer proteins

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4^−/− and Myd88^−/−, but not TRIF^−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.     

Effective chemokine secretion by dendritic cells and expansion of cross-presenting CD4-/CD8+ dendritic cells define a protective phenotype in the mouse model of coxsackievirus myocarditis

Enteroviruses such as coxsackievirus B3 (CVB3) are able to induce lethal acute and chronic myocarditis. In resistant C57BL/6 mice, CVB3 myocarditis is abrogated by T-cell-dependent mechanisms, whereas major histocompatibility complex (MHC)-matched permissive A.BY/SnJ mice develop chronic myocarditis based on virus persistence. To define the role of T-cell-priming dendritic cells (DCs) in the outcome of CVB3 myocarditis, DCs were analyzed in this animal model in the course of CVB3 infection. In both mouse strains, DCs were found to be infectible with CVB3; however, formation of infectious virions was impaired. In DCs derived from C57BL/6 mice, significantly higher quantities of interleukin-10 (IL-10) and the proinflammatory cytokines IL-6 and tumor necrosis factor alpha were measured compared to those from A.BY/SnJ mice. Additionally, the chemokines interferon-inducible protein 10 (IP-10) and RANTES were secreted by DCs from resistant C57BL/6 mice earlier in infection and at significantly higher levels. The protective role of IP-10 in CVB3 myocarditis was confirmed in IP-10^−/− mice, which had increased myocardial injury compared to the immunocompetent control animals. Also, major differences in resistant and permissive mice were found in DC subsets, with C57BL/6 mice harboring more cross-priming CD4⁻ CD8⁺ DCs. As CD4⁻ CD8⁺ DCs are known to express 10 times more Toll-like receptor 3 (TLR3) than other DC subsets, we followed the course of CVB3 infection in TLR3^−/− mice. These mice developed a fulminant acute myocarditis and secreted sustained low amounts of type I interferons; secretion of IP-10 and RANTES was nearly abrogated in DCs. We conclude that MHC-independent genetic factors involving DC-related IP-10 secretion and TLR3 expression are beneficial in the prevention of chronic coxsackievirus myocarditis.     

Disruption of Multivesicular Body Vesicles Does Not Affect Major Histocompatibility Complex (MHC) Class II-Peptide Complex Formation and Antigen Presentation by Dendritic Cells

The antigen processing compartments in antigen-presenting cells (APCs) have well known characteristics of multivesicular bodies (MVBs). However, the importance of MVB integrity to APC function remains unknown. In this study, we have altered the ultrastructure of the MVB by perturbing cholesterol content genetically through the use of a deletion of the lipid transporter Niemann-Pick type C1 (NPC1). Immunofluorescence and electron microscopic analyses reveal that the antigen processing compartments in NPC1^−/− dendritic cells (DCs) have an abnormal ultrastructure in that the organelles are enlarged and the intraluminal vesicles are almost completely absent and those remaining are completely disorganized. MHC-II is restricted to the limiting membrane of these enlarged MVBs where it colocalizes with the peptide editor H2-DM. Curiously, proteolytic removal of the chaperone protein Invariant chain from MHC-II, degradation of internalized foreign antigens, and antigenic-peptide binding to nascent MHC-II are normal in NPC1^−/− DCs. Antigen-pulsed NPC1^−/− DCs are able to effectively activate antigen-specific CD4 T cells in vitro, and immunization of NPC1^−/− mice reveals surprisingly normal CD4 T cell activation in vivo. Our data thus reveal that the localization of MHC-II on the intraluminal vesicles of multivesicular antigen processing compartments is not required for efficient antigen presentation by DCs.     

Signaling of High Mobility Group Box 1 (HMGB1) through Toll-like Receptor 4 in Macrophages Requires CD14

High mobility group box 1 (HMGB1) is a DNA-binding protein that possesses cytokinelike, proinflammatory properties when released extracellularly in the C23–C45 disulfide form. HMGB1 also plays a key role as a mediator of acute and chronic inflammation in models of sterile injury. Although HMGB1 interacts with multiple pattern recognition receptors (PRRs), many of its effects in injury models occur through an interaction with toll-like receptor 4 (TLR4). HMGB1 interacts directly with the TLR4/myeloid differentiation protein 2 (MD2) complex, although the nature of this interaction remains unclear. We demonstrate that optimal HMGB1-dependent TLR4 activation in vitro requires the coreceptor CD14. TLR4 and MD2 are recruited into CD14-containing lipid rafts of RAW264.7 macrophages after stimulation with HMGB1, and TLR4 interacts closely with the lipid raft protein GM1. Furthermore, we show that HMGB1 stimulates tumor necrosis factor (TNF)-α release in WT but not in TLR4^−/−, CD14^−/−, TIR domain-containing adapter-inducing interferon-β (TRIF)^−/− or myeloid differentiation primary response protein 88 (MyD88)^−/− macrophages. HMGB1 induces the release of monocyte chemotactic protein 1 (MCP-1), interferon gamma–induced protein 10 (IP-10) and macrophage inflammatory protein 1α (MIP-1α) in a TLR4- and CD14-dependent manner. Thus, efficient recognition of HMGB1 by the TLR4/MD2 complex requires CD14.     

Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4^lps-d), mice deficient in TLR9 (TLR9^−/−) and TLR4/9 double mutant mice (TLR4^lps-d/TLR9^−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4^lps-d/TLR9^−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4^lps-d/TLR9^−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response.     

Accelerated Calvarial Healing in Mice Lacking Toll-Like Receptor 4

The bone and immune systems are closely interconnected. The immediate inflammatory response after fracture is known to trigger a healing cascade which plays an important role in bone repair. Toll-like receptor 4 (TLR4) is a member of a highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate the systemic inflammatory response induced by exposed bone components during long-bone fracture. Here we tested the hypothesis that TLR4 activation affects the healing of calvarial defects. A 1.8 mm diameter calvarial defect was created in wild-type (WT) and TLR4 knockout (TLR4^−/−) mice. Bone healing was tested using radiographic, histologic and gene expression analyses. Radiographic and histomorphometric analyses revealed that calvarial healing was accelerated in TLR4^−/− mice. More bone was observed in TLR4^−/− mice compared to WT mice at postoperative days 7 and 14, although comparable healing was achieved in both groups by day 21. Bone remodeling was detected in both groups on postoperative day 28. In TLR4^−/− mice compared to WT mice, gene expression analysis revealed that higher expression levels of IL-1β, IL-6, TNF-α,TGF-β1, TGF-β3, PDGF and RANKL and lower expression level of RANK were detected at earlier time points (≤ postoperative 4 days); while higher expression levels of IL-1β and lower expression levels of VEGF, RANK, RANKL and OPG were detected at late time points (> postoperative 4 days). This study provides evidence of accelerated bone healing in TLR4^−/− mice with earlier and higher expression of inflammatory cytokines and with increased osteoclastic activity. Further work is required to determine if this is due to inflammation driven by TLR4 activation.     

Distinct Roles for Dectin-1 and TLR4 in the Pathogenesis of Aspergillus fumigatus Keratitis

Aspergillus species are a major worldwide cause of corneal ulcers, resulting in visual impairment and blindness in immunocompetent individuals. To enhance our understanding of the pathogenesis of Aspergillus keratitis, we developed a murine model in which red fluorescent protein (RFP)-expressing A. fumigatus (Af293.1RFP) conidia are injected into the corneal stroma, and disease progression and fungal survival are tracked over time. Using Mafia mice in which c-fms expressing macrophages and dendritic cells can be induced to undergo apoptosis, we demonstrated that the presence of resident corneal macrophages is essential for production of IL-1β and CXCL1/KC, and for recruitment of neutrophils and mononuclear cells into the corneal stroma. We found that β-glucan was highly expressed on germinating conidia and hyphae in the cornea stroma, and that both Dectin-1 and phospho-Syk were up-regulated in infected corneas. Additionally, we show that infected Dectin-1^−/− corneas have impaired IL-1β and CXCL1/KC production, resulting in diminished cellular infiltration and fungal clearance compared with control mice, especially during infection with clinical isolates expressing high β-glucan. In contrast to Dectin 1^−/− mice, cellular infiltration into infected TLR2^−/−, TLR4^−/−, and MD-2^−/− mice corneas was unimpaired, indicating no role for these receptors in cell recruitment; however, fungal killing was significantly reduced in TLR4^−/− mice, but not TLR2^−/− or MD-2^−/− mice. We also found that TRIF^−/− and TIRAP^−/− mice exhibited no fungal-killing defects, but that MyD88^−/− and IL-1R1^−/− mice were unable to regulate fungal growth. In conclusion, these data are consistent with a model in which β-glucan on A.fumigatus germinating conidia activates Dectin-1 on corneal macrophages to produce IL-1β, and CXCL1, which together with IL-1R1/MyD88-dependent activation, results in recruitment of neutrophils to the corneal stroma and TLR4-dependent fungal killing.     

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.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

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.     

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.     

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.     

Toll-Like Receptor 4 Mediates the Response of Epithelial and Stromal Cells to Lipopolysaccharide in the Endometrium

Background

Ascending infections of the female genital tract with bacteria causes pelvic inflammatory disease (PID), preterm labour and infertility. Lipopolysaccharide (LPS) is the main component of the cell wall of Gram-negative bacteria. Innate immunity relies on the detection of LPS by Toll-like receptor 4 (TLR4) on host cells. Binding of LPS to TLR4 on immune cells stimulates secretion of pro-inflammatory cytokines such as IL-6, chemokines such as CXCL1 and CCL20, and prostaglandin E₂. The present study tested the hypothesis that TLR4 on endometrial epithelial and stromal cells is essential for the innate immune response to LPS in the female genital tract.

Methodology/Principal Findings

Wild type (WT) mice expressed TLR4 in the endometrium. Intrauterine infusion of purified LPS caused pelvic inflammatory disease, with accumulation of granulocytes throughout the endometrium of WT but not Tlr4^−/− mice. Intra-peritoneal infusion of LPS did not cause PID in WT or Tlr4^−/− mice, indicating the importance of TLR4 in the endometrium for the detection of LPS in the female genital tract. Stromal and epithelial cells isolated from the endometrium of WT but not Tlr4^−/− mice, secreted IL-6, CXCL1, CCL20 and prostaglandin E₂ in response to LPS, in a concentration and time dependent manner. Co-culture of combinations of stromal and epithelial cells from WT and Tlr4^−/− mice provided little evidence of stromal-epithelial interactions in the response to LPS.

Conclusions/Significance

The innate immune response to LPS in the female genital tract is dependent on TLR4 on the epithelial and stromal cells of the endometrium.     

CD14 Mediates Toll-like Receptor 4 (TLR4) Endocytosis and Spleen Tyrosine Kinase (Syk) and Interferon Regulatory Transcription Factor 3 (IRF3) Activation in Epithelial Cells and Impairs Neutrophil Infiltration and Pseudomonas aeruginosa Killing in Vivo

In the current study, we examined the role of CD14 in regulating LPS activation of corneal epithelial cells and Pseudomonas aeruginosa corneal infection. Our findings demonstrate that LPS induces Toll-like receptor 4 (TLR4) internalization in corneal epithelial cells and that blocking with anti-CD14 selectively inhibits TLR4 endocytosis, spleen tyrosine kinase (Syk) and IRF3 phosphorylation, and production of CCL5/RANTES and IFN-β, but not IL-8. Using a murine model of P. aeruginosa corneal infection, we show that although infected CD14^−/− corneas produce less CCL5, they exhibit significantly increased CXC chemokine production, neutrophil recruitment to the corneal stroma, and bacterial clearance than C57BL/6 mice. We conclude that CD14 has a critical role in mediating TLR4 signaling through IRF3 in resident corneal epithelial cells and macrophages and thereby modulates TLR4 cell surface activation of the MyD88/NF-κB/AP-1 pathway and production of CXC chemokines and neutrophil infiltration to infected tissues.     

Critical Role of TLR4 in Human Metapneumovirus Mediated Innate Immune Responses and Disease Pathogenesis

Human metapneumovirus (hMPV) is one of the main causes of acute respiratory tract infections in children, elderly and immunocompromised patients. The mammalian Toll-like receptors (TLR) were identified as critical regulators of innate immunity to a variety of microbes, including viruses. We have recently shown that hMPV-induced cytokine, chemokine and type I interferon secretion in dendritic cells occurs via TLR4, however, its role in hMPV-induced disease is unknown. In this study, wild-type(WT) and TLR4-deficient mice (TLR4^−/−) were infected with hMPV and examined for clinical disease parameters, such as body weight loss and airway obstruction, viral clearance, lung inflammation, dendritic cell maturation, T-cell proliferation and antibody production. Our results demonstrate that absence of TLR4 in hMPV-infected mice significantly reduced the inflammatory response as well as disease severity, shown by reduced body weight loss and airway obstruction and hyperresponsiveness (AHR), compared to WT mice. Levels of cytokines and chemokines were also significantly lower in the TLR4^−/− mice. Accordingly, recruitment of inflammatory cells in the BAL, lungs, as well as in lymph nodes, was significantly reduced in the TLR4^−/− mice, however, viral replication and clearance, as well as T-cell proliferation and neutralizing antibody production, were not affected. Our findings indicate that TLR4 is important for the activation of the innate immune response to hMPV, however it does play a role in disease pathogenesis, as lack of TLR4 expression is associated with reduced clinical manifestations of hMPV disease, without affecting viral protection.