TLR2-dependent MyD88 signaling contributes to early host defense in murine Enterococcus faecium peritonitis

The incidence of infections with Enterococcus faecium is increasing worldwide. TLRs have been implicated in the recognition of pathogens and the initiation of an adequate innate immune response. We here sought to determine the roles of MyD88, the common adaptor protein involved in TLR signaling, TLR2, TLR4, and CD14 in host defense against E. faecium peritonitis. MyD88 knockout (KO) mice demonstrated an impaired early response to E. faecium peritonitis, as reflected by higher bacterial loads in peritoneal fluid and liver accompanied by a markedly attenuated neutrophil influx into the abdominal cavity. In vitro, not only MyD88 KO macrophages but also TLR2 KO and CD14 KO macrophages displayed a reduced responsiveness to E. faecium. In accordance, transfection of TLR2 rendered human embryonic kidney 293 cells responsive to E. faecium, which was enhanced by cotransfection of CD14. TLR2 KO mice showed higher bacterial loads in peritoneal fluid after in vivo infection with E. faecium and a diminished influx of neutrophils, whereas CD14 KO mice had an unaltered host response. E. faecium phagocytosis and killing were not affected by MyD88, TLR2, or CD14 deficiency. TLR4 did not play a role in the immune response to E. faecium in vitro or in vivo. These data suggest that MyD88 contributes to the effective clearance of E. faecium during peritonitis at least in part via TLR2 and by facilitating neutrophil recruitment to the site of the infection.     

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.     

Endogenous tissue-type plasminogen activator is protective during Escherichia coli-induced abdominal sepsis in mice

Sepsis is associated with enhanced production of tissue-type plasminogen activator (tPA). We investigated the function of endogenous tPA in the immune responses to Escherichia coli-induced abdominal sepsis using tPA gene-deficient (tPA(-/-)) and normal wild-type (WT) mice. tPA(-/-) mice demonstrated an impaired defense against E. coli peritonitis as indicated by higher bacterial loads at the primary site of the infection, enhanced dissemination, and reduced survival. The protective function of tPA was independent of plasmin since plasminogen gene-deficient (Plg(-/-)) mice were indistinguishable from WT mice. Relative to WT mice, tPA(-/-) mice demonstrated similar neutrophil counts in the peritoneal cavity despite much higher bacterial loads and higher local concentrations of neutrophil attracting chemokines, suggesting a reduced migratory response. In line, tPA(-/-) mice demonstrated a reduced thioglycolate-induced neutrophil influx into the peritoneal cavity and i.p. injection of WT mice with a replication-defective adenoviral vector expressing tPA caused an enhanced cell migration to the peritoneal cavity during E. coli peritonitis. These findings identify a novel protective function of tPA in abdominal sepsis caused by E. coli that seems independent of its role in the generation of plasmin.     

Evaluating vaccinia virus cytokine co-expression in TLR GKO mice

Using Toll-like receptor (TLR) and MyD88 gene knock-out (GKO) mice the effect of TLRs and MyD88 on virus replication, interferon (IFN)-β production, natural killer (NK) cell and CD8T cell responses were assessed following ectromelia virus (ECTV) and recombinant vaccinia virus (rVV) infection. The capacity for rVVs encoding cytokines to restore immune function in MyD88(-/-) mice was clearly demonstrated. Results showed that TLR2(-/-), TLR4(-/-)and TLR7(-/-) mice survived ECTV infection whereas MyD88(-/-) and TLR9(-/-)mice, in contrast, were highly susceptible. Next, following infection with rVV, MyD88(-/-) mice elicited reduced serum IFN-β, NK cell and CD8T cell responses compared with wild-type mice, whereas TLR9(-/-) mice showed elevated CD8T cell responses. When MyD88(-/-)mice were infected with rVV co-expressing IFN-β these mice were able to restore IFN-β levels and CD8T cell responses but not NK cell activation. Interestingly, even though rVV co-expressing interleukin (IL)-2 enhanced NK cell activation in MyD88(-/-) mice, this was not associated with an antiviral effect, as observed in normal mice. Surprisingly, co-infection with rVV IL-2/rVV IL-12, but not rVV IL-2/rVV IFN-β, restored the attenuated phenotype of rVV IL-2 in MyD88(-/-) mice indicating that the IL-2/IL-12 combination promotes antiviral responses. Our results clearly show that the CD8T cell defect observed in MyD88(-/-) mice to vaccinia virus infection can be restored by rVV-encoding IFN-β demonstrating the critical role of this cytokine in T cell mediated immunity and illustrates that the model can provide an effective platform for the elucidation of cytokine immunobiology.     

TLR2, TLR4 and the MYD88 signaling pathway are crucial for neutrophil migration in acute kidney injury induced by sepsis

The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-), TLR4(-/-) and MyD88(-/-) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2(-/-), TLR4(-/-) and MyD88(-/-) mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88(-/-) mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1(+low) cells migration compared with the knockout mice and decreased in GR1(+high) cells migration into the peritoneal cavity. The TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.     

The development of early host response to Pseudomonas aeruginosa lung infection is critically dependent on myeloid differentiation factor 88 in mice

Toll-like receptors (TLR) induce distinct patterns of host responses through myeloid differentiation factor 88 (MyD88)-dependent and/or -independent pathways, depending on the nature of the pathogen. Pseudomonas aeruginosa is a cause of serious lung infection in immunocompromised individuals and cystic fibrosis patients. The role of the TLR-MyD88 pathway in P. aeruginosa-induced lung infection in vivo was examined in this study. MyD88-/- mice demonstrated an impaired clearance of P. aeruginosa from the lung. Little or no neutrophil recruitment was observed in the airways of MyD88-/- mice following P. aeruginosa lung infection. This observation was associated with a reduced production of inflammatory mediators that affect neutrophil recruitment, including macrophage-inflammatory protein-2, tumor necrosis factor, and interleukin-1beta in the airways of MyD88-/- mice. Similarly, MyD88-/- mice showed inhibited NF-kappaB activation in the lung following P. aeruginosa infection. Interestingly, P. aeruginosa infection induced a 7.5-fold increase of TLR2 mRNA expression in the lungs of MyD88+/+ mice. Furthermore, host responses to P. aeruginosa lung infection in TLR2-/- and TLR4 mutant mice were partially inhibited compared with the responses of respective control mice. Taken together, our results indicate that the MyD88-dependent pathway is essential for the development of early host responses to P. aeruginosa infection, leading to the clearance of this bacterium, and that TLR2 and TLR4 are involved in this process.     

Cutting edge: MyD88 is required for resistance to Toxoplasma gondii infection and regulates parasite-induced IL-12 production by dendritic cells

Host resistance to the intracellular protozoan Toxoplasma gondii is highly dependent on early IL-12 production by APC. We demonstrate here that both host resistance and T. gondii-induced IL-12 production are dramatically reduced in mice lacking the adaptor molecule MyD88, an important signaling element used by Toll-like receptor (TLR) family members. Infection of MyD88-deficient mice with T. gondii resulted in uncontrolled parasite replication and greatly reduced plasma IL-12 levels. Defective IL-12 responses to T. gondii Ags (soluble tachyzoite Ag (STAg)) were observed in MyD88(-/-) peritoneal macrophages, neutrophils, and splenic dendritic cells (DC). In contrast, DC from TLR2- or TLR4-deficient animals developed normal IL-12 responses to STAg. In vivo treatment with pertussis toxin abolished the residual IL-12 response displayed by STAg-stimulated DC from MyD88(-/-) mice. Taken together, these data suggest that the induction of IL-12 by T. gondii depends on a unique mechanism involving both MyD88 and G protein-coupled signaling pathways.     

T and B cells are not required for clearing Staphylococcus aureus in systemic infection despite a strong TLR2-MyD88-dependent T cell activation

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2-MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2-MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4(+) T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3(-/-), Rag2(-/-), and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2-MyD88 and thereby promotes T(H)1 and T(H)17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.     

TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B

Recombinant hemagglutinin B (rHagB), a virulence factor of the periodontal pathogen Porphyromonas gingivalis, has been shown to induce protective immunity against bacterial infection. Furthermore, we have demonstrated that rHagB is a TLR4 agonist for dendritic cells. However, it is not known how rHagB dendritic cell stimulation affects the activation and differentiation of T cells. Therefore, we undertook the present study to examine the role of TLR4 signaling in shaping the CD4(+) T cell response following immunization of mice with rHagB. Immunization with this Ag resulted in the induction of specific CD4(+) T cells and Ab responses. In TLR4(-/-) and MyD88(-/-) but not Toll/IL-1R domain-containing adapter inducing IFN-β-deficient (TRIF(Lps2)) mice, there was an increase in the Th2 CD4(+) T cell subset, a decrease in the Th1 subset, and higher serum IgG(1)/IgG(2) levels of HagB-specific Abs compared with those in wild-type mice. These finding were accompanied by increased GATA-3 and Foxp3 expression and a decrease in the activation of CD4(+) T cells isolated from TLR4(-/-) and MyD88(-/-) mice. Interestingly, TLR4(-/-) CD4(+) T cells showed an increase in IL-2/STAT5 signaling. Whereas TRIF deficiency had minimal effects on the CD4(+) T cell response, it resulted in increased IFN-γ and IL-17 production by memory CD4(+) T cells. To our knowledge, these results demonstrate for the first time that TLR4 signaling, via the downstream MyD88 and TRIF molecules, exerts a differential regulation on the CD4(+) T cell response to HagB Ag. The gained insight from the present work will aid in designing better therapeutic strategies against P. gingivalis infection.     

Toll-like receptor 9 signaling has anti-inflammatory effects on the early phase of Helicobacter pylori-induced gastritis

Helicobacter pylori (H. pylori)-induced immune responses in the gastric mucosa are skewed toward T helper (Th) 1 phenotype, which is characterized by predominant production of tumor necrosis factor (TNF)-α and interferon (IFN)-γ by helper T cells. Toll-like receptors (TLRs) play an essential role in mucosal defense against microbes through the recognition of bacterial molecules. Among the members of the TLR family, TLR9 recognizes bacterial unmethylated CpG DNA sites, and signal transduction of TLR9 induces production of a variety of cytokines, including type-I IFN (IFN-α/β). We investigated the expression and role of TLR9 in H. pylori-induced gastritis in mice. Expression of TLR9 mRNA in the gastric tissue increased after infection with H. pylori. TLR9 was mainly expressed in the macrophages, dendritic cells, and CD3⁺ cells in the gastric mucosa. Neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA were higher in TLR9 knockout (KO) mice than in wild-type mice at 2 and 4 months after H. pylori inoculation. These differences in inflammatory parameters between H. pylori-infected wild-type and TLR9 KO mice disappeared 6 months after H. pylori inoculation. Expression of interleukin-4 mRNA, typical Th2 cytokine, in the gastric tissue did not differ between H. pylori-infected wild-type and TLR9 KO mice. Expression level of IFN-α/β mRNA in the TLR9 KO mice was lower than that in wild-type mice by 4 months after inoculation. Administration of IFN-α reduced H. pylori infection-induced increase in neutrophil infiltration and the expression levels of TNF-α and IFN-γ mRNA in TLR9 KO mice. Our findings suggest that TLR9 signaling plays important roles in the suppression of H. pylori-induced gastritis in the early phase via downregulation of Th1-type cytokines modulated by IFN-α.     

Early induction of CCL7 downstream of TLR9 signaling promotes the development of robust immunity to cryptococcal infection

We investigated mechanisms by which TLR9 signaling promoted the development of the protective response to Cryptococcus neoformans in mice with cryptococcal pneumonia. The afferent (week 1) and efferent (week 3) phase immune parameters were analyzed in the infected wild-type (TLR9(+/+)) and TLR-deficient (TLR9(-/-)) mice. TLR9 deletion diminished 1) accumulation and activation of CD11b(+) dendritic cells (DCs), 2) the induction of IFN-γ and CCR2 chemokines CCL7, CCL12, but not CCL2, at week 1, and 3) pulmonary accumulation and activation of the major effector cells CD4(+) and CD8(+) T cells, CD11b(+) lung DCs, and exudate macrophages at week 3. The significance of CCL7 induction downstream of TLR9 signaling was investigated by determining whether CCL7 reconstitution would improve immunological parameters in C. neoformans-infected TLR9(-/-) mice. Early reconstitution with CCL7 1) improved accumulation and activation of CD11b(+) DCs at week 1, 2) restored early IFN-γ production in the lungs, and 3) restored the accumulation of major effector cell subsets. CCL7 administration abolished the difference in lung fungal burdens between TLR9(+/+) and TLR9(-/-) mice at week 3; however, significant reduction of fungal burdens between PBS- and CCL7-treated mice has not been observed, suggesting that additional mechanism(s) apart from early CCL7 induction contribute to optimal fungal clearance in TLR9(+/+) mice. Collectively, we show that TLR9 signaling during the afferent phase contributes to the development of protective immunity by promoting the early induction of CCL7 and IFN-γ and the subsequent early recruitment and activation of DCs and additional effector cells in mice with cryptococcal pneumonia.     

TLR9-dependent and independent pathways drive activation of the immune system by Propionibacterium acnes

Propionibacterium acnes is usually a relatively harmless commensal. However, under certain, poorly understood conditions it is implicated in the etiology of specific inflammatory diseases. In mice, P. acnes exhibits strong immunomodulatory activity leading to splenomegaly, intrahepatic granuloma formation, hypersensitivity to TLR ligands and endogenous cytokines, and enhanced resistance to infection. All these activities reach a maximum one week after P. acnes priming and require IFN-γ and TLR9. We report here the existence of a markedly delayed (1-2 weeks), but phenotypically similar TLR9-independent immunomodulatory response to P. acnes. This alternative immunomodulation is also IFN-γ dependent and requires functional MyD88. From our experiments, a role for MyD88 in the IFN-γ-mediated P. acnes effects seems unlikely and the participation of the known MyD88-dependent receptors, including TLR5, Unc93B-dependent TLRs, IL-1R and IL-18R in the development of the alternative response has been excluded. However, the crucial role of MyD88 can partly be attributed to TLR2 and TLR4 involvement. Either of these two TLRs, activated by bacteria and/or endogenously generated ligands, can fulfill the required function. Our findings hint at an innate immune sensitizing mechanism, which is potentially operative in both infectious and sterile inflammatory disorders.     

Cutting edge: myeloid differentiation factor 88 deficiency improves resistance against sepsis caused by polymicrobial infection

Toll-like receptors (TLRs) are important for the activation of innate immune cells upon encounter of microbial pathogens. The present study investigated the potential roles of TLR2, TLR4, and the signaling protein myeloid differentiation factor 88 (MyD88) in polymicrobial septic peritonitis. Whereas both TLR2 and TLR4 were dispensable for host defense against septic peritonitis, MyD88-deficient mice were protected in this infection model. Recruitment of neutrophils to the septic focus and bacterial clearance were normal in MyD88-deficient mice. In contrast, the systemic inflammatory response was strongly attenuated in the absence of MyD88. Surprisingly, MyD88 deficiency did not alter cytokine and chemokine production in spleen, but markedly reduced the inflammatory response in liver and lung. Production of monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1alpha was entirely independent of MyD88. These results imply a central role of MyD88 for the systemic immune pathology of polymicrobial sepsis and show that cytokine production in spleen and induction of certain chemokines are MyD88 independent.     

Toll-like receptor-4 is required for intestinal response to epithelial injury and limiting bacterial translocation in a murine model of acute colitis

Inflammatory bowel disease (IBD) arises from a dysregulated mucosal immune response to luminal bacteria. Toll-like receptor (TLR)4 recognizes LPS and transduces a proinflammatory signal through the adapter molecule myeloid differentiation marker 88 (MyD88). We hypothesized that TLR4 participates in the innate immune response to luminal bacteria and the development of colitis. TLR4-/- and MyD88-/- mice and littermate controls were given 2.5% dextran sodium sulfate (DSS) for 5 or 7 days followed by a 7-day recovery. Colitis was assessed by weight loss, rectal bleeding, and histopathology. Immunostaining was performed for macrophage markers, chemokine expression, and cell proliferation markers. DSS treatment of TLR4-/- mice was associated with striking reduction in acute inflammatory cells compared with wild-type mice despite similar degrees of epithelial injury. TLR4-/- mice experienced earlier and more severe bleeding than control mice. Similar results were seen with MyD88-/- mice, suggesting that this is the dominant downstream pathway. Mesenteric lymph nodes from TLR4-/- and MyD88-/- mice more frequently grew gram-negative bacteria. Altered neutrophil recruitment was due to diminished macrophage inflammatory protein-2 expression by lamina propria macrophages in TLR4-/- and MyD88-/- mice. The similarity in crypt epithelial damage between TLR4-/- or MyD88-/- and wild-type mice was seen despite decreased epithelial proliferation in knockout mice. TLR4 through the adapter molecule MyD88 is important in intestinal response to injury and in limiting bacterial translocation. Despite the diversity of luminal bacteria, other TLRs do not substitute for the role of TLR4 in this acute colitis model. A defective innate immune response may result in diminished bacterial clearance and ultimately dysregulated response to normal flora.     

Matrix metalloproteinase-9 deficiency impairs host defense against abdominal sepsis

Matrix metalloproteinase (MMP)-9 is involved in extracellular matrix degradation and leukocyte migration. To determine the role of MMP-9 in the innate immune response to peritonitis, MMP-9 gene-deficient (MMP-9(-/-)) and normal wild-type mice were i.p. infected with Escherichia coli. MMP-9 mRNA and pro-MMP-9 protein levels increased rapidly upon induction of peritonitis. Although MMP-9(-/-) neutrophils showed a normal phagocytosis of E. coli in vitro, MMP-9(-/-) mice displayed a reduced resistance against E. coli peritonitis, as indicated by an enhanced bacterial outgrowth in the peritoneal cavity and increased dissemination of the infection. Furthermore, the cytokine response to LPS was not influenced by MMP-9 deficiency. However, during E. coli peritonitis, MMP-9(-/-) mice showed much higher peritoneal chemokine and cytokine levels compared with wild-type mice. Despite the increased local chemokine concentrations, MMP-9(-/-) mice displayed a diminished recruitment of leukocytes to the site of infection, indicating that cellular migration was impaired. Moreover, MMP-9(-/-) mice developed more severe distant organ damage during infection. These data suggest that MMP-9 is an essential component of an effective host response to E. coli peritonitis.     

MyD88 regulation of Fusarium keratitis is dependent on TLR4 and IL-1R1 but not TLR2

The fungal pathogens Fusarium solani and Fusarium oxysporum cause severe corneal disease in the United States and worldwide and were the causative organisms in a recent outbreak of contact lens-associated keratitis. To characterize innate immunity in Fusarium keratitis, we developed a murine model in which conidia are injected into the corneal stroma. Immunocompetent C57BL/6 mice rapidly developed severe corneal opacification associated with neutrophil infiltration and clearance of Fusarium hyphae. In contrast, neutrophil infiltration was delayed in MyD88-/- mice, resulting in uncontrolled growth of Fusarium hyphae in the corneal stroma and anterior chamber, and eventually resulting in corneal perforation. Corneal opacification scores in TLR2-/-, TLR4-/-, and TLR2/4-/- mice were similar to those of C57BL/6 mice; however, TLR4-/- and TLR2/4-/- mice had impaired antifungal responses. The phenotype of infected IL-1R1-/- mice was similar to that of MyD88-/- mice, with uncontrolled fungal growth resulting in corneal perforation. IL-1R1-/- mice also produced significantly less CXCL1/KC in the corneal stroma compared with C57BL/6 mice consistent with delayed neutrophil recruitment to the corneal stroma. Together, these findings indicate that IL-1R1 and MyD88 regulate CXC chemokine production and neutrophil recruitment to the cornea, and that TLR4 has an important role in controlling growth and replication of these pathogenic fungi.     

TLRs 2 and 4 are not involved in hypersusceptibility to acute Pseudomonas aeruginosa lung infections

TLRs are implicated in defense against microorganisms. Animal models have demonstrated that the susceptibility to a number of Gram-negative pathogens is linked to TLR4, and thus LPS of many Gram-negative bacteria have been implicated as virulence factors. To assess the role of this pathogen-associated molecular pattern as it is exposed on intact Pseudomonas aeruginosa, the susceptibility of mice lacking TLR4 or both TLR2 and TLR4 was examined in a model of acute Pseudomonas pneumonia. These mutant mice were not hypersusceptible to the Pseudomonas challenge and mounted an effective innate response that cleared the organism despite low levels of TNF-alpha and KC in the airways. Bacterial and neutrophil counts in the lung were similar in control and TLR-deficient mice at 6 and 24 h after infection. MyD88(-/-) mice were, however, hypersusceptible, with 100% of mice dying within 48 h with a lower dose of P. aeruginosa. Of note there were normal levels of IL-6 and G-CSF in the airways of TLR mutant mice that were absent from the MyD88(-/-) mice. Thus, the susceptibility of mice to P. aeruginosa acute lung infection does not go through TLR2 or TLR4, implying that Pseudomonas LPS is not the most important virulence factor in acute pneumonia caused by this organism. Furthermore, G-CSF treatment of infected MyD88(-/-) mice results in improved clearance and survival. Thus, the resistance to infection in TLR2/TLR4(-/-) mice may be linked to G-CSF and possibly IL-6 production.     

Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice

Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice. In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice. Similar results of early LPS-induced PMN infiltration and enhanced clearance of E. coli were seen in Toll-like receptor (TLR) 4-deficient mice. Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice. In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance.