MyD88-dependent responses involving toll-like receptor 2 are important for protection and clearance of Legionella pneumophila in a mouse model of Legionnaires' disease

Legionella pneumophila is a gram-negative facultative intracellular parasite of macrophages. Although L. pneumophila is the causative agent of a severe pneumonia known as Legionnaires' disease, it is likely that most infections caused by this organism are cleared by the host innate immune system. It is predicted that host pattern recognition proteins belonging to the Toll-like receptor (TLR) family are involved in the protective innate immune responses. We examined the role of TLR-mediated responses in L. pneumophila detection and clearance using genetically altered mouse hosts in which the macrophages are permissive for L. pneumophila intracellular replication. Our data demonstrate that cytokine production by bone marrow-derived macrophages (BMMs) in response to L. pneumophila infection requires the TLR adapter protein MyD88 and is reduced in the absence of TLR2 but not in the absence of TLR4. Bacterial growth ex vivo in BMMs from MyD88-deficient mice was not enhanced compared to bacterial growth ex vivo in BMMs from heterozygous littermate controls. Wild-type mice were able to clear L. pneumophila from the lung, whereas respiratory infection of MyD88-deficient mice caused death that resulted from robust bacterial replication and dissemination. In contrast to an infection with virulent L. pneumophila, MyD88-deficient mice were able to clear infections with L. pneumophila dotA mutants, indicating that MyD88-independent responses in the lung are sufficient to clear bacteria that are unable to replicate intracellularly. In vivo growth of L. pneumophila was enhanced in the lungs of TLR2-deficient mice, which resulted in a delay in bacterial clearance. No significant differences were observed in the growth and clearance of L. pneumophila in the lungs of TLR4-deficient mice and heterozygous littermate control mice. Our data indicate that MyD88 is crucial for eliciting a protective innate immune response against virulent L. pneumophila and that TLR2 is one of the pattern recognition receptors involved in initiating this MyD88-dependent response.     

The role of MyD88 and TLR4 in the LPS-mimetic activity of Taxol

Taxol can mimic bacterial lipopolysaccharide (LPS) by activating mouse macrophages in a cell cycle-independent, LPS antagonist-inhibitable manner. Macrophages from C3H/HeJ mice, which have a spontaneous mutation in Toll-like receptor 4 (TLR4), are hyporesponsive to both LPS and Taxol, suggesting that LPS and Taxol may share a signaling pathway involving TLR4. To determine whether TLR4 and its interacting adaptor molecule MyD88 are necessary for Taxol's LPS mimetic actions, we examined Taxol responses of primary macrophages from genetically defective mice lacking either TLR4 (C57BL/10ScNCr) or MyD88 (MyD88 knockout). When stimulated with Taxol, macrophages from wild-type mice responded robustly by secreting both TNF and NO, while macrophages from either TLR4-deficient C57BL/10ScNCr mice or MyD88 knockout mice produced only minimal amounts of TNF and NO. Taxol-induced NF-kappa B-driven luciferase activity was reduced after transfection of RAW 264.7 macrophages with a dominant negative version of mouse MyD88. Taxol-induced microtubule-associated protein kinase (MAPK) activation and NF-kappa B nuclear translocation were absent from TLR4-null macrophages, but were preserved in MyD88 knockout macrophages with a slight delay in kinetics. Neither Taxol-induced NF-kappa B activation, nor I kappa B degradation was affected by the presence of phosphatidylinositol 3-kinase inhibitors. These results suggest that Taxol and LPS not only share a TLR4/MyD88-dependent pathway in generating inflammatory mediators, but also share a TLR4-dependent/MyD88-independent pathway leading to activation of MAPK and NF-kappa B.     

MyD88 deficiency leads to decreased NK cell gamma interferon production and T cell recruitment during Chlamydia muridarum genital tract infection, but a predominant Th1 response and enhanced monocytic inflammation are associated with infection resolution

We have previously shown that MyD88 knockout (KO) mice exhibit delayed clearance of Chlamydia muridarum genital infection compared to wild-type (WT) mice. A blunted Th1 response and ineffective suppression of the Th2 response were also observed in MyD88 KO mice. The goal of the present study was to investigate specific mechanisms whereby absence of MyD88 leads to these effects and address the compensatory mechanisms in the genital tract that ultimately clear infection in the absence of MyD88. It was observed that NK cells recruited to the genital tract in MyD88 KO mice failed to produce gamma interferon (IFN-γ) mRNA and protein. This defect was associated with decreased local production of interleukin-17 (IL-17), IL-18, and tumor necrosis factor alpha (TNF-α) but normal levels of IL-12p70. Additionally, recruitment of CD4 T cells to the genital tract was reduced in MyD88 KO mice compared to that in WT mice. Although chronic infection in MyD88 KO mice resulted in oviduct pathology comparable to that of WT mice, increased histiocytic inflammation was observed in the uterine horns. This was associated with increased CCL2 levels and recruitment of macrophages as a potential compensatory mechanism. Further deletion of TLR4-TRIF signaling in MyD88 KO mice, using TLR4/MyD88 double-KO mice, did not further compromise host defense against chlamydiae, suggesting that compensatory mechanisms are Toll-like receptor (TLR) independent. Despite some polarization toward a Th2 response, a Th1 response remained predominant in the absence of MyD88, and it provided equivalent protection against a secondary infection as observed in WT mice.     

Mechanism of pathogen-specific TLR4 activation in the mucosa: fimbriae, recognition receptors and adaptor protein selection

The mucosal host defence discriminates pathogens from commensals, and prevents infection while allowing the normal flora to persist. Paradoxically, Toll-like receptors (TLR) control the mucosal defence against pathogens, even though the TLR recognise conserved molecules like LPS, which are shared between pathogens and commensals. This study proposes a mechanism of pathogen-specific mucosal TLR4 activation, involving adhesive ligands and their host cell receptors. TLR4 signalling was activated in CD14-negative, LPS-unresponsive epithelial cells by P fimbriated, uropathogenic Escherichia coli but not by a mutant lacking fimbriae. Epithelial TLR4 signalling in vivo involved the glycosphingolipid receptors for P fimbriae and the adaptor proteins Toll/IL-1R (TIR) domain-containing adaptor inducing IFN-beta (TRIF)/TRIF-related adaptor molecule (TRAM), but myeloid differentiation protein 88 (MyD88)/TIR domain-containing adaptor protein were not required for the epithelial response. Substituting the P fimbriae with type 1 fimbriae changed TLR4 signalling from the TRIF to the MyD88 adaptor pathway. In addition, the adaptor proteins and the fimbrial type were found to influence bacterial clearance. Trif(-/-) and Tram(-/-) mice remained infected with P fimbriated E. coli but cleared the type 1 fimbriated strain, while Myd88(-/-) mice became carriers of both the P and the type 1 fimbriated bacteria. Thus, TLR4 may be engaged specifically by pathogens, when the proper cell surface receptors are engaged by virulence ligands.     

Cellular responses to bacterial cell wall components are mediated through MyD88-dependent signaling cascades

MyD88 is an adaptor molecule essential for signaling via the Toll-like receptor (TLR)/IL-1 receptor family. TLR4 is a member of the TLR family and a point mutation in the Tlr4 gene causes hyporesponsiveness to lipopolysaccharide (LPS) in C3H/HeJ mice. We have previously shown that both TLR4- and MyD88-deficient mice are hyporesponsive to LPS. In this study we examined the responsiveness of these two knockout mice to various bacterial cell wall components. Cells from TLR4-deficient mice responded to several kinds of LPS, peptidoglycan and crude cell wall preparation from Gram-positive bacteria and mycobacterial lysates. In contrast, macrophages and splenocytes from MyD88-deficient mice did not respond to any of the bacterial components we tested. These results show that MyD88 is essential for the cellular response to bacterial cell wall components.     

Interleukin-1 receptor-associated kinase 4 is essential for initial host control of Brucella abortus infection

Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Recent studies have revealed that Toll-like receptor (TLR)-initiated immune response to Brucella spp. depends on myeloid differentiation factor 88 (MyD88) signaling. Therefore, we decided to study the role of the interleukin-1 receptor-associated kinase 4 (IRAK-4) in host innate immune response against B. abortus. After Brucella infection, it was shown that the number of CFU in IRAK-4(-/-) mice was high compared to that in IRAK-4(+/-) animals only at 1 week postinfection. At 3 and 6 weeks postinfection, IRAK-4(-/-) mice were able to control the infection similarly to heterozygous animals. Furthermore, the type 1 cytokine profile was evaluated. IRAK-4(-/-) mice showed lower production of systemic interleukin-12 (IL-12) and gamma interferon (IFN-γ). Additionally, a reduced percentage of CD4(+) and CD8(+) T cells expressing IFN-γ was observed compared to IRAK-4(+/-). Further, the production of IL-12 and tumor necrosis factor alpha (TNF-α) by macrophages and dendritic cells from IRAK-4(-/-) mice was abolished at 24 h after stimulation with B. abortus. To investigate the role of IRAK-4 in mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways, macrophages were stimulated with B. abortus, and the signaling components were analyzed by protein phosphorylation. Extracellular signal-regulated kinase 1 (ERK1) and ERK2 and p38 as well as p65 NF-κB phosphorylation was profoundly impaired in IRAK-4(-/-) and MyD88(-/-) macrophages activated by Brucella. In summary, the results shown in this study demonstrated that IRAK-4 is critical to trigger the initial immune response against B. abortus but not at later phases of infection.     

MyD88-dependent signaling contributes to protection following Bacillus anthracis spore challenge of mice: implications for Toll-like receptor signaling

Bacillus anthracis is a spore-forming, gram-positive organism that is the causative agent of the disease anthrax. Recognition of Bacillus anthracis by the host innate immune system likely plays a key protective role following infection. In the present study, we examined the role of TLR2, TLR4, and MyD88 in the response to B. anthracis. Heat-killed Bacillus anthracis stimulated TLR2, but not TLR4, signaling in HEK293 cells and stimulated tumor necrosis factor alpha (TNF-alpha) production in C3H/HeN, C3H/HeJ, and C57BL/6J bone marrow-derived macrophages. The ability of heat-killed B. anthracis to induce a TNF-alpha response was preserved in TLR2-/- but not in MyD88-/- macrophages. In vivo studies revealed that TLR2-/- mice and TLR4-deficient mice were resistant to challenge with aerosolized Sterne strain spores but MyD88-/- mice were as susceptible as A/J mice. We conclude that, although recognition of B. anthracis occurs via TLR2, additional MyD88-dependent pathways contribute to the host innate immune response to anthrax infection.     

Toll-like receptor 2 mediates inflammatory cytokine induction but not sensitization for liver injury by Propioni- bacterium acnes

Recognition of Gram-positive bacteria by Toll-like receptor 2 (TLR2) induces activation of proinflammatory pathways. In mice, sensitization with the Gram-positive Propionibacterium acnes followed by a challenge with the TLR4 ligand, lipopolysaccharide (LPS), results in fulminant hepatic failure. Here, we investigated the role of TLR2 in liver sensitization to LPS-induced injury. Stimulation of Chinese hamster ovary cells and peritoneal macrophages with heat-killed P. acnes required expression of TLR2 but not of TLR4, suggesting that P. acnes was a TLR2 ligand. Cell activation by P. acnes was myeloid differentiation primary-response protein 88 (MyD88)-dependent, and it was augmented by coexpression of CD14 in mouse peritoneal macrophages. In vitro, P. acnes behaved as a TLR2 ligand and induced TLR4 hetero- and TLR2 homotolerance in peritoneal macrophages. In vivo priming of wild-type mice with P. acnes, but not with the selective TLR2 ligands peptidoglycan and lipotheicoic acid, resulted in hepatocyte necrosis, hyperelevated serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL)-6, interferon-gamma (IFN-gamma), and IL-12 (p40/p70), and increased RNA expression of proinflammatory cytokines (IL-12p40, IL-1alpha, IL-6, IL-1beta, IL-18, IFN-gamma) in the liver after a LPS challenge. Furthermore, P. acnes priming sensitized TLR2-deficient (TLR2-/-) but not MyD88-/- mice to LPS-induced injury, evidenced by hepatocyte necrosis, increased levels of serum TNF-alpha, IFN-gamma, IL-6, and liver proinflammatory cytokine mRNA expression. IFN-gamma, a cytokine sensitizing to endotoxin, was induced by P. acnes in splenocytes of TLR2-/- and TLR9-/- but not MyD88-/- mice. These results suggest that although P. acnes triggers TLR2-mediated cell activation, TLR2-independent but MyD88-dependent mechanisms mediate in vivo sensitization by P. acnes for LPS-induced liver injury.     

Deficiencies of myeloid differentiation factor 88, Toll-like receptor 2 (TLR2), or TLR4 produce specific defects in macrophage cytokine secretion induced by Helicobacter pylori

Helicobacter pylori is a gram-negative microaerophilic bacterium that colonizes the gastric mucosa, leading to disease conditions ranging from gastritis to cancer. Toll-like receptors (TLRs) play a central role in innate immunity by their recognition of conserved molecular patterns on bacteria, fungi, and viruses. Upon recognition of microbial components, these TLRs associate with several adaptor molecules, including myeloid differentiation factor 88 (MyD88). To investigate the contribution of the innate immune system to H. pylori infection, bone marrow-derived macrophages from mice deficient in TLR2, TLR4, TLR9, and MyD88 were infected with H. pylori SS1 and SD4 for 24 or 48 h. We demonstrate that MyD88 was essential for H. pylori induction of all cytokines investigated except alpha interferon (IFN-alpha). The secretion of IFN-alpha was substantially increased from cells deficient in MyD88. H. pylori induced interleukin-12 (IL-12) and IL-10 through TLR4/MyD88 signaling. In addition, H. pylori induced less IL-6 and IL-1beta in TLR2-deleted macrophages, suggesting that the MyD88 pathway activated by TLR2 stimulation is responsible for H. pylori induction of the host proinflammatory response (IL-6 and IL-1beta). These observations are important in light of a recent report on IL-6 and IL-1beta playing a role in the development of H. pylori-related gastric cancer. In conclusion, our study demonstrates that H. pylori activates TLR2 and TLR4, leading to the secretion of distinct cytokines by macrophages.     

MyD88 mediates neutrophil recruitment initiated by IL-1R but not TLR2 activation in immunity against Staphylococcus aureus

MyD88 is an important signaling adaptor for both TLR and IL-1R family members. Here, we evaluated the role of TLR2/MyD88 and IL-1R/MyD88 signaling in host defense against S. aureus by using a cutaneous infection model in conjunction with bioluminescent bacteria. We found that lesions of S. aureus-infected MyD88- and IL-1R-deficient mice were substantially larger with higher bacterial counts compared with wild-type mice. In contrast, TLR2-deficient mice had lesions that were only moderately larger with minimally higher bacterial counts. In addition, MyD88- and IL-1R- but not TLR2-deficient mice had severely decreased recruitment of neutrophils to the site of infection. This neutrophil recruitment was not dependent upon IL-1R/MyD88 signaling by recruited bone marrow-derived cells, suggesting that resident skin cells utilize IL-1R/MyD88 signaling to promote neutrophil recruitment.     

Toll-like receptor 4 protects against lethal Leptospira interrogans serovar icterohaemorrhagiae infection and contributes to in vivo control of leptospiral burden

The roles of innate immune responses in protection from or pathogenesis of severe leptospirosis remain unclear. We examined the role of Toll-like receptors (TLRs) in mouse infection and macrophage responses to Leptospira. C3H/HeJ mice (TLR4 deficient) and C3H/HeJ-SCID mice, but not C3H/OuJ mice (TLR4 intact), died after intraperitoneal infection with Leptospira interrogans serovar Icterohaemorrhagiae. Death in both C3H/HeJ mouse strains was associated with jaundice and pulmonary hemorrhage, similar to the patient from whom the isolate was obtained. In chronic sublethal infection, TLR4-deficient mice harbored more leptospires in liver, lung, and kidney than control mice. Heat-killed Leptospira stimulated macrophages to secrete proinflammatory cytokines, tumor necrosis factor alpha, interleukin-6, and macrophage inflammatory protein 2 not inhibited by polymyxin B, suggesting that leptospiral lipopolysaccharide (LPS) did not drive these responses. Anti-TLR4 and anti-MD-2 but not anti-CD14 monoclonal antibodies inhibited cytokine production. Peritoneal macrophages from CD14-/- and TLR2-/- mice exhibited no defect in cytokine responses to Leptospira compared to controls. Macrophages from C3H/HeJ, TLR4-/-, and MyD88-/- mice secreted far-lower levels of cytokines than wild-type macrophages in response to Leptospira. TLR4 plays a crucial role in protection from acute lethal infection and control of leptospiral burden during sublethal chronic infection. Cytokine responses in macrophages correlated with leptospiral clearance. These TLR4-dependent but CD14/TLR2-independent responses are likely mediated by a leptospiral ligand(s) other than LPS.     

Containment of aerogenic Mycobacterium tuberculosis infection in mice does not require MyD88 adaptor function for TLR2, -4 and -9

The role of Toll-like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLR-mediated pathogen recognition and MyD88-dependent signaling events on anti-mycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple- and MyD88-deficient mice. After aerosol infection, both TLR2/4/9-deficient and wild-type mice expressed pro-inflammatory cytokines promoting antigen-specific T cells and the production of IFN-gamma to similar extents. Moreover, TLR2/4/9-deficient mice expressed IFN-gamma-dependent inducible nitric oxide synthase and LRG-47 in infected lungs. MyD88-deficient mice expressed pro-inflammatory cytokines and were shown to expand IFN-gamma-producing antigen-specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9-deficient mice controlled Mtb replication. IFN-gamma-dependent restriction of mycobacterial growth was severely impaired only in Mtb-infected MyD88, but not in TLR2/4/9-deficient bone marrow-derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, -4, and -9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti-mycobacterial defense.     

Unresponsiveness of MyD88-deficient mice to endotoxin.

MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.     

Central role of toll-like receptor 4 signaling and host defense in experimental pneumonia caused by Gram-negative bacteria

Toll-like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide. However, the precise role of TLR4 in regulating gene expression in response to an infection caused by gram-negative bacteria has not been fully elucidated. The role of TLR4 signaling in coordinating gene expression was assessed by gene expression profiling in lung tissue in a mouse model of experimental pneumonia with a low-dose infection of Klebsiella pneumoniae. We analyzed four mouse strains: C57BL/6 mice, which are resistant to bacterial dissemination; 129/SvJ mice, which are susceptible; C3H/HeJ mice, which are susceptible and have defective TLR4 signaling; and their respective control strain, C3H/HeN (intermediate resistance). At 4 h after infection, C57BL/6 and C3H/HeN mice demonstrated the greatest number of genes, with 67 shared induced genes which were TLR4 dependent and highly associated with the resistance phenotype. These genes included cytokine and chemokine genes required for neutrophil activation or recruitment, growth factor receptors, MyD88 (a critical adaptor protein for TLR signaling), and adhesion molecules. TLR4 signaling accounted for over 74% of the gene expression in the C3H background. These data suggest that early TLR4 signaling controls the vast majority of gene expression in the lung in response to an infection caused by gram-negative bacteria and that this subsequent gene expression determines survival of the host.     

Involvement of CD14, toll-like receptors 2 and 4, and MyD88 in the host response to the fungal pathogen Cryptococcus neoformans in vivo

The major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses against C. neoformans. MyD88(-/-) mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with live C. neoformans. CD14(-/-) mice had reduced survival when infected i.v., while TLR2(-/-) mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge with C. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14(-/-), TLR2(-/-), and MyD88(-/-) mice. MyD88(-/-) mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs of C. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88(-/-) mice correlating with increased numbers of lung CFU and serum and lung GXM levels.     

TLR2 as an essential molecule for protective immunity against Toxoplasma gondii infection

To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.     

Lethal encephalitis in myeloid differentiation factor 88-deficient mice infected with herpes simplex virus 1

Herpes simplex virus 1 (HSV-1), a large DNA virus from the Herpesviridae family, is the major cause of sporadic lethal encephalitis and blindness in humans. Recent studies have shown the importance of Toll-like receptors (TLRs) in the immune response to HSV-1 infection. Myeloid differentiation factor 88 (MyD88) is a critical adaptor protein that is downstream to mediated TLR activation and is essential for the production of inflammatory cytokines. Here, we studied the relationship between MyD88 and HSV-1 using a purified HSV-1 isolated from a natural oral recurrent human infection. We observed the activation of TLR-2 by HSV-1 in vitro using Chinese hamster ovary cells stably transfected with a reporter gene. Interestingly, we found that only peritoneal macrophages from MyD88-/- mice, but not macrophages from TRL2-/- or from wild-type mice, were unable to produce tumor necrosis factor-alpha in response to HSV-1 exposure. Additionally, although TLR2-/- mice showed no enhanced susceptibility to intranasal infection with HSV-1, MyD88-/- mice were highly susceptible to infection and displayed viral migration to the brain, severe neuropathological signs of encephalitis, and 100% mortality by day 10 after infection. Together, our results suggest that innate resistance to HSV-1 is mediated by MyD88 and may rely on activation of multiple TLRs.     

Endotoxin-induced uveitis is primarily dependent on radiation-resistant cells and on MyD88 but not TRIF

TLR4 activation by LPS (endotoxin) is mediated by the MyD88 and TRIF intracellular signaling pathways. We determined the relative activation of these pathways in murine ocular tissue after LPS exposure. Additionally, we explored whether BM-derived or non-BM-derived cells were the major contributors to EIU. Mice deficient in TRIF or MyD88 and their congenic (WT) controls received 250 ng ultrapure LPS ivt at 0 h. Ocular inflammation was assessed by histological analysis at 4, 6, and 24 h, and additionally, in MyD88(-/-) mice, intravital microscopy was performed at 4 h and 6 h to assess adherent, rolling, and infiltrating cells in the iris vasculature and tissue. Cytokines associated with the MyD88 and TRIF intracellular signaling pathways were analyzed in ocular tissue at 4 h. BM chimeric mice (WT→WT, TLR4(-/-)→WT, WT→TLR4(-/-)) received 250 ng LPS by ivt injection, and ocular tissues were examined by histology at 6 h. Lack of MyD88 resulted in a markedly diminished cellular response and reduced production of MyD88-related cytokines 4 h post-LPS treatment. In contrast, lack of TRIF led to reduced production of TRIF-related cytokines and no change in the cellular response to LPS. Therefore, the MyD88 pathway appears to be the dominant TLR4 pathway in EIU. Only WT → TLR4(-/-) chimeric mice were resistant to EIU, and this suggests, surprisingly, that non-BM-derived (radiation-resistant) cells in the eye play a greater role than BM-derived cells.     

ASC is essential for LPS-induced activation of procaspase-1 independently of TLR-associated signal adaptor molecules

Toll-like receptors (TLRs) initiate a signalling cascade via association with an adaptor molecule, myeloid differentiation factor 88 (MyD88) and/or TIR domain-containing adaptor inducing-IFN-beta (Trif), to induce various pro-inflammatory cytokines for microbial eradication. After stimulation of TLR4 with lipopolysaccharide (LPS), both IL-1beta and IL-18 are processed, depending on the activation of caspase-1, although its mechanism remains unclear. ASC is an adapter protein possibly involved in the activation of procaspase-1. To unravel the requirement of ASC, we generated Asc(-/-) mice. Upon stimulation with LPS, Asc(-/-) macrophages failed in the processing of procaspase-1 and maturation of pro-IL-1beta and pro-IL-18, but normally produced other pro-inflammatory cytokines including TNF-alpha and IL-6. MyD88(-/-) and Trif(-/-) macrophages showed normal activation of caspase-1, demonstrating a dispensable role for MyD88 and Trif. After, LPS-challenged Asc(-/-) mice lacked serum elevation of IL-1beta and IL-18. Moreover, the Asc(-/-) mice exhibited neither acute liver injury nor lethal shock. These results demonstrate critical roles for ASC in the release of IL-1beta/IL-18 via activation of caspase-1 and provide new insights into the inflammatory responses for host defence and diseases.