TLR/MyD88 and liver X receptor alpha signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis

Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)(-/-) mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor alpha (LXRalpha) signaling pathways. We infected ApoE(-/-) mice and ApoE(-/-) mice that also lacked TLR2, TLR4, MyD88, or LXRalpha intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-alpha. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE(-/-) mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE(-/-) mice was further enhanced in ApoE(-/-)LXRalpha(-/-) double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-alpha. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRalpha appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.     

Involvement of MyD88 in host defense and the down-regulation of anti-heat shock protein 70 autoantibody formation by MyD88 in Toxoplasma gondii-infected mice

This study investigated the influence of TLR (toll-like receptor)4, TLR2, and MyD88 in Toxoplasma gondii-infected wild-type (WT) mice and TLR4-, TLR2-, and MyD88-deficient mice. Ninety-five percent of MyD88-deficient mice died 10-16 days after intraperitoneal infection with 100 cysts of T. gondii Fukaya strain, whereas 95-100% of TLR4- and TLR2-deficient mice and WT C57BL/6 (B6) mice survived for more than 7 wk after T. gondii infection. The distribution of T. gondii in various organs of TLR4-, TLR2-, and MyD88-deficient mice and WT B6 mice was assessed 2 wk after T. gondii intraperitoneal infection using quantitative competitive polymerase chain reaction. In MyD88-deficient mice, high levels of T. gondii load were observed in the brain, tongue, heart, lungs, spleen, liver, mesenteric lymph node, and kidneys after infection. The T. gondii load was significantly increased in the lungs in both TLR4- and TLR2-deficient mice compared with WT B6 mice. High levels of anti-mouse heat shock protein (mHSP)70 autoantibody and anti-T. gondii HSP70 antibody production were detected in the sera from MyD88-deficient mice.     

Myeloid differentiation factor 88 (MyD88) is required for murine resistance to Candida albicans and is critically involved in Candida -induced production of cytokines

We have studied the role of myeloid differentiation factor 88 (MyD88), the universal Toll-like receptor (TLR) adaptor protein, in murine defenses against Candida albicans. MyD88-deficient mice, experimentally infected in vivo, had a very significant impaired survival, and a higher tissue fungal burden when compared with control mice. The recruitment of neutrophils to the site of infection was also significantly diminished in MyD88-\- mice. In vitro production of proinflammatory cytokines such as TNF-alpha, IFN-gamma and IL-12p70, by antigen-stimulated splenocytes from mice intravenously infected with the low-virulence C. albicans PCA2 strain, could not be detected in MyD88-\- mice. This default of production of Th1 cytokines in MyD88-deficient mice correlated with a greatly diminished frequency of IFN-gamma-producing CD4 + T lymphocytes. Also, the frequency of IFN-gamma-producing CD8 + T lymphocytes was lower in MyD88-\- mice than in control mice. Although C. albicans-specific antibody titers in PCA2-infected mice appeared more quickly in MyD88-\- mice than in control mice, the MyD88-\- group was not able to maintain the Candida-specific IgM nor IgG titers at the third week of infection. The complexity of antigens recognized by sera from MyD88-\- mice was quite similar to that from infected control mice. Taken together, these data show that MyD88-\- mice are extremely susceptible to C. albicans infections, suggesting that MyD88-dependent signaling pathways are essential for both the innate and adaptive immune responses to C. albicans.     

TLR2 synergizes with both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Streptococcus pneumoniae

We previously demonstrated that induction of splenic cytokine and chemokine secretion in response to Streptococcus pneumoniae (Pn) is MyD88-, but not critically TLR2-dependent, suggesting a role for additional TLRs. In this study, we investigated the role of TLR2, TLR4, and/or TLR9 in mediating this response. We show that a single deficiency in TLR2, TLR4, or TLR9 has only modest, selective effects on cytokine and chemokine secretion, whereas substantial defects were observed in TLR2(-/-)xTLR9(-/-) and TLR2(-/-)xTLR4(-/-) mice, though not as severe as in MyD88(-/-) mice. Chloroquine, which inhibits the function of intracellular TLRs, including TLR9, completely abrogated detectable cytokine and chemokine release in spleen cells from TLR2(-/-)xTLR4(-/-) mice, similar to what is observed for mice deficient in MyD88. These data demonstrate significant synergy between TLR2 and both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Pn.     

Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2(-/-) and TLR4(-/-) animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88(-/-) mice failed to control acute and chronic M. avium growth and succumbed 9-14 wk postinfection. Infected TLR2(-/-) mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88(-/-) animals, while TLR4(-/-) mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88(-/-) mice revealed massive destruction of lung tissue not present in WT, TLR2(-/-), or TLR4(-/-) mice. In addition, MyD88(-/-) and TLR2(-/-), but not TLR4(-/-), mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88(-/-) and TLR2(-/-) macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88(-/-) mice. Similarly, MyD88(-/-) mice displayed a profound defect in IFN-gamma response that was not evident in TLR2(-/-) or TLR4(-/-) mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-gamma responses.     

Chlamydia pneumoniae-induced foam cell formation requires MyD88-dependent and -independent signaling and is reciprocally modulated by liver X receptor activation

Chlamydia pneumoniae is detected by macrophages and other APCs via TLRs and can exacerbate developing atherosclerotic lesions, but how that occurs is not known. Liver X receptors (LXRs) centrally control reverse cholesterol transport, but also negatively modulate TLR-mediated inflammatory pathways. We isolated peritoneal macrophages from wild-type, TLR2, TLR3, TLR4, TLR2/4, MyD88, TRIF, MyD88/TRIF, and IFN regulatory factor 3 (IRF3) KO mice, treated them with live or UV-killed C. pneumoniae in the presence or absence of oxidized LDL, then measured foam cell formation. In some experiments, the synthetic LXR agonist GW3965 was added to macrophages infected with C. pneumoniae in the presence of oxidized LDL. Both live and UV-killed C. pneumoniae induced IRF3 activation and promoted foam cell formation in wild-type macrophages, whereas the genetic absence of TLR2, TLR4, MyD88, TRIF, or IRF3, but not TLR3, significantly reduced foam cell formation. C. pneumoniae-induced foam cell formation was significantly reduced by the LXR agonist GW3965, which in turn inhibited C. pneumoniae-induced IRF3 activation, suggesting a bidirectional cross-talk. We conclude that C. pneumoniae facilitates foam cell formation via activation of both MyD88-dependent and MyD88-independent (i.e., TRIF-dependent and IRF3-dependent) pathways downstream of TLR2 and TLR4 signaling and that TLR3 is not involved in this process. This mechanism could at least partly explain why infection with C. pneumoniae accelerates the development of atherosclerotic plaque and lends support to the proposal that LXR agonists might prove clinically useful in suppressing atherogenesis.     

Role of TLR2, TLR4, and MyD88 in murine ozone-induced airway hyperresponsiveness and neutrophilia.

Exposure to air pollutants such as ozone (O(3)) induces airway hyperresponsiveness (AHR) and airway inflammation. Toll-like receptors (TLR) are first-line effector molecules in innate immunity to infections and signal via adapter proteins, including myeloid differentiation factor-88 (MyD88). We investigated the sensing of ozone by TLR2, TLR4, and MyD88. Ozone induced AHR in wild-type (WT) C57BL/6 mice, but AHR was absent in TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice. Bronchoalveolar lavage neutrophilia induced by ozone was inhibited at 3 h but not at 24 h in TLR2(-/-) and TLR4(-/-) mice, while in MyD88(-/-) mice, this was inhibited at 24 h. We investigated the expression of inflammatory cytokines and TLR2, TLR4, and MyD88 in these mice. Ozone induced time-dependent increases in inflammatory gene expression of keratinocyte chemoattractant (KC) and IL-6 and of TLR2, TLR4, and MyD88 in WT mice. IL-6 and KC expression induced by ozone was inhibited in TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice. Expression of MyD88 was increased in TLR2(-/-) and TLR4(-/-) mice, while induction of TLR2 or TLR4 was reduced in TLR2(-/-) and TLR4(-/-) mice, respectively. TLR2 and TLR4 mediate AHR induced by oxidative stress such as ozone, while the adapter protein MyD88, but not TLR2 or TLR4, is important in mediating ozone-induced neutrophilia. TLR2 and TLR4 may also be important in regulating the speed of neutrophilic response. Therefore, ozone may induce murine AHR and neutrophilic inflammation through the activation of the Toll-like receptor pathway that may sense noninfectious stimuli such as oxidative stress.     

MyD88 functions as a negative regulator of TLR3/TRIF-induced corneal inflammation by inhibiting activation of c-Jun N-terminal kinase

The adaptor molecule MyD88 is necessary for responses to all Toll-like receptors except TLR3 and a subset of TLR4 signaling events, which are mediated by the adaptor molecule TRIF. To determine the role of TRIF in host inflammatory responses, corneal epithelium of C57BL/6, TLR3(-/-), TRIF(-/-), and MyD88(-/-) mice was abraded and stimulated with the synthetic TLR3 ligand poly(I:C). We found that poly(I:C) induced a pronounced cellular infiltration into the corneal stroma, which was TLR3- and TRIF-dependent. Unexpectedly, the inflammatory response was exacerbated in MyD88(-/-) mice, with enhanced neutrophil and F4/80(+) cell infiltration into the corneal stroma and elevated corneal haze, which is an indicator of loss of corneal transparency. To determine whether MyD88-dependent inhibition of TLR3/TRIF responses is a general phenomenon, we examined cytokine production by MyD88(-/-) bone marrow-derived macrophages; however, no significant difference was observed between MyD88(+/+) or MyD88(-/-) macrophages. In contrast, human corneal epithelial cells (HCECs) transfected with MyD88 small interfering RNA had significantly increased (2.5-fold) CCL5/RANTES production compared with control HCECs, demonstrating a negative regulatory role for MyD88 in TLR3/TRIF responses in these cells. Finally, knockdown of MyD88 in HCECs resulted in increased phosphorylation of c-Jun N-terminal kinase (JNK), but not p38, IRF-3, or NF-kappaB. Consistent with this finding, the JNK inhibitor SP600125, but not p38 inhibitor SB203580, ablated this response. Taken together, these findings demonstrate a novel JNK-dependent inhibitory role for MyD88 in the TLR3/TRIF activation pathway.     

Absence of MyD88 results in enhanced TLR3-dependent phosphorylation of IRF3 and increased IFN-β and RANTES production

Toll-like receptors are a group of pattern-recognition receptors that play a crucial role in "danger" recognition and induction of the innate immune response against bacterial and viral infections. TLR3 has emerged as a key sensor of viral dsRNA, resulting in the induction of the anti-viral molecule, IFN-β. Thus, a clearer understanding of the biological processes that modulate TLR3 signaling is essential. Previous studies have shown that the TLR adaptor, Mal/TIRAP, an activator of TLR4, inhibits TLR3-mediated IFN-β induction through a mechanism involving IRF7. In this study, we sought to investigate whether the TLR adaptor, MyD88, an activator of all TLRs except TLR3, has the ability to modulate TLR3 signaling. Although MyD88 does not significantly affect TLR3 ligand-induced TNF-α induction, MyD88 negatively regulates TLR3-, but not TLR4-, mediated IFN-β and RANTES production; this process is mechanistically distinct from that employed by Mal/TIRAP. We show that MyD88 inhibits IKKε-, but not TBK1-, induced activation of IRF3. In doing so, MyD88 curtails TLR3 ligand-induced IFN-β induction. The present study shows that while MyD88 activates all TLRs except TLR3, MyD88 also functions as a negative regulator of TLR3. Thus, MyD88 is essential in restricting TLR3 signaling, thereby protecting the host from unwanted immunopathologies associated with the excessive production of IFN-β. Our study offers a new role for MyD88 in restricting TLR3 signaling through a hitherto unknown mechanism whereby MyD88 specifically impairs IKKε-mediated induction of IRF3 and concomitant IFN-β and RANTES production.     

Interleukin-10 activates Toll-like receptor 4 and requires MyD88 for cardiomyocyte survival

Toll-like receptors (TLRs) are important in a variety of inflammatory diseases including acute cardiac disorders. TLR4 innate signaling regulates the synthesis of anti-inflammatory cytokine, interleukin-10 (IL-10) upon TLR4 agonists’ re-stimulation. Anti-apoptotic action of IL-10 in cardiac dysfunction is generally accepted but its protective mechanism through TLR4 is not yet understood. We studied the effect of IL-10 in the activation of TLR4 downstream signals leading to cardiomyocytes survival. IL-10 caused a significant increase in the expression of CD14, MyD88 and TLR4. TLR4 activation led to the translocation of the interferon regulatory factor 3 (IRF3) into the nucleus. Phosphorylation of IRF3 enhanced mRNA synthesis for IL-1β but not TNF-α and was elevated even after removal of IL-10 stimulation. Furthermore, degradation of inhibitory kappa B (IκB) kinase (Ikk) suggested that IκBβ was the main activating kinase for IRF3-regulated NF-κB activation and phosphorylation of p65. Phosphorylated NF-κB p65 was translocated into the nucleus. Concomitantly, an increase in Bcl-xL activity inhibited Bax and the proteolytic activity of caspase 3 as well as a decrease in PARP cleavage. An inhibition of MyD88, modulated the above listed responses to IL-10 as there was a decrease in TLR4 and IRF3 and an increase in TNF-α mRNA. This was associated with a decrease in NF-κB p65, Bcl-xL mRNA and protein levels as well as there was an activation of Bax and PARP cleavage independent of caspase 3 activation. These data in cardiomyocytes suggest that IL-10 induced anti-apoptotic signaling involves upregulation of TLR4 through MyD88 activation.     

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.     

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.     

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.     

TLR9 and MyD88 are crucial for the development of protective immunity to malaria

Effective resolution of malaria infection by avoiding pathogenesis requires regulated pro- to anti-inflammatory responses and the development of protective immunity. TLRs are known to be critical for initiating innate immune responses, but their roles in the regulation of immune responses and development of protective immunity to malaria remain poorly understood. In this study, using wild-type, TLR2(-/-), TLR4(-/-), TLR9(-/-), and MyD88(-/-) mice infected with Plasmodium yoelii, we show that TLR9 and MyD88 regulate pro/anti-inflammatory cytokines, Th1/Th2 development, and cellular and humoral responses. Dendritic cells from TLR9(-/-) and MyD88(-/-) mice produced significantly lower levels of proinflammatory cytokines and higher levels of anti-inflammatory cytokines than dendritic cells from wild-type mice. NK and CD8(+) T cells from TLR9(-/-) and MyD88(-/-) mice showed markedly impaired cytotoxic activity. Furthermore, mice deficient in TLR9 and MyD88 showed higher Th2-type and lower Th1-type IgGs. Consequently, TLR9(-/-) and MyD88(-/-) mice exhibited compromised ability to control parasitemia and were susceptible to death. Our data also show that TLR9 and MyD88 distinctively regulate immune responses to malaria infection. TLR9(-/-) but not MyD88(-/-) mice produced significant levels of both pro- and anti-inflammatory cytokines, including IL-1β and IL-18, by other TLRs/inflammasome- and/or IL-1R/IL-18R-mediated signaling. Thus, whereas MyD88(-/-) mice completely lacked cell-mediated immunity, TLR9(-/-) mice showed low levels of cell-mediated immunity and were slightly more resistant to malaria infection than MyD88(-/-) mice. Overall, our findings demonstrate that TLR9 and MyD88 play central roles in the immune regulation and development of protective immunity to malaria, and have implications in understanding immune responses to other pathogens.     

IFN-alphabeta-mediated inflammatory responses and antiviral defense in liver is TLR9-independent but MyD88-dependent during murine cytomegalovirus infection

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-alphabeta. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-alphabeta in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-alpha in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-alpha production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-alpha, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-alpha production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-alpha in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-gamma, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88(-/-) mice. Additionally, MyD88(-/-) but not TLR9(-/-) mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.     

A toll-like receptor 2 pathway regulates the Ppargc1a/b metabolic co-activators in mice with Staphylococcal aureus sepsis

Activation of the host antibacterial defenses by the toll-like receptors (TLR) also selectively activates energy-sensing and metabolic pathways, but the mechanisms are poorly understood. This includes the metabolic and mitochondrial biogenesis master co-activators, Ppargc1a (PGC-1α) and Ppargc1b (PGC-1β) in Staphylococcus aureus (S. aureus) sepsis. The expression of these genes in the liver is markedly attenuated inTLR2(-/-) mice and markedly accentuated in TLR4(-/-) mice compared with wild type (WT) mice. We sought to explain this difference by using specific TLR-pathway knockout mice to test the hypothesis that these co-activator genes are directly regulated through TLR2 signaling. By comparing their responses to S. aureus with WT mice, we found that MyD88-deficient and MAL-deficient mice expressed hepatic Ppargc1a and Ppargc1b normally, but that neither gene was activated in TRAM-deficient mice. Ppargc1a/b activation did not require NF-kβ, but did require an interferon response factor (IRF), because neither gene was activated in IRF-3/7 double-knockout mice in sepsis, but both were activated normally in Unc93b1-deficient (3d) mice. Nuclear IRF-7 levels in TLR2(-/-) and TLR4(-/-) mice decreased and increased respectively post-inoculation and IRF-7 DNA-binding at the Ppargc1a promoter was demonstrated by chromatin immunoprecipitation. Also, a TLR2-TLR4-TRAM native hepatic protein complex was detected by immunoprecipitation within 6 h of S. aureus inoculation that could support MyD88-independent signaling to Ppargc1a/b. Overall, these findings disclose a novel MyD88-independent pathway in S. aureus sepsis that links TLR2 and TLR4 signaling in innate immunity to Ppargc1a/b gene regulation in a critical metabolic organ, the liver, by means of TRAM, TRIF, and IRF-7.     

Redundant Toll-like receptor signaling in the pulmonary host response to Pseudomonas aeruginosa

Activation of pulmonary defenses against Pseudomonas aeruginosa requires myeloid differentiation factor 88 (MyD88), an adaptor for Toll-like receptor (TLR) signaling. To determine which TLRs mediate recognition of P. aeruginosa, we measured cytokine responses of bone marrow cells from wild-type mice and mice lacking TLR2 (TLR2(-/-)), TLR4 (TLR4(-/-)), TLR2 and TLR4 (TLR2/4(-/-)), or MyD88 (MyD88(-/-)) to wild-type P. aeruginosa and to fliC P. aeruginosa, which lacks the TLR5 ligand flagellin. Mice also were challenged with aerosolized bacteria to determine cytokine responses, lung inflammation, and bacterial clearance. TNF induction required MyD88 and was absent in TLR2/4(-/-) cells in response to fliC but not wild-type P. aeruginosa, whereas TLR2(-/-) cells exhibited augmented responses. In vivo, TLR4(-/-) mice responded to wild-type P. aeruginosa with reduced cytokine production and inflammation, but intact bacterial clearance, while TLR2(-/-) mice had partially impaired cytokine responses and delayed bacterial killing despite normal inflammation. When challenged with fliC, MyD88(-/-) mice failed to mount early cytokine and inflammatory responses or control bacterial replication, resulting in necrotizing lung injury and lethal disseminated infection. TLR4(-/-) and TLR2/4(-/-) mice responded to fliC infection with severely limited inflammatory and cytokine responses but intact bacterial clearance. TLR2(-/-) mice had partially reduced cytokine responses but augmented inflammation and preserved bacterial killing. These data indicate that TLR4- and flagellin-induced signals mediate most of the acute inflammatory response to Pseudomonas and that TLR2 has a counterregulatory role. However, MyD88-dependent pathways, in addition to those downstream of TLR2, TLR4, and TLR5, are required for pulmonary defense against P. aeruginosa.     

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.     

MyD88-dependent and -independent murine cytomegalovirus sensing for IFN-alpha release and initiation of immune responses in vivo

Antiviral immunity requires early and late mechanisms in which IFN-alpha and IL-12 play major roles. However, the initial events leading to their production remain largely unclear. Given the crucial role of TLR in innate recognition, we investigated their role in antiviral immunity in vivo. Upon murine CMV (MCMV) infection, both MyD88-/- and TLR9-/- mice were more susceptible and presented increased viral loads compared with C57BL/6, TLR2-/-, TLR3-/-, or TLR4-/- mice. However, in terms of resistance to infection, IFN-alpha production and in many other parameters of early inflammatory responses, the MyD88-/- mice showed a more defective response than TLR9-/- mice. In the absence of the TLR9/MyD88 signaling pathway, cytokine production was dramatically impaired with a complete abolition of bioactive IL-12p70 serum release contrasting with a high flexibility for IFN-alpha release, which is initially (36 h) plasmacytoid dendritic cell- and MyD88-dependent, and subsequently (44 h) PDC-, MyD88-independent and, most likely, TLR-independent. NK cells from MCMV-infected MyD88-/- and TLR9-/- mice displayed a severely impaired IFN-gamma production, yet retained enhanced cytotoxic activity. In addition, dendritic cell activation and critical inflammatory cell trafficking toward the liver were still effective. In the long term, except for isotype switching to MCMV-specific IgG1, the establishment of Ab responses was not significantly altered. Thus, our results demonstrate a critical requirement of TLR9 in the process of MCMV sensing to assure rapid antiviral responses, coordinated with other TLR-dependent and -independent events that are sufficient to establish adaptive immunity.