MyD88, but not toll-like receptors 4 and 2, is required for efficient clearance of Brucella abortus

It is not clear how the host initially recognizes and responds to infection by gram-negative pathogenic Brucella spp. It was previously shown (D. S. Weiss, B. Raupach, K. Takeda, S. Akira, and A. Zychlinsky, J. Immunol. 172:4463-4469, 2004) that the early macrophage response against gram-negative bacteria is mediated by Toll-like receptor 4 (TLR4), which signals in response to lipopolysaccharide (LPS). Brucella, however, has a noncanonical LPS which does not have potent immunostimulatory activity. We evaluated the kinetics of TLR4 activation and the cytokine response in murine macrophages after Brucella infection. We found that during infection of macrophages, Brucella avoids activation of TLR4 at 6 h but activates TLR4, TLR2, and myeloid differentiation factor 88 (MyD88) at 24 h postinfection. Interestingly, even though its activation is delayed, MyD88 is important for host defense against Brucella infection in vivo, since MyD88(-/-) mice do not clear the bacteria as efficiently as wild-type, TLR4(-/-), TLR2(-/-), or TLR4/TLR2(-/-) mice.     

MyD88-deficient mice display a profound loss in resistance to Mycobacterium tuberculosis associated with partially impaired Th1 cytokine and nitric oxide synthase 2 expression

Mycobacterium tuberculosis possesses agonists for several Toll-like receptors (TLRs), yet mice with single TLR deletions are resistant to acute tuberculosis. MyD88(-/-) mice were used to examine whether TLRs play any role in protection against aerogenic M. tuberculosis H37Rv infection. MyD88(-/-) mice failed to control mycobacterial replication and rapidly succumbed. Moreover, expressions of interleukin 12, tumor necrosis factor alpha, gamma interferon, and nitric oxide synthase 2 were markedly decreased in the knockout animals. These results argue that resistance to M. tuberculosis must depend on MyD88-dependent signals mediated by an as-yet-undetermined TLR or a combination of TLRs.     

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.     

Toll-like receptors participate in macrophage activation and intracellular control of Leishmania (Viannia) panamensis

Toll-like receptors (TLRs) play a central role in macrophage activation and control of parasitic infections. Their contribution to the outcome of Leishmania infection is just beginning to be deciphered. We examined the interaction of Leishmania panamensis with TLRs in the activation of host macrophages. L. panamensis infection resulted in upregulation of TLR1, TLR2, TLR3, and TLR4 expression and induced tumor necrosis factor alpha (TNF-α) secretion by human primary macrophages at comparable levels and kinetics to those of specific TLR ligands. The TLR dependence of the host cell response was substantiated by the absence of TNF-α production in MyD88/TRIF(-/-) murine bone marrow-derived macrophages and mouse macrophage cell lines in response to promastigotes and amastigotes. Systematic screening of TLR-deficient macrophages revealed that TNF-α production was completely abrogated in TLR4(-/-) macrophages, consistent with the increased intracellular parasite survival at early time points of infection. TNF-α secretion was significantly reduced in macrophages lacking endosomal TLRs but was unaltered by a lack of TLR2 or MD-2. Together, these findings support the participation of TLR4 and endosomal TLRs in the activation of host macrophages by L. panamensis and in the early control of infection.     

Normal development of the gut-associated lymphoid tissue except Peyer's patch in MyD88-deficient mice

MyD88 is a key adaptor molecule for signalling via Toll-like receptors (TLRs) and the response to gut commensal microbes. To investigate the role of TLRs/MyD88 pathway in the development of the gut-associated lymphoid tissue (GALT), we examined the development of Peyer's patches (PPs) and cryptopatch (CP), and also one of effector compartment, intraepithelial lymphocyte (IEL) in MyD88-/-, TLR2-/- and TLR4-/- mice. In MyD88-/- mice, the organogenesis of PPs was not disturbed. However, PPs in 2-week-old MyD88-/- mice were significantly smaller than those in MyD88+/- mice. Also, in 2-week-old TLR4-/-, but not TLR2-/- mice, PPs did not develop rapidly. The development of PPs in MyD88-/- and TLR4-/- mice was completely recovered in 10 weeks. PP cells from MyD88-/- mice showed significant decrease in proliferation when stimulated with lipopolysaccharide. The development of CP and IEL was also normal in 10-week-old MyD88-/- mice. These results suggest that the TLRs/MyD88 pathway might be involved in the development of PPs only at early postnatal stage, and TLRs/MyD88-independent signalling is critically involved in the development of GALT in adult mice.     

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

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

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

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

MyD88 signaling is required for efficient innate and adaptive immune responses to Paracoccidioides brasiliensis infection

The mechanisms that govern the initial interaction between Paracoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88(-/-) C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells. MyD88(-/-) macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88(-/-) mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88(-/-) mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88(-/-) mice; the lesions replaced extensive areas of several organs. Therefore, MyD88(-/-) mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses against P. brasiliensis.     

MyD88 is critical for the development of innate and adaptive immunity during acute lymphocytic choriomeningitis virus infection

We investigated the roles of Toll-like receptor 2 (TLR2) and myeloid differentiation factor 88 (MyD88) in the course of a lymphocytic choriomeningitis virus (LCMV) infection and revealed the following: (i) studies of transfected cells and murine peritoneal macrophages demonstrated that TLR2 and MyD88 are essential for the initial pro-inflammatory cytokine response (human IL-8, mouse IL-6) to LCMV; (ii) TLR2 knockout (KO) mice and MyD88 KO mice challenged with LCMV produced less IL-6 and monocyte chemotactic protein-1 in the serum than wild-type mice; (iii) in contrast to inflammatory cytokines, the production of type 1 IFN (IFN-alpha) in response to LCMV was MyD88 independent; (iv) MyD88 plays an essential role in antiviral CD8(+) T cell responses, CD8(+) T cells in MyD88 KO mice were defective in their expression of intracellular antiviral cytokines; and (v) the failure of MyD88 KO mice to activate CD8(+) T cells was accompanied by persistent viral infection in MyD88 KO mice. We demonstrate that TLR-mediated responses are important in the innate immune response to LCMV and that MyD88 is essential for the control of the LCMV infection and the maturation/activation of virus-specific CD8(+) T cells.     

Glycopeptidolipids from Mycobacterium avium promote macrophage activation in a TLR2- and MyD88-dependent manner

The Toll-like receptors (TLRs) are key components in the immune response against numerous pathogens. Previous studies have indicated that TLR2 plays an essential role in promoting immune responses against mycobacterial infections. Prior work has also shown that mice deficient in TLR2 are more susceptible to infection by Mycobacterium tuberculosis, Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium avium. Therefore, it is important to define the molecules expressed by pathogenic mycobacteria, which bind the various TLRs. Although a number of TLR agonists have been characterized for M. tuberculosis, no specific TLR ligand has been identified in M. avium. We have found that glycopeptidolipids (GPLs), which are highly expressed surface molecules on M. avium, can stimulate the nuclear factor-kappaB pathway as well as mitogen-activated protein kinase p38 and Jun N-terminal kinase activation and production of proinflammatory cytokines when added to murine bone marrow-derived macrophages. This stimulation was dependent on TLR2 and myeloid differentiation primary-response protein 88 (MyD88) but not TLR4. M. avium express apolar and serovar-specific (ss)GPLs, and it is the expression of the latter that determines the serotype of a particular M. avium strain. It is interesting that the ssGPLs activated macrophages in a TLR2- and MyD88-dependent manner, and no macrophage activation was observed when using apolar GPLs. ssGPLs also differed in their ability to activate macrophages with Serovars 1 and 2 stimulating inhibitor of kappaB p38 and phosphorylation and tumor necrosis factor alpha (TNF-alpha) secretion, while Serovar 4 failed to stimulate p38 activation and TNF-alpha production. Our studies indicate that ssGPLs can function as TLR2 agonists and promote macrophage activation in a MyD88-dependent pathway.     

MyDths and un-TOLLed truths: sensor, instructive and effector immunity to tuberculosis

Controversy exists concerning the role of Toll-like receptors and MyD88 in immunity to tuberculosis (TB). This mini-review argues that (i) Toll-like receptors are not essential for an effective immune response against TB, (ii) MyD88 is essential, but not because it transduces signals from TLRs, (iii) adaptive immunity to TB is largely TLR/MyD88-independent. Some of the discrepancies may be resolved by cogent attribution of distinct immune functions to the individual components of the TLR/MyD88 system. In mice, TLRs and MyD88 are fully dispensable in sensing Mtb infection and instructing T cell-mediated adaptive immunity, and while TLRs are also redundant during macrophage effector immunity, MyD88 is essential for efficient killing of mycobacteria. This distinction should help to molecularly pinpoint the MyD88-dependent, yet TLR-independent critical mechanisms of macrophage activation involved in intracellular growth restriction of Mtb. Disrupted IL-1R and/or IFN-gamma signaling pathways likely play a much more prominent role in explaining the exquisite susceptibility of MyD88-deficient mice to TB than the function of MyD88 as a TLR adaptor.     

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.     

TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses through signaling pathways mediated by Toll-interleukin 1 receptor (TIR) domain-containing adaptors such as MyD88, TIRAP and TRIF. MyD88 is a common adaptor that is essential for proinflammatory cytokine production, whereas TRIF mediates the MyD88-independent pathway from TLR3 and TLR4. Here we have identified a fourth TIR domain-containing adaptor, TRIF-related adaptor molecule (TRAM), and analyzed its physiological function by gene targeting. TRAM-deficient mice showed defects in cytokine production in response to the TLR4 ligand, but not to other TLR ligands. TLR4- but not TLR3-mediated MyD88-independent interferon-beta production and activation of signaling cascades were abolished in TRAM-deficient cells. Thus, TRAM provides specificity for the MyD88-independent component of TLR4 signaling.     

Distinct roles for MyD88 and Toll-like receptors 2, 5, and 9 in phagocytosis of Borrelia burgdorferi and cytokine induction

The contribution of Toll-like receptors (TLRs) to phagocytosis of Borrelia burgdorferi has not been extensively studied. We show that bone marrow-derived macrophages (BMDM) from MyD88(-/-) mice or Raw cells transfected with a dominant-negative MyD88 were unable to efficiently internalize B. burgdorferi. Knockouts of TLR2 and TLR9 or knockdown of TLR5 by small interfering RNA produced no defects in phagocytosis of B. burgdorferi. Production of inflammatory cytokines was greatly diminished in MyD88(-/-) BMDM but only partially affected in TLR2(-/-) BMDM or knockdown of TLR5 and unaffected in TLR9(-/-) BMDM. Cytochalasin D reduced cytokine induction, but not to the level of the MyD88(-/-) BMDM. Addition of cytochalasin D to TLR2(-/-) BMDM inhibited inflammatory responses to B. burgdorferi to the level of MyD88(-/-) BMDM, consistent with a role for TLR2 in both recognition of extracellular products and lysosomal sampling by TLR2 after processing of the organism. Cytochalasin D had no impact on cytokine production in cells undergoing TLR5 knockdown. These results suggest that MyD88, but not TLR2, TLR5, and TLR9, is important for the uptake of B. burgdorferi and that MyD88 affects inflammatory responses through both its effects on phagocytosis and its role in transducing signals from TLR2 and TLR5.     

The Toll-like receptor adaptor proteins MyD88 and Mal/TIRAP contribute to the inflammatory and destructive processes in a human model of rheumatoid arthritis

The widespread distribution of Toll-like receptors (TLRs) and their ligands raises the question whether they contribute to the production of inflammatory and tissue destructive molecules in rheumatoid arthritis (RA). We examined the expression and function of TLR2 and TLR4 and their downstream signaling adaptors MyD88 and Mal/TIRAP in synovial membrane cultures from RA tissue. Both TLR2 and TLR4 were detected by flow cytometry, and stimulation with TLR2 and TLR4 ligands augmented the spontaneous production of tumor necrosis factor-alpha, interleukin (IL)-6, and IL-8, indicating that TLR2 and TLR4 are functional in these cultures. In addition, overexpression of dominant-negative forms of MyD88 and Mal/TIRAP significantly down-regulated the spontaneous production of cytokines tumor necrosis factor-alpha, IL-6, and vascular endothelial growth factor, and enzymes MMP-1, MMP-2, MMP-3, and MMP-13 in RA synovial membrane cell cultures. Because TLR2 and TLR4 require both MyD88 and Mal/TIRAP for signaling, this study suggests that TLR function may regulate the expression of these factors in the RA synovium. Conditioned media from synovial membrane cell cultures stimulated human macrophages in a MyD88- and Mal-dependent manner, suggesting the release of a TLR ligand(s) from these cells. Thus, TLRs not only protect against infection but may also promote the inflammatory and destructive process in RA.     

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.     

Essential role for TLR4 and MyD88 in the development of chronic intestinal nematode infection

Expulsion of the gastrointestinal nematode Trichuris muris is mediated by a T helper 2 type response involving IL-4 and IL-13. Here we show that Th1 response-associated susceptibility is dependent on activation signals mediated by MyD88 and Toll-like receptor 4 (TLR4). TLR4- and MyD88-deficient mice are highly resistant to chronic T. muris infection and develop strong antigen-specific Th2 responses in mucosa-associated lymphoid tissues. Hence, TLR4 and MyD88 are involved not only in the development of pro-inflammatory responses against bacterial pathogens but are also crucially involved in responses against multicellular organisms such as helminths. These results provide the first demonstration of the critical role of TLR4 and MyD88 in bridging the innate and acquired immune response during gastrointestinal nematode infection.     

Pathological role of Toll-like receptor signaling in cerebral malaria

Toll-like receptors (TLRs) recognize malaria parasites or their metabolites; however, their physiological roles in malaria infection in vivo are not fully understood. Here, we show that myeloid differentiation primary response gene 88 (MyD88)-dependent TLR signaling mediates brain pathogenesis of severe malaria infection, namely cerebral malaria (CM). A significant number of MyD88-, but not TIR domain containing adaptor-inducing IFN-beta (TRIF)-deficient or wild-type (WT) mice survived CM caused by Plasmodium berghei ANKA (PbA) infection. Although systemic parasitemia was comparable, sequestration of parasite and hemozoin load in the brain blood vessels was significantly lower in MyD88-deficient mice compared with those in TRIF-deficient or WT mice. Moreover, brain-specific pathological changes were associated with MyD88-dependent infiltration of CD8+, CCR5+ T cells and CD11c+ dendritic cells, including CD11c+, NK1.1+ and B220+ cells, and up-regulation of genes such as Granzyme B, Lipocalin 2, Ccl3 and Ccr5. Further studies using mice lacking various TLRs suggest that TLR2 and TLR9, but not TLR4, 5 and 7, were involved in CM. These results strongly suggest that TLR2- and/or TLR9-mediated, MyD88-dependent brain pathogenesis may play a critical role in CM, the lethal complication during PbA infection.