Mycobacterial infection in MyD88-deficient mice

MyD88 is an adaptor protein that plays a major role in TLR/IL-1 receptor family signaling. To understand the role of MyD88 in the development of murine tuberculosis in vivo, MyD88 knockout (KO) mice aerially were infected with Mycobacterium tuberculosis. Infected MyD88 mice were not highly susceptible to M. tuberculosis infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration which were larger than those in wild-type (WT) mice (P < 0.01). The pulmonary tissue levels of mRNA for iNOS and IL-18 were slightly lower, but levels of mRNA for IL-1 beta, IL-2, IL-4, IL-6, IL-10, IFN-gamma, and TGF-beta were higher in MyD88 KO mice. IFN-gamma, TNF-alpha, IL-1 beta, and IL-12 also were high in the sera of MyD88 KO mice. There were no statistically significant differences in the expression of TNF-alpha, IL-12, and ICAM-1 mRNA between MyD88 KO and WT mice. Thus, MyD88 deficiency did not influence the development of murine tuberculosis. NF-kappa B activity was similar in the alveolar macrophages from the lung tissues of MyD88 KO and WT mice. Also, there may be a TLR2-specific, MyD88-independent IL-1 receptor/TLR-mediated pathway to activate NF-kappa B in the host defense against mycobacterial infection.     

Dual signaling of MyD88 and TRIF is critical for maximal TLR4-induced dendritic cell maturation

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways, as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study, we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation, neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs, whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors, of which type I IFNs were one of the active components, played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types, MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore, signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs, with MyD88 playing a larger role than TRIF. In sum, in our experimental systems, TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation.     

MyD88 and IFN-alphabeta differentially control maturation of bystander but not Salmonella-associated dendritic cells or CD11cintCD11b+ cells during infection

The interface between dendritic cells (DCs) and T cells is critical to elicit effective immunity against pathogens. The maturation state of DCs determines the quality of the interaction and governs the type of response. DCs can be matured directly through activating Toll-like receptors (TLRs) or indirectly by cytokines. We explore the role of the TLR adaptor MyD88 on DC maturation during Salmonella infection. Using Salmonella expressing GFP, we also examine the phenotype and function of bacteria-associated DCs matured in the absence of bacteria-mediated TLR signalling. MyD88 was required for upregulation of CD80 on DCs during infection, whereas CD86 and CD40 were upregulated independently of MyD88, although requiring a higher bacterial burden in the MLN. MyD88-independent upregulation was mediated by IFN-αβ produced during infection. In infected MyD88^−/−IFN-αβR^−/− mice, which lack most bacteria-driven TLR signalling, indirect DC maturation was abolished. In contrast, DCs containing Salmonella upregulated co-stimulatory molecules independently of MyD88 and IFN-αβ, revealing a pathway of phenotypic maturation active in infected DCs. However, despite high co-stimulatory molecule expression, Salmonella -containing DCs from MyD88^−/− or MyD88^−/−IFN-αβR^−/− mice had a compromised capacity to activate T cells. Thus, bacterial stimulation of TLRs influences DC function at multiple levels that modulates their capacity to direct antibacterial immunity.     

Central role of MyD88-dependent dendritic cell maturation and proinflammatory cytokine production to control Brucella abortus infection

Brucella abortus is a facultative intracellular bacterium that infects humans and domestic animals. The enhanced susceptibility to virulent B. abortus observed in MyD88 knockout (KO) mice led us to investigate the mechanisms involved in MyD88-dependent immune responses. First, we defined the role of MyD88 in dendritic cell (DC) maturation. In vitro as well as in vivo, B. abortus-exposed MyD88 KO DCs displayed a significant impairment on maturation as observed by expression of CD40, CD86, and MHC class II on CD11c+ cells. In addition, IL-12 and TNF-alpha production was totally abrogated in MyD88 KO DCs and macrophages. Furthermore, B. abortus-induced IL-12 production was found to be dependent on TLR2 in DC, but independent on TLR2 and TLR4 in macrophages. Additionally, we investigated the role of exogenous IL-12 and TNF-alpha administration on MyD88 KO control of B. abortus infection. Importantly, IL-12, but not TNF-alpha, was able to partially rescue host susceptibility in MyD88 KO-infected animals. Furthermore, we demonstrated the role played by TLR9 during virulent B. abortus infection. TLR9 KO-infected mice showed 1 log Brucella CFU higher than wild-type mice. Macrophages and DC from TLR9 KO mice showed reduced IL-12 and unaltered TNF-alpha production when these cells were stimulated with Brucella. Together, these results suggest that susceptibility of MyD88 KO mice to B. abortus is due to impaired DC maturation and lack of IL-12 synthesis. Additionally, DC activation during Brucella infection plays an important regulatory role by stimulating and programming T cells to produce IFN-gamma.     

MyD88-dependent TLR1/2 signals educate dendritic cells with gut-specific imprinting properties

Gut-associated dendritic cells (DC) synthesize all-trans retinoic acid, which is required for inducing gut-tropic lymphocytes. Gut-associated DC from MyD88(-/-) mice, which lack most TLR signals, expressed low levels of retinal dehydrogenases (critical enzymes for all-trans retinoic acid biosynthesis) and were significantly impaired in their ability to induce gut-homing T cells. Pretreatment of extraintestinal DC with a TLR1/2 agonist was sufficient to induce retinal dehydrogenases and to confer these DC with the capacity to induce gut-homing lymphocytes via a mechanism dependent on MyD88 and JNK/MAPK. Moreover, gut-associated DC from TLR2(-/-) mice, or from mice in which JNK was pharmacologically blocked, were impaired in their education to imprint gut-homing T cells, which correlated with a decreased induction of gut-tropic T cells in TLR2(-/-) mice upon immunization. Thus, MyD88-dependent TLR2 signals are necessary and sufficient to educate DC with gut-specific imprinting properties and contribute in vivo to the generation of gut-tropic T cells.     

Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection

Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns. We previously showed that TLR2 recognizes Gram-positive bacterial components whereas TLR4 recognizes LPS, a component of Gram-negative bacteria. MyD88 is shown to be an adaptor molecule essential for TLR family signaling. To investigate the role of TLR family in host defense against Gram-positive bacteria, we infected TLR2- and MyD88-deficient mice with Staphylococcus aureus. Both TLR2- and MyD88-deficient mice were highly susceptible to S. aureus infection, with more enhanced susceptibility in MyD88-deficient mice. Peritoneal macrophages from MyD88-deficient mice did not produce any detectable levels of cytokines in response to S. aureus. In contrast, TLR2-deficient macrophages produced reduced, but significant, levels of the cytokines, and TLR4-deficient macrophages produced the same amounts as wild-type cells, indicating that S. aureus is recognized not only by TLR2, but also by other TLR family members except for TLR4.     

MyD88-mediated instructive signals in dendritic cells regulate pulmonary immune responses during respiratory virus infection

Respiratory syncytial virus (RSV) is the leading cause of respiratory disease in infants worldwide. The induction of innate immunity and the establishment of adaptive immune responses are influenced by the recognition of pathogen-associated molecular patterns by TLRs. One of the primary pathways for TLR activation is by MyD88 adapter protein signaling. The present studies indicate that MyD88 deficiency profoundly impacts the pulmonary environment in RSV-infected mice characterized by the accumulation of eosinophils and augmented mucus production. Although there was little difference in CD4 T cell accumulation, there was also a significant decrease in conventional dendritic cells recruitment to the lungs of MyD88(-/-) mice. The exacerbation of RSV pathophysiology in MyD88(-/-) mice was associated with an enhanced Th2 cytokine profile that contributed to an inappropriate immune response. Furthermore, bone marrow-derived dendritic cells (BMDC) isolated from MyD88(-/-) mice were incapable of producing two important Th1 instructive signals, IL-12 and delta-like4, upon RSV infection. Although MyD88(-/-) BMDCs infected with RSV did up-regulate costimulatory molecules, they did not up-regulate class II as efficiently and stimulated less IFN-gamma from CD4(+) T cells in vitro compared with wild-type BMDCs. Finally, adoptive transfer of C57BL/6 BMDCs into MyD88(-/-) mice reconstituted Th1 immune responses in vivo, whereas transfer of MyD88(-/-) BMDCs into wild-type mice skewed the RSV responses toward a Th2 phenotype. Taken together, our data indicate that MyD88-mediated pathways are essential for the least pathogenic responses to this viral pathogen through the regulation of important Th1-associated instructive signals.     

Lymphocytoid choriomeningitis virus activates plasmacytoid dendritic cells and induces a cytotoxic T-cell response via MyD88

Toll-like receptors (TLRs) and retinoic acid-inducible gene I-like helicases (RLHs) are two major machineries recognizing RNA virus infection of innate immune cells. Intracellular signaling for TLRs and RLHs is mediated by their cytoplasmic adaptors, i.e., MyD88 or TRIF and IPS-1, respectively. In the present study, we investigated the contributions of TLRs and RLHs to the cytotoxic T-lymphocyte (CTL) response by using lymphocytoid choriomeningitis virus (LCMV) as a model virus. The generation of virus-specific cytotoxic T lymphocytes was critically dependent on MyD88 but not on IPS-1. Type I interferons (IFNs) are known to be important for the development of the CTL response to LCMV infection. Serum levels of type I IFNs and proinflammatory cytokines were mainly dependent on the presence of MyD88, although IPS-1^−/− mice showed a decrease in IFN-α levels but not in IFN-β and proinflammatory cytokine levels. Analysis of Ifna6^+/GFP reporter mice revealed that plasmacytoid dendritic cells (DCs) are the major source of IFN-α in LCMV infection. MyD88^−/− mice were highly susceptible to LCMV infection in vivo. These results suggest that recognition of LCMV by plasmacytoid DCs via TLRs is responsible for the production of type I IFNs in vivo. Furthermore, the activation of a MyD88-dependent innate mechanism induces a CTL response, which eventually leads to virus elimination.     

Critical role of TRIF and MyD88 in Mycobacterium tuberculosis Hsp70-mediated activation of dendritic cells

As a potent immune regulator, heat shock protein 70 derived from Mycobacterium tuberculosis (Mtb Hsp70) has adjuvant effect and activates immune cells such as macrophages and dendritic cells (DCs). Although Toll-like receptors (TLRs) are known to involve in DCs activation by Mtb Hsp70, there is still a controversy and the underlying mechanism is not well understood. In this study, we examined whether TRIF and MyD88, the core adaptor molecules for TLRs signaling, regulate Mtb Hsp70-induced DCs activation. Although Mtb Hsp70 produced substantial level of cytokines (IL-6, IL-12p40, and TNF-α) in TRIF-deficient DCs in a dose-dependent manner, each level was significantly lower than that in WT cells. The cytokines production was almost abolished in MyD88-deficient DCs. Consistent with cytokine results, Mtb Hsp70-induced activation of NF-κB and MAPKs was also impaired in both TRIF- and MyD88-deficient DCs, as compared with WT cells. Inhibitor assay revealed that NF-κB, ERK, and JNK, but not p38, regulate Mtb Hsp70-induced production of cytokines. In addition, the up-regulation of co-stimulatory molecules and MHC class II was mostly TRIF-dependent in DCs in response Mtb Hsp70, whereas MyD88 was only partially involved. Finally, mixed leukocytes reaction (MLR) assay revealed that both TRIF and MyD88 are critical for DCs ability promoted by Mtb Hsp70 to differentiate naïve T cells into effector T cells of producing IFN-γ. Our findings suggest that both TRIF and MyD88 are essential for the activation and maturation of DCs in response to Mtb Hsp70.     

MyD88-deficient bone marrow cells accelerate onset and reduce survival in a mouse model of amyotrophic lateral sclerosis

Increasing evidence suggests that neurotoxicity of secreted superoxide dismutase 1 (SOD1) mutants is associated with amyotrophic lateral sclerosis (ALS). We show here that mutant SOD1 protein activates microglia via a myeloid differentiation factor 88 (MyD88)–dependent pathway. This inflammatory response is also associated with a marked recruitment of bone marrow–derived microglia (BMDM) in the central nervous system. We then generated chimeric SOD1^G37R and SOD1^G93A mice by transplantation of bone marrow (BM) cells from MyD88-deficient or green fluorescent protein (GFP)–expressing mice. SOD1^G37R mice receiving MyD88^−/− BM cells exhibit a significantly earlier disease onset and shorter lifespan compared with mice transplanted with control GFP cells. This compelling beneficial effect of MyD88-competent BMDM is a previously unrecognized natural innate immune mechanism of neuroprotection in a mouse model of late-onset motor neuron disease.     

Neisserial porin-induced dendritic cell activation is MyD88 and TLR2 dependent

Neisserial porins have been shown to act as B cell mitogens and immune adjuvants. PorA and PorB are the major outer membrane porin proteins of the human pathogen Neisseria meningitidis. We have shown that the mechanism of the immunopotentiating capability of porin involves up-regulation of the T cell costimulatory ligand, CD86. Due to neisserial porin's ability to activate B cells and potentiate immune responses, we hypothesized that porin also employs the potent immune stimulatory function of dendritic cells (DC). We examined the ability of purified N. meningitidis PorB to induce maturation of murine splenic and bone marrow-derived DC. PorB treatment induced DC maturation, as demonstrated by increased expression of CD86 and class I and II MHC molecules. In addition, PorB not only enhanced the allostimulatory activity of DC, but also augmented the ability of DC to stimulate T cells in an Ag-specific manner. PorB-matured DC secreted the inflammatory cytokine IL-6, which may have implications for the adjuvant property of porin. Induction of IL-6 by PorB is also significant because IL-6 is one of a number of cytokines produced during infection with N. meningitidis and may be involved in the inflammatory process observed during infection and disease. We previously demonstrated the requirement of MyD88 and TLR2 for PorB-induced B cell activation. In the present study, MyD88 and TLR2 were also essential for PorB-induced DC activation. This work is significant for elucidating the mechanism(s) of neisserial porin's immune stimulatory activity.     

Uropathogenic Escherichia coli block MyD88-dependent and activate MyD88-independent signaling pathways in rat testicular cells

Uropathogenic Escherichia coli (UPEC) is the most common etiological cause of urogenital tract infections and represents a considerable cause of immunological male infertility. We examined TLR 1-11 expression profiles in testicular cells and the functional response to infection with UPEC. All testicular cell types expressed mRNAs for at least two TLRs and, in particular, synthesis of TLR4 was induced in testicular macrophages (TM), Sertoli cells (SC), peritubular cells (PTC), and peritoneal macrophages (PM) after UPEC exposure. Even though MyD88-dependent pathways were activated as exemplified by phosphorylation of mitogen-activated protein kinases in TM, SC, PTC, and PM and by the degradation of IkappaBalpha and the nuclear translocation of NF-kappaB in PTC and PM, treatment with UPEC did not result in secretion of the proinflammatory cytokines IL-1alpha, IL-6, and TNF-alpha in any of the investigated cells. Moreover, stimulated production of these cytokines by nonpathogenic commensal E. coli or LPS in PM was completely abolished after coincubation with UPEC. Instead, in SC, PTC, TM, and PM, UPEC exposure resulted in activation of MyD88-independent signaling as documented by nuclear transfer of IFN-related factor-3 and elevated expression of type I IFNs alpha and beta, IFN-gamma-inducible protein 10, MCP-1, and RANTES. We conclude that in this in vitro model UPEC can actively suppress MyD88-dependent signaling at different levels to prevent proinflammatory cytokine secretion by testicular cells. Thus, testicular innate immune defense is shifted to an antiviral-like MyD88-independent response.     

TLR ligands can activate dendritic cells to provide a MyD88-dependent negative signal for Th2 cell development

During infection, CD4(+) Th cell responses polarize to become primarily Th1 or Th2. Th1 cells, which make IFN-gamma, are crucial for immunity to many bacterial and protozoal infections, whereas Th2 cells, which make IL-4, IL-5, and IL-13, are important for resistance to helminth infections. Polarized Th1 responses are induced by dendritic cells (DCs), which respond to pathogen-derived TLR ligands to produce IL-12 and related cytokines that are instrumental in Th1 cell outgrowth, and coordinately process and present Ag in the context of MHC class II to activate naive Th cells. In this study we show that in addition to providing positive signals for Th1 cell development, mouse DCs activated by TLR engagement can also provide a potent negative signal that prevents the development of Th2 cells. Production of this signal, which is not IL-12, IL-18, IL-23, IL-27, or IFN-gamma and is not provided via Th1 cells, is dependent upon a MyD88-dependent, TNF receptor-associated factor-6-independent signaling pathway in DCs. The signal is released from DCs in response to activation via TLR ligands and exerts an effect directly on Th cells rather than through a third-party cell. Our findings indicate that DCs can provide potent negative as well as positive instruction for Th response polarization, and that these instructional signals are distinct and independent.     

Atg5 regulates formation of MyD88 condensed structures and MyD88-dependent signal transduction

MyD88 is known as an essential adaptor protein for Toll-like receptors (TLRs). Previous studies have shown that transfected MyD88 forms condensed structures in the cytoplasm. However, upon TLR stimulation, there is little formation of endogenous MyD88 condensed structures. Thus, the formation of MyD88 condensed structures is tightly suppressed, but the mechanism and significance of this suppression are currently unknown. Here we show that Atg5, a key regulatory protein of autophagy, inhibits the formation of MyD88 condensed structures. We found that endogenous MyD88 had already formed condensed structures in Atg5-deficient cells and that the formation of condensed structures was further enhanced by TLR stimulation. This suppressive effect of Atg5 may not be associated with autophagic processes because MyD88 itself was not degraded and because TLR stimulation did not induce LC3 punctate formation and LC3 conversion. Immunoprecipitation analysis revealed that Atg5 could interact with MyD88. Furthermore, Atg5 deficiency increased formation of the MyD88–TRAF6 signaling complex induced by TLR stimulation, and it enhanced activation of NF-κB signaling but not MAPKs and Akt. These findings indicate that Atg5 regulates the formation of MyD88 condensed structures through association with MyD88 and eventually exerts a modulatory effect on MyD88-dependent signaling.     

鸡髓样分化因子88的原核表达及单克隆抗体制备

目的:克隆、表达、纯化鸡髓样分化因子88(MyD88),制备其单克隆抗体。方法:从脾脏cDNA中扩增857bp的MyD88基因片段,插入pMAL-c5X表达载体,转化大肠杆菌BL21(DE3)获得表达菌株,IPTG诱导表达,用SDS-PAGE分析MBP(麦芽糖结合蛋白)-MyD88重组融合蛋白的表达,切胶纯化目的蛋白;免疫BALB/c小鼠,制备针对MyD88的单克隆抗体,Western印迹检测抗体特异性,制备腹水并进行抗体亚型鉴定和效价测定。结果:构建了鸡MyD88原核表达载体pMAL-MyD88,并在大肠杆菌中获得高表达,目的蛋白以可溶性和包涵体两种形式存在;建立了3株抗鸡MyD88单克隆抗体细胞株,制备了腹水,亚型分别为IgG1、IgG1和IgG2a,轻链均为κ,腹水抗体的效价均为1∶2×105。结论:在原核表达系统中表达、纯化了重组鸡MyD88,制备了针对鸡MyD88的单克隆抗体,为后续的MyD88定量和功能研究奠定了基础。     

MyD88-dependent activation of B220-CD11b+LY-6C+ dendritic cells during Brucella melitensis infection

IFN-gamma is a key cytokine controlling Brucella infection. One of its major function is the stimulation of Brucella-killing effector mechanisms, such as inducible NO synthase (iNOS)/NOS2 activity, in phagocytic cells. In this study, an attempt to identify the main cellular components of the immune response induced by Brucella melitensis in vivo is made. IFN-gamma and iNOS protein were analyzed intracellularly using flow cytometry in chronically infected mice. Although TCRbeta(+)CD4(+) cells were the predominant source of IFN-gamma in the spleen, we also identified CD11b(+)LY-6C(+)LY-6G(-)MHC-II(+) cells as the main iNOS-producing cells in the spleen and the peritoneal cavity. These cells appear similar to inflammatory dendritic cells recently described in the mouse model of Listeria monocytogenes infection and human psoriasis: the TNF/iNOS-producing dendritic cells. Using genetically deficient mice, we demonstrated that the induction of iNOS and IFN-gamma-producing cells due to Brucella infection required TLR4 and TLR9 stimulation coupled to Myd88-dependent signaling pathways. The unique role of MyD88 was confirmed by the lack of impact of Toll-IL-1R domain-containing adaptor inducing IFN-beta deficiency. The reduction of IFN-gamma(+) and iNOS(+) cell frequency observed in MyD88-, TLR4-, and TLR9-deficient mice correlated with a proportional lack of Brucella growth control. Taken together, our results provide new insight into how immune responses fight Brucella infection.     

Autoimmunity stimulated by adoptively transferred dendritic cells is initiated by both alphabeta and gammadelta T cells but does not require MyD88 signaling

Vaccination of nonautoimmune prone mice with syngeneic dendritic cells (DC) readily induces anti-DNA autoantibodies but does not trigger systemic disease. We observed that anti-DNA autoantibody generation absolutely required alphabeta T cells and that gammadelta T cells also contributed to the response, but that regulatory T cells restrained autoantibody production. Although both NZB/W F(1) mice and DC vaccinated C57/BL6 mice produced autoantibodies against dsDNA, vaccinated mice had higher levels of Abs against H1 histone and lower levels of antinucleosome Abs than NZB/W F(1) mice. Despite a 100-fold increase in IL-12 and Th1 skewing to a foreign Ag, OVA, synergistic TLR activation of DC in vitro failed to augment anti-DNA Abs or promote class switching beyond that induced by LPS alone. TLR stimulation was not absolutely required for the initial loss of B cell tolerance because anti-DNA levels were similar when wild-type (WT) or MyD88-deficient DC were used for vaccination or WT and MyD88-deficient recipients were vaccinated with WT DC. In contrast, systemic administration of LPS, augmented anti-DNA Ab levels and promoted class switching, and this response was dependent on donor DC signaling via MyD88. LPS also augmented responses in the MyD88-deficient recipients, suggesting that LPS likely exerts its effects on both transferred DC and host B cells in vivo. These results indicate that both the alphabeta and gammadelta subsets are necessary for promoting autoantibody production by DC vaccination, and that although TLR/MyD88 signaling is not absolutely required for initiation, this pathway does promote augmentation, and Th1-mediated skewing, of anti-DNA autoantibodies.     

TLR4 and Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta, but not MyD88, regulate Escherichia coli-induced dendritic cell maturation and apoptosis in vivo

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2(-/-), TLR4(-/-) and TIR domain-containing adapter-inducing IFN-beta (TRIF)(-/-) mice. In contrast, DC apoptosis is reduced only in TLR4(-/-) and TRIF(-/-) mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.