IL-4-dependent Th2 collateral priming to inhaled antigens independent of Toll-like receptor 4 and myeloid differentiation factor 88

Allergic asthma is an inflammatory lung disease thought to be initiated and directed by type 2 helper T cells responding to environmental Ags. The mechanisms by which allergens induce Th2-adaptive immune responses are not well understood, although it is now clear that innate immune signals are required to promote DC activation and Th2 sensitization to inhaled proteins. However, the effect of ongoing Th2 inflammation, as seen in chronic asthma, on naive lymphocyte activation has not been explored. It has been noted that patients with atopic disorders demonstrate an increased risk of developing sensitivities to new allergens. This suggests that signals from an adaptive immune response may facilitate sensitization to new Ags. We used a Th2-adoptive transfer murine model of asthma to identify a novel mechanism, termed "collateral priming," in which naive CD4(+) T cells are activated by adaptive rather than innate immune signals. Th2 priming to newly encountered Ags was dependent on the production of IL-4 by the transferred Th2 population but was independent of Toll-like receptor 4 signaling and the myeloid differentiation factor 88 Toll-like receptor signaling pathway. These results identify a novel mechanism of T cell priming in which an Ag-specific adaptive immune response initiates distinct Ag-specific T cell responses in the absence of classical innate immune system triggering signals.     

Effects of myeloid differentiation primary response gene 88 (MyD88) activation on Helicobacter infection in vivo and induction of a Th17 response

Background:  Helicobacter pylori is a spiral‐shaped Gram‐negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. Materials and Methods:  Wild type (WT) and MyD88‐deficient (MyD88^?/?) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real‐time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. Results:  mRNA expression levels of the Th17 cytokines interleukin‐17A (IL‐17A) and IL‐22 were markedly up‐regulated in WT compared with MyD88^?/? mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17‐dependent intestinal antimicrobial peptide lipocalin‐2 was linked with increased bacterial burden in the absence of MyD88 signaling. Conclusion:  We provide evidence showing that MyD88‐dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.     

Lipopolysaccharide and double-stranded RNA up-regulate toll-like receptor 2 independently of myeloid differentiation factor 88

Toll-like receptor 2 (TLR2) is a signaling receptor for a variety of microbial products, including bacterial lipoproteins and peptidoglycan, and is central in initiating immune responses toward Gram-positive bacteria, spirochetes, and mycobacteria. The mechanisms behind regulation of TLR2 protein expression are still not well understood. By using a newly developed monoclonal antibody against mouse TLR2, we detected TLR2 protein expression on macrophages, neutrophils, and dendritic cells. Endogenous macrophage TLR2 localized mostly to the cell membrane, with particular accumulation around phagosomes containing zymosan. Treatment of macrophages with the TLR2 antibody diminished cellular response to lipoproteins and down-regulated membrane TLR2. Marked up-regulation of surface TLR2 was observed on macrophages in response to whole bacteria, lipoproteins, lipopolysaccharide, poly(I-C) (double-stranded RNA), R848, and CpG DNA, and this up-regulation appeared to be a very sensitive marker for the presence of microbial products. Up-regulation of TLR2 in response to stimuli correlated with an increased response to secondary lipoprotein exposure following a low concentration of primary lipoprotein challenge. By comparison, exposure to a larger primary challenge induced a hyporeactive state. Most interestingly, lipopolysaccharide- and double-stranded RNA-induced up-regulation of surface TLR2 in macrophages was found to be MyD88-independent, whereas the up-regulation in response to lipoproteins, R848, and CpG DNA was absent in MyD88-deficient cells. We conclude that complex mechanisms regulate expression and signaling via TLR2. Up-regulation of TLR2 in the presence of low, yet clinically relevant amounts of microbial products may be an important mechanism by which the immune system boosts its response to a beginning infection.     

Burn injury promotes antigen-driven Th2-type responses in vivo

Severe injury induces detrimental changes in immune function, often leaving the host highly susceptible to developing life-threatening opportunistic infections. Advances in our understanding of how injury influences host immune responses suggest that injury causes a phenotypic imbalance in the regulation of Th1- and Th2-type immune responses. We report in this study, using a TCR transgenic CD4(+) T cell adoptive transfer approach, that injury skews T cell responses toward increased Th2-type reactivity in vivo without substantially limiting Ag-driven CD4(+) T cell expansion. The increased Th2-type response did not occur unless injured mice were immunized with specific Ag, suggesting that the phenotypic switch is Ag dependent. These findings establish that severe injury induces fundamental changes in the induction of Ag-specific CD4(+) Th cell responses favoring the development of Th2-type immune reactivity in vivo.     

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

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

Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice

Listeria monocytogenes (LM), a facultative intracellular Gram-positive bacterium, often causes lethal infection of the host. In this study we investigated the molecular mechanism underlying LM eradication in the early phase of infection. Upon infection with LM, both IL-12 and IL-18 were produced, and then they synergistically induced IFN-gamma production, leading to normal LM clearance in the host. IFN-gamma knockout (KO) mice were highly susceptible to LM infection. IL-12/IL-18 double knockout mice were also highly susceptible. Their susceptibility was less than that of IFN-gamma KO mice, but more than that of single IL-12 or IL-18 KO mice. Mice deficient in myeloid differentiation factor 88 (MyD88), an essential adaptor molecule used by signal transduction pathways of all members of the Toll-like receptor (TLR) family, showed an inability to produce IL-12 and IFN-gamma following LM infection and were most susceptible to LM. Furthermore, MyD88-deficient, but not IFN-gamma-deficient, Kupffer cells could not produce TNF-alpha in response to LM in vitro, indicating the importance of MyD88-dependent TNF-alpha production for host defense. As TLR2 KO, but not TLR4 KO, mice showed partial impairment in their capacity to produce IL-12, IFN-gamma, and TNF-alpha, TLR2 activation partly contributed to the induction of IL-12-mediated IFN-gamma production. These results indicated a critical role for TLRs/MyD88-dependent IL-12/TNF-alpha production and for IL-12- and IL-18-mediated IFN-gamma production in early phase clearance of LM.     

正常妊娠大鼠的Th2型免疫反应

采用流式细胞仪,^3H-TdR掺入和酶联免疫打点(enzyme-linked immunospot,ELISPOT)方法研究妊娠免疫学指标的改变。妊娠晚期大鼠脾脏单个核细胞表面分子主要组织相容性抗原Ⅱ(MHCⅡ)明显下调,外周血单个核细胞表达CD11c明显减少,共激活分子B7-1和B7-2未见改变;脾脏和外周血单个核细胞中Th2细胞因子IL-10、IL-4表达增多,TGFD阳性细胞数也明显增加,而Th1细胞因子IFNγ的产生未受抑制。此外,脾脏和外周血中单个核细胞的抗原特异性增殖未见改变,而腹腔淋巴结细胞的增殖明显升高。脾脏单个核细胞在妊娠晚期分泌较少的抗原特异性抗体。提示妊娠期性激素具有免疫调节作用,可能与怀孕时Th1细胞介导的自身免疫性疾病得到缓解有关。     

TcpC inhibits toll-like receptor signaling pathway by serving as an E3 ubiquitin ligase that promotes degradation of myeloid differentiation factor 88

TcpC is a virulence factor of uropathogenic E. coli (UPEC). It was found that TIR domain of TcpC impedes TLR signaling by direct association with MyD88. It has been a long-standing question whether bacterial pathogens have evolved a mechanism to manipulate MyD88 degradation by ubiquitin-proteasome pathway. Here, we show that TcpC is a MyD88-targeted E3 ubiquitin ligase. Kidney macrophages from mice with pyelonephritis induced by TcpC-secreting UPEC showed significantly decreased MyD88 protein levels. Recombinant TcpC (rTcpC) dose-dependently inhibited protein but not mRNA levels of MyD88 in macrophages. Moreover, rTcpC significantly promoted MyD88 ubiquitination and accumulation in proteasomes in macrophages. Cys12 and Trp106 in TcpC are crucial amino acids in maintaining its E3 activity. Therefore, TcpC blocks TLR signaling pathway by degradation of MyD88 through ubiquitin-proteasome system. Our findings provide not only a novel biochemical mechanism underlying TcpC-medicated immune evasion, but also the first example that bacterial pathogens inhibit MyD88-mediated signaling pathway by virulence factors that function as E3 ubiquitin ligase.     

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.     

Toll-like receptor 2 pathway drives streptococcal cell wall-induced joint inflammation: critical role of myeloid differentiation factor 88

The IL-1R/Toll-like receptor (TLR) superfamily of receptors has a key role in innate immunity and inflammation. In this study, we report that streptococcal cell wall (SCW)-induced joint inflammation is predominantly dependent on TLR-2 signaling, since TLR-2-deficient mice were unable to develop either joint swelling or inhibition of cartilage matrix synthesis. Myeloid differentiation factor 88 (MyD88) is a Toll/IL-1R domain containing adaptor molecule known to have a central role in both IL-1R/IL-18R and TLR signaling. Mice deficient for MyD88 did not develop SCW-induced arthritis; both joint swelling and disturbance of cartilage chondrocyte anabolic function was completely abolished. Local levels of proinflammatory cytokines and chemokines in synovial tissue washouts were strongly reduced in MyD88-deficient mice. Histology confirmed the pivotal role of MyD88 in acute joint inflammation. TLR-2-deficient mice still allow influx of inflammatory cells into the joint cavity, although the number of cells was markedly reduced. No influx of inflammatory cells was seen in joints of MyD88-deficient mice. In addition, cartilage matrix proteoglycan loss was completely absent in MyD88 knockout mice. These findings clearly demonstrated that MyD88 is a key component in SCW-induced joint inflammation. Since agonists of the Toll-like pathway are abundantly involved in both septic and rheumatoid arthritis, targeting of MyD88 may be a novel therapy in inflammatory joint diseases.     

Neutrophils clear bacteria associated with parasitic nematodes augmenting the development of an effective Th2-type response

Infection with the parasitic nematode Nippostrongylus brasiliensis induces a potent Th2 response; however, little is known about early stages of the innate response that may contribute to protective immunity. To examine early events in this response, chemokine expression in the draining lymph node was examined after N. brasiliensis inoculation. Pronounced increases of several chemokines, including CCL2, were observed. Compared with wild-type mice, elevations in a Gr-1bright population in the draining lymph node was significantly decreased in CCL2-/- mice after N. brasiliensis inoculation. Further flow cytometric and immunofluorescent analysis showed that in wild-type mice, Gr-1+ cells transiently entered and exited the draining lymph node shortly after N. brasiliensis inoculation. The Gr-1bright population was comprised of neutrophils expressing TGF-beta and TNF-alpha. Following Gr-1+ cell depletion, N. brasiliensis infection resulted in transient, but significantly increased levels of IFN-gamma, increased serum IgG2a, reduced Th2 cytokines and serum IgE, greatly increased mortality, and delayed worm expulsion. Furthermore, bacteria were readily detected in vital organs. Infection of Gr-1+ cell-depleted mice with N. brasiliensis larvae that were pretreated with antibiotics prevented bacterial dissemination, Th1 inflammatory responses, and decreases in host survival. This study indicates that parasitic nematodes can be an important vector of potentially harmful bacteria, which is typically controlled by CCL2-dependent neutrophils that ensure the optimal development of Th2 immune responses and parasite resistance.     

Cellular activation,phagocytosis, and bactericidal activity against group B streptococcus involve parallel myeloid differentiation factor 88-dependent and independent signaling pathways

Group B streptococci (GBS) vigorously activate inflammatory responses. We reported previously that a secreted GBS "factor" activates phagocytes via Toll-like receptor (TLR)2 and TLR6, but that GBS cell walls activate cells independently of these receptors. We hypothesized that the phagocytic immune functions in response to GBS, such as inflammation, uptake, and elimination of bacteria, occur through a coordinated engagement of TLRs, along with the coreceptors CD14 and CD11b/CD18. Using various knockout mice we show that GBS-induced activation of p38 and NF-kappaB depends upon the expression of the cytoplasmic TLR adapter protein, myeloid differentiation factor 88 (MyD88), but not TLR2 and/or TLR4. Macrophages with deletions of CD14 and complement receptor 3 had a normal cytokine response to whole bacteria, although the response to GBS factor was abrogated in CD14-null cells. The intracellular formation of bactericidal oxygen species proved to be MyD88 dependent; however, uptake of GBS, a prerequisite for intracellular killing by O(2) radicals, occurred independently of MyD88. While deletion of complement receptor 3 greatly diminished the uptake of opsonized GBS, it did not affect the formation of bactericidal O(2) radicals or inflammatory signaling intermediates. We conclude that the inflammatory, bactericidal, and phagocytic responses to GBS occur via parallel but independent processes.     

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri

Myeloid differentiation factor 88 (MyD88) is a universal and essential adapter for the TLR/IL-1R family. In this report, the first mollusk Myd88 ortholog (named as CfMyd88) was cloned from Zhikong scallop (Chlamys farreri). The full-length cDNA of CfMyd88 was of 1554 bp, including a 5'-terminal untranslated region (UTR) of 427 bp, a polyA tail, and an open reading frame (ORF) of 1104 bp encoding a polypeptide of 367 amino acids containing the typical TLR and IL-1R-related (TIR) domain and death domain (DD). Homology analysis revealed that the predicted amino acid sequence of CfMyd88 was homologous to a variety of previously identified Myd88s with more than 30% identity. The temporal expressions of CfMyd88 mRNA in the mixed primary cultured haemocytes stimulated by lipopolysaccharide (LPS) and peptidoglycans (PGN) were measured by real-time RT-PCR system. The mRNA expression of CfMyd88 decreased after stimulation with both LPS and PGN, and the lowest level was about 1/3 times (at 6 h) and 1/10 times (at 9 h) to that in the control group, respectively. The expression then recovered and was upregulated to two-fold at 9 h after LPS stimulation or to the original level at 12 h after PGN stimulation. The results suggest that the MyD88-dependent signaling pathway exists in scallop and was involved in the defense system.     

Dendritic cell-derived IL-12 promotes B cell induction of Th2 differentiation: a feedback regulation of Th1 development.

B cells convert what are normally conditions for Th1 differentiation into an environment suitable for Th2 development. This capacity is dependent on CD40 as B cells from CD40-/- mice do not elicit Th2 differentiation. To elucidate the basis of this effect, we surveyed cytokine RNA made by naive B cells after activation with anti-Ig and anti-CD40. Resting B cells make TGF-beta message only, however, 4 days after activation, RNA encoding IL-6, IL-10, and TNF-alpha was found. The expression of these messages was accelerated by 2 days in the presence of IL-12. The relevance of these observations to T cell differentiation was investigated: addition of OVA peptide to splenic cells from DO.11.10 transgenic mice causes most T cells to make IFN-gamma. Coactivation of B cells in these cultures reduces the number of IFN-gamma-producing T cells and increases the number synthesizing IL-4. Abs to IL-6 and IL-10 block the IL-4 enhancement. Dissection of the component APC demonstrated that interaction of B cells with IL-12-producing dendritic cells is crucial for B cell-mediated IL-4 enhancement: Thus, B cells preactivated in the presence of dendritic cells from IL-12-/- mice show little IL-4-inducing activity when used to activate T cells. This immune regulation is initiated by IL-12 and therefore represents a feedback loop to temper its own dominant effect (IFN-gamma induction).     

Plasmodium berghei infection in mice induces liver injury by an IL-12- and toll-like receptor/myeloid differentiation factor 88-dependent mechanism.

Malaria, caused by infection with Plasmodium spp., is a life cycle-specific disease that includes liver injury at the erythrocyte stage of the parasite. In this study, we have investigated the mechanisms underlying Plasmodium berghei-induced liver injury, which is characterized by the presence of apoptotic and necrotic hepatocytes and dense infiltration of lymphocytes. Although both IL-12 and IL-18 serum levels were elevated after infection, IL-12-deficient, but not IL-18-deficient, mice were resistant to liver injury induced by P. berghei. Neither elevation of serum IL-12 levels nor liver injury was observed in mice deficient in myeloid differentiation factor 88 (MyD88), an adaptor molecule shared by Toll-like receptors (TLRs). These results demonstrated a requirement of the TLR-MyD88 pathway for induction of IL-12 production during P. berghei infection. Hepatic lymphocytes from P. berghei-infected wild-type mice lysed hepatocytes from both uninfected and infected mice. The hepatocytotoxic action of these cells was blocked by a perforin inhibitor but not by a neutralizing anti-Fas ligand Ab and was up-regulated by IL-12. Surprisingly, these cells killed hepatocytes in an MHC-unrestricted manner. However, CD1d-deficient mice that lack CD1d-restricted NK T cells, were susceptible to liver injury induced by P. berghei. Collectively, our results indicate that the liver injury induced by P. berghei infection of mice induces activation of the TLR-MyD88 signaling pathway which results in IL-12 production and activation of the perforin-dependent cytotoxic activities of MHC-unrestricted hepatic lymphocytes.     

Toxoplasma gondii triggers myeloid differentiation factor 88-dependent IL-12 and chemokine ligand 2 (monocyte chemoattractant protein 1) responses using distinct parasite molecules and host receptors

Toll-like receptors (TLR) that signal through the common adaptor molecule myeloid differentiation factor 88 (MyD88) are essential in proinflammatory cytokine responses to many microbial pathogens. In this study we report that Toxoplasma gondii triggers neutrophil IL-12 and chemokine ligand 2 (CCL2; monocyte chemoattractant protein 1) production in strict dependence upon functional MyD88. Nevertheless, the responses are distinct. Although we identify TLR2 as the receptor triggering CCL2 production, parasite-induced IL-12 release did not involve this TLR. The production of both IL-12 and CCL2 was increased after neutrophil activation with IFN-gamma. However, the synergistic effect of IFN-gamma on IL-12, but not CCL2, was dependent upon Stat1 signal transduction. Although IL-10 was a potent down-regulator of Toxoplasma-triggered neutrophil IL-12 release, the cytokine had no effect on parasite-induced CCL2 production. Soluble tachyzoite Ag fractionation demonstrated that CCL2- and IL-12 inducing activities are biochemically distinct. Importantly, Toxoplasma cyclophilin-18, a molecule previously shown to induce dendritic cell IL-12, was not involved in neutrophil IL-12 production. Our results show for the first time that T. gondii possesses multiple molecules triggering distinct MyD88-dependent signaling cascades, that these pathways are independently regulated, and that they lead to distinct profiles of cytokine production.     

Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-alpha and IL-6. Administration of anti-TNF-alpha Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-alpha as the molecular mediator of bacterial clearance and septic shock.