TLR Adaptor MyD88 Research Articles

TLR3 Signaling is Required for Robust Extrafollicular B Cell Response (EF) in Influenza A/PR/8 Infection

Abstract Following influenza A virus (IAV) infection B cells rapidly proliferate and differentiate into plasma cells (PCs) in extrafollicular foci (EF) within mediastinal lymph nodes (medLN), resulting in protective antibody secretion. We had shown that co-expression of both TLR adaptor proteins, TRIF and MyD88, is required for robust EF responses. Only two TLRs, TLR3 and 4, signal using TRIF. As shown previously TLR3 and 7, but not TLR4, expression were upregulated by medLN B cells within 2 days post infection (dpi) in a Type-I IFN-dependent manner, suggesting that TLR3 might utilize TRIF in B cells during IAV infection. To test this we generated MyD88 × TLR3 double-deficient (DKO) mice. Flow cytometry on MedLN at 7 dpi showed significantly reduced EF B cells (CD19 loB220 loCD38 loCD24 hi) and CD138 +plasma cells (PCs), including IAV-specific PCs, as well as reduced serum IAV-specific IgG and IgG 2Cin DKO compared to C57BL/6 or single- MyD88 and TLR3 deficient mice. IAV infection of mixed bone marrow chimeras indicated that the TLR3-TRIF axis contributes to EF formation at least in part in a B cell-intrinsic manner. In vitro stimulation of B cells with the TLR3 agonist, Poly: IC, caused expression of several activation/costimulatory molecules but did not induce proliferation. Together, the data suggest that TLR3-mediated B cell is a non-redundant driver of rapid protective antibody responses. Supported by NIH/NIAID R01AI117890 (NB) and NIH T32 HL007013-40 (JHL and EJK).

Absence of Non-Canonical, Inhibitory MYD88 Splice Variants in B Cell Lymphomas Correlates With Sustained NF-κB Signaling.

Gain-of-function mutations of the TLR adaptor and oncoprotein MyD88 drive B cell lymphomagenesis via sustained NF-κB activation. In myeloid cells, both short and sustained TLR activation and NF-κB activation lead to the induction of inhibitory MYD88 splice variants that restrain prolonged NF-κB activation. We therefore sought to investigate whether such a negative feedback loop exists in B cells. Analyzing MYD88 splice variants in normal B cells and different primary B cell malignancies, we observed that MYD88 splice variants in transformed B cells are dominated by the canonical, strongly NF-κB-activating isoform of MYD88 and contain at least three novel, so far uncharacterized signaling-competent splice isoforms. Sustained TLR stimulation in B cells unexpectedly reinforces splicing of NF-κB-promoting, canonical isoforms rather than the ‘MyD88s’, a negative regulatory isoform reported to be typically induced by TLRs in myeloid cells. This suggests that an essential negative feedback loop restricting TLR signaling in myeloid cells at the level of alternative splicing, is missing in B cells when they undergo proliferation, rendering B cells vulnerable to sustained NF-κB activation and eventual lymphomagenesis. Our results uncover MYD88 alternative splicing as an unappreciated promoter of B cell lymphomagenesis and provide a rationale why oncogenic MYD88 mutations are exclusively found in B cells.

The Bile Acids Specifically Modulate Colonic MUC2 and Tight Junction Protein Expression in the Human Colon Cancer Cell Line

Abstract Objectives Alterations to mucin secretion and epithelial tight junctions can compromise the ability of the epithelium to act as a barrier for the host to prevent pathogenic attack. Bile acids are synthesized in hepatocytes and released into the intestine, further modified by gut bacteria. Although many studies have investigated the changes of intestinal bile acids in the pathogenesis of various immune disorders, there are few reports about its function in preventing or interventing the dysfunction of the intestinal barrier. In this study, we sought to investigate the effects of the colonic bile acids on MUC2 and tight junction protein expression, which are crucial to colonic barrier. Methods Regulation of MUC2 and tight junction protein expression was assayed in the human colon cancer LS174T and T84 cells. The cells were treated with deoxycholic acid (DCA), lithocholic acid (LCA), 3-oxo-DCA, 3-oxo-LCA, isoDCA and isoLCA (100 μM or 200 μM), respectively. Proliferation of the cells was investigated with the MTT assay. mRNA expression of MUC2, ZO-1, occludin, claudin1 were measured by RT-PCR. Nuclear bile acid receptor FXR and TGR5, toll-like receptors and TLR adaptor MyD88, and genes (CDX2, AGR2, MyD88) related to mucin synthesis and secretion were also measured. Results In comparison with the untreated control, DCA, 3-oxo-DCA, isoDCA and isoLCA (100 μM) significantly upregulated the ZO-1, occludin and bile acid receptor FXR gene expression in the T84 cell. LCA, 3-oxo-LCA and isoLCA upregulated MUC2 expression at 200 μM, but showed no significant effect at 100 μM. DCA only significantly upregulated MUC2 expression at 200 μM, but isoDCA upregulated MUC2 expression independent of concentration in the LS174T cell. The expression of CDX2, AGR2, MyD88 was consistent with MUC2. Conclusions Bile acids at various concentrations specifically modulate MUC2 and tight junction protein expression, and thereby alter the colonic barrier function. This regulatory effect of bile acids could be mediated by activating bile acid receptors FXR. Funding Sources This work was financially supported by Ministry of Science and Technology of China (10000 Talent Project).

Vaccinia Virus Immunomodulator A46: Destructive Interactions with MAL and MyD88 Shown by Negative-Stain Electron Microscopy

Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the Toll/interleukin-1 receptor (TIR) domain-containing TLR adaptor proteins MAL, MyD88, TRAM, and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88. Using targeted mutagenesis and protein-protein crosslinking, we show that A46 interacts with MAL and MyD88 through several facets, including residues on helices α1 and α7 and the C-terminal flexible region. We propose a model in which A46 targets the MAL and MyD88 signalosome intra-strand interfaces and gradually destroys their assemblies in a concentration-dependent manner.

Vaccinia Virus Immunomodulator A46: Destructive Interactions with MAL and MyD88 Shown by Negative-Stain EM

Vaccinia virus A46 is an anti-inflammatory and non-anti-apoptotic, two-domain member of the poxviral Bcl-2-like protein family that inhibits the cellular innate immune response at the level of the TIR domain-containing TLR adaptor proteins MAL, MyD88, TRAM and TRIF. The mechanism of interaction of A46 with its targets has remained unclear. The TIR domains of MAL and MyD88 have been shown to signal by forming filamentous assemblies. We show a clear concentration-dependent destruction of both of these assemblies by A46 by means of negative-stain electron microscopy from molar ratios of 1:15 for MAL and 1:30 for MyD88. Using targeted mutagenesis and protein-protein cross-linking, we show that A46 interacts with MAL and MyD88 through several facets, including residues on helices α1 and α7 and the C-terminal flexible region. We propose a model in which A46 targets the MAL and MyD88 signalosome intra-strand interfaces and gradually destroys their assemblies in a concentration-dependent manner.

Single-cell analyses reveal functional classification of dendritic cells and their potential roles in inflammatory disease.

Dendritic cells (DCs) have crucial roles in immune-related diseases. However, it is difficult to explore DCs because of their rareness and heterogeneity. Although previous studies had been performed to detect the phenotypic characteristics of DC populations, the functional diversity has been ignored. Using a combination of flow cytometry, single-cell quantitative PCR, and bioinformatic analysis, we depicted the DC panorama with not only phenotypic but also functional markers. Functional classification of DCs in mouse lymphoid tissue (spleen) and nonlymphoid tissue (liver) was performed. The results revealed that expression of macrophage scavenger receptor 1 ( MSR1) and C-C motif chemokine receptors ( CCR) 1, CCR2, and CCR4 were elevated in liver DCs, suggesting increased lipid uptake and migration abilities. The enriched expression of costimulatory molecule CD80, TLR9, and TLR adaptor MYD88 in spleen DCs indicated a more-mature phenotype, enhanced pathogen recognition, and T-cell stimulation abilities. Furthermore, we compared DCs in the atherosclerotic mouse models with healthy controls. In addition to the quantitative increase in DCs in the liver and spleen of the apolipoprotein E-knockout ( ApoE-/-) mice, the functional expression patterns of the DCs also changed at the single-cell level. These results promote our understanding of the participation of DCs in inflammatory diseases and have potential applications in DC clinical assessment.-Shi, Q., Zhuang, F., Liu, J.-T., Li, N., Chen, Y.-X., Su, X.-B., Yao, A.-H., Yao, Q.-P., Han, Y., Li, S.-S., Qi, Y.-X., Jiang, Z.-L. Single-cell analyses reveal functional classification of dendritic cells and their potential roles in inflammatory disease.

Astilbin from Smilax glabra Roxb. Attenuates Inflammatory Responses in Complete Freund's Adjuvant-Induced Arthritis Rats.

Astilbin, a flavonoid compound, was isolated from the rhizome of Smilax glabra Roxb. (with red cross-section) grown in Guizhou Province, China. We accessed its effect and potential mechanism on attenuation of the inflammatory response in CFA-induced AA rats. Our results showed that daily oral administration of astilbin at 5.3 mg/kg reduced joint damage in the hind paw of AA rats. Accordingly, astilbin exhibited remarkable inhibitory effects on TNF-α, IL-1β, and IL-6 mRNA expression. Significant decrease of serum cytokine levels of TNF-α, IL-1β, and IL-6 was also observed in astilbin-treated AA rats compared to the vehicle-treated AA rats. The reduced expression of these cytokines was associated with protein activity suppression of three key molecular targets in the pathogenesis of RA, including IKKβ, NF-κB p65 subunit, and TLR adaptor MyD88. Furthermore, the therapeutic effects of astilbin on the inhibition of cytokines production as well as the reduction of inflammatory response in AA rats are close to a commonly used antirheumatic drug, leflunomide. Collectively, our data suggest that the action mechanism of astilbin, as an anti-inflammatory agent for RA treatment, is associated with modulating the production of proinflammatory cytokines and inhibiting the expression of key elements in NF-κB signaling pathway mediated by TLR.

Mycobacterium tuberculosis hijacks alveolar macrophages to translocate from the airway to the lung parenchyma.

Abstract Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a chronic bacterial infection of the lung that affects one third of the world’s population. While lung alveolar macrophages (AM) are thought to be the first host cell infected with Mtb, it remains unclear how bacteria leave AM in the airway and disseminate to the lung parenchyma. Using fluorescent Mtb to identify infected cells and non-surgical intra-tracheal antibody delivery to label airway-resident cells, we found that airway-resident AM are indeed the first cells infected by Mtb. Interestingly, infected AM become increasingly resistant to airway labeling over time, with only 30% of infected AM localizing to the airway by Day 21 post-infection, suggesting that infected AM exit the airway and translocate to the lung parenchyma as infection progresses. In contrast, uninfected AM continue to predominantly label with the airway antibody, suggesting that migration of AM into the lung parenchyma is specific to infected cells. In the absence of the TLR adaptor MyD88, but not IL12R or type I IFN, infected AM are partially impaired in their ability to migrate, implicating MyD88 signaling in this trafficking pathway. Surprisingly, AM infected with the attenuated ΔdrrA strain that lacks the virulence lipid PDIM are impaired in translocation to the parenchyma, identifying a possible role for this lipid in promoting AM migration. Conversely, AM infected with the hypervirulent SA161 strain translocated to the lung parenchyma more rapidly 10 days post-infection. Combined, these studies provide insights into the cellular mechanisms of Mtb entry into the lung parenchyma following aerosol infection, a key step in the pathogenesis of tuberculosis.

Cutting Edge: ABIN-1 Protects against Psoriasis by Restricting MyD88 Signals in Dendritic Cells

Psoriasis is a chronic, inflammatory skin disease caused by a combination of environmental and genetic factors. The Tnip1 gene encodes A20 binding and inhibitor of NF-κB-1 (ABIN-1) protein and is strongly associated with susceptibility to psoriasis in humans. ABIN-1, a widely expressed ubiquitin-binding protein, restricts TNF- and TLR-induced signals. In this study, we report that mice lacking ABIN-1 specifically in dendritic cells (DCs), ABIN-1(fl) CD11c-Cre mice, exhibit perturbed immune homeostasis. ABIN-1-deficient DCs display exaggerated NF-κB and MAPK signaling and produce more IL-23 than do normal cells in response to TLR ligands. Challenge of ABIN-1(fl) CD11c-Cre mice with topical TLR7 ligand leads to greater numbers of Th17 and TCRγδ T cells and exacerbated development of psoriaform lesions. These phenotypes are reversed by DC-specific deletion of the TLR adaptor MyD88. These studies link ABIN-1 with IL-23 and IL-17, and they provide cellular and molecular mechanisms by which ABIN-1 regulates susceptibility to psoriasis.

The phosphatase and tensin homolog PTEN improves murine polymicrobial sepsis outcome by decreasing MyD88 expression. (P1344)

Abstract Sepsis is systemic inflammatory response caused by an infectious agent and may progress to multiple organ dysfunction and death. Therapeutic strategies to inhibit excessive inflammation in sepsis are needed. The lipid and protein phosphatase PTEN is a well-known tumor suppressor, but its role in host defense is poorly studied. Here, we investigated whether PTEN activation is involved in TLR activation in vivo and in vitro. PTEN expression is decreased in cells from the peritoneal cavity 24 h after cecum ligation and puncture (CLP). In vivo pretreatment with the PTEN inhibitor bpV(HOpic) 150 ug/Kg for 1 hour before cecum and ligation puncture decreases animals survival, associated with high bacterial number in the peritoneal cavity when compared to untreated mice. In vivo PTEN inhibition increases expression of the TLR adaptor MyD88 during sepsis. In vitro, LPS (TLR4) and PAM3Cys (TLR2) increase PTEN phosphorylation in thyoglicolate-elicited macrophages (MΦs). PTEN mRNA silencing further enhances MyD88 expression and TLR-2 and -4-induced IRAK and NFkB p65 phosphorylation. Transfection of MΦs with adenovirus expressing constitutively active PTEN decreases MyD88 expression. Together, our data show that PTEN is a key determinant of macrophage TLR activation by decreasing MyD88 expression, leading to reduced systemic inflammatory response and therefore improving sepsis outcome.

In vivo PPAR-gamma activation inhibits MyD88 expression to protect mice from polymicrobial sepsis. (P4425)

Abstract Sepsis is a disorder characterized by systemic inflammatory response caused by an infectious agent and may progress to multiple organ dysfunction and death. Overwhelming inflammatory response is believed to underlie tissue damage and preceding multiple organ disfunction syndrome. Thus, therapeutic strategies to inhibit excessive inflammation in sepsis is needed. The nuclear receptor peroxisossome proliferating factor (PPAR)-g exhibit anti-inflammatory properties in different immune cells. We hypothesize that prolonged PPAR-g activation decreases systemic inflammatory responses, improving sepsis outcome. We employed a model of polymicrobial sepsis (cecum and ligation puncture-CLP) to evaluate whether prolonged PPAR-g activation inhibits inflammation and improves animal survival. PPAR-g mRNA expression is increased in both peritoneal and lung cells at 6 h after CLP. Our data show that the PPAR-g agonist pioglitazone treatment for 18 h enhances bacterial clearance in the peritoneal cavity; which was associated with enhanced neutrophil migration to the infectious focus. These data correlates with lower expression of the TLR adaptor MyD88 in pioglitazone in vivo and peritoneal macrophages in vitro. Together, our findings suggest that PPAR-g activation impairs TLR responses, by inhibiting expression of MyD88 and preventing overwhelming inflammatory response.

MyD88 is critically involved in immune tolerance breakdown at environmental interfaces of Foxp3-deficient mice

Tregs expressing the transcription factor Foxp3 suppress self-reactive T cells, prevent autoimmunity, and help contain immune responses to foreign antigens, thereby limiting the potential for inadvertent tissue damage. Mutations in the FOXP3 gene result in Treg deficiency in mice and humans, which leads to the development of a multisystem autoimmune inflammatory disease. The contribution of dysregulated innate immune responses to the pathogenesis of Foxp3 deficiency disease is unknown. In this study, we examined the role of microbial signals in the pathogenesis of Foxp3 deficiency disease by studying Foxp3 mutant mice that had concurrent deficiencies in TLR signaling pathways. Global deficiency of the common TLR adaptor MyD88 offered partial protection from Foxp3 deficiency disease. Specifically, it protected from disease at the environmental interfaces of the skin, lungs, and gut. In contrast, systemic disease, in the form of unrestrained lymphoproliferation, continued unabated. The effect of MyD88 deficiency at environmental interfaces involved the disruption of chemokine gradients that recruit effector T cells and DCs, resulting in their entrapment in secondary lymphoid tissues. These results suggests that Tregs have a key role in maintaining tolerance at host-microbial interfaces by restraining tonic MyD88-dependent proinflammatory signals. Moreover, microbial factors may play a substantial role in the pathogenesis of human autoimmune disease resulting from Treg deficiency.

Activation of the NLRP3 Inflammasome by Group B Streptococci

Group B Streptococcus (GBS) is a frequent agent of life-threatening sepsis and meningitis in neonates and adults with predisposing conditions. We tested the hypothesis that activation of the inflammasome, an inflammatory signaling complex, is involved in host defenses against this pathogen. We show in this study that murine bone marrow-derived conventional dendritic cells responded to GBS by secreting IL-1β and IL-18. IL-1β release required both pro-IL-1β transcription and caspase-1-dependent proteolytic cleavage of intracellular pro-IL-1β. Dendritic cells lacking the TLR adaptor MyD88, but not those lacking TLR2, were unable to produce pro-IL-1β mRNA in response to GBS. Pro-IL-1β cleavage and secretion of the mature IL-1β form depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) sensor and the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain adaptor. Moreover, activation of the NLRP3 inflammasome required GBS expression of β-hemolysin, an important virulence factor. We further found that mice lacking NLRP3, apoptosis-associated speck-like protein, or caspase-1 were considerably more susceptible to infection than wild-type mice. Our data link the production of a major virulence factor by GBS with the activation of a highly effective anti-GBS response triggered by the NLRP3 inflammasome.

Activation of TLR2 Enhances Tight Junction Barrier in Epidermal Keratinocytes

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.

A composite MyD88/CD40 switch synergistically activates mouse and human dendritic cells for enhanced antitumor efficacy

The in vivo therapeutic efficacy of DC-based cancer vaccines is limited by suboptimal DC maturation protocols. Although delivery of TLR adjuvants systemically boosts DC-based cancer vaccine efficacy, it could also increase toxicity. Here, we have engineered a drug-inducible, composite activation receptor for DCs (referred to herein as DC-CAR) comprising the TLR adaptor MyD88, the CD40 cytoplasmic region, and 2 ligand-binding FKBP12 domains. Administration of a lipid-permeant dimerizing ligand (AP1903) induced oligomerization and activation of this fusion protein, which we termed iMyD88/CD40. AP1903 administration to vaccinated mice enabled prolonged and targeted activation of iMyD88/CD40-modified DCs. Compared with conventionally matured DCs, AP1903-activated iMyD88/CD40-DCs had increased activation of proinflammatory MAPKs. AP1903-activated iMyD88/CD40-transduced human or mouse DCs also produced higher levels of Th1 cytokines, showed improved migration in vivo, and enhanced both antigen-specific CD8+ T cell responses and innate NK cell responses. Furthermore, treatment with AP1903 in vaccinated mice led to robust antitumor immunity against preestablished E.G7-OVA lymphomas and aggressive B16.F10 tumors. Thus, the iMyD88/CD40 unified "switch" effectively and safely replaced exogenous adjuvant cocktails, allowing remote and sustained DC activation in vivo. DC "licensing" through iMyD88/CD40 may represent a mechanism by which to exploit the natural synergy between the TLR and CD40 signaling pathways in DCs using a single small molecule drug and could augment the efficacy of antitumor DC-based vaccines.

TLR3‐mediated IFN‐β gene induction is negatively regulated by the TLR adaptor MyD88 adaptor‐like

There is limited insight into the mechanisms involved in the counterregulation of TLR. Given the important role of TLR3/TIR domain-containing adaptor-inducing IFN-β (TRIF)-dependent signalling in innate immunity, novel insights into its modulation is of significance in the context of many physiological and pathological processes. Herein, we sought to perform analysis to definitively assign a mechanistic role for MyD88 adaptor-like (Mal), an activator of TLR2/4 signalling, in the negative regulation of TLR3/TRIF signalling. Biochemical and functional analysis demonstrates that Mal negatively regulates TLR3, but not TLR4, mediated IFN-β production. Co-immunoprecipitation experiments demonstrate that Mal associates with IRF7 (IRF, IFN regulatory factor), not IRF3, and Mal specifically blocks IRF7 activation. In doing so, Mal impedes TLR3 ligand-induced IFN-β induction. Interestingly, Mal does not affect the induction of IL-6 and TNF-α upon TLR3 ligand engagement. Together, these data show that the TLR adaptor Mal interacts with IRF7 and, in doing so, impairs IFN-β induction through the positive regulatory domains I-III enhancer element of the IFN-β gene following poly(I:C) stimulation. Our findings offer a new mechanistic insight into TLR3/TRIF signalling through a hitherto unknown mechanism whereby Mal inhibits poly(I:C)-induced IRF7 activation and concomitant IFN-β production. Thus, Mal is essential in restricting TLR3 signalling thereby protecting the host from unwanted immunopathologies associated with excessive IFN-β production.

TLR Adaptor MyD88 Is Essential for Pathogen Control during OralToxoplasma gondiiInfection but Not Adaptive Immunity Induced by a Vaccine Strain of the Parasite

TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88(-/-) mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-gamma production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4(+) T cell IFN-gamma reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88(-/-) mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-gamma responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule.

Syk kinase is required for collaborative cytokine production induced through Dectin-1 and Toll-like receptors.

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF, MIP-1α and MIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk- and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

TLR Signaling Mediated by MyD88 Is Required for a Protective Innate Immune Response by Neutrophils to Citrobacter rodentium

Enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium are classified as attaching and effacing pathogens based on their ability to adhere to intestinal epithelium via actin-filled membranous protrusions (pedestals). Infection of mice with C. rodentium causes breach of the colonic epithelial barrier, a vigorous Th1 inflammatory response, and colitis. Ultimately, an adaptive immune response leads to clearance of the bacteria. Whereas much is known about the adaptive response to C. rodentium, the role of the innate immune response remains unclear. In this study, we demonstrate for the first time that the TLR adaptor MyD88 is essential for survival and optimal immunity following infection. MyD88(-/-) mice suffer from bacteremia, gangrenous mucosal necrosis, severe colitis, and death following infection. Although an adaptive response occurs, MyD88-dependent signaling is necessary for efficient clearance of the pathogen. Based on reciprocal bone marrow transplants in conjunction with assessment of intestinal mucosal pathology, repair, and cytokine production, our findings suggest a model in which TLR signaling in hemopoietic and nonhemopoietic cells mediate three distinct processes: 1) induction of an epithelial repair response that maintains the protective barrier and limits access of bacteria to the lamina propria; 2) production of KC or other chemokines that attract neutrophils and thus facilitate killing of bacteria; and 3) efficient activation of an adaptive response that facilitates Ab-mediated clearance of the infection. Taken together, these experiments provide evidence for a protective role of innate immune signaling in infections caused by attaching and effacing pathogens.

MyD88- and Bruton’s Tyrosine Kinase-Mediated Signals Are Essential for T Cell-Independent Pathogen-Specific IgM Responses

Bacteremia is one of the leading causes of death by infectious disease. To understand the immune mechanisms required for the rapid control of bacteremia, we studied Borrelia hermsii, a bacterial pathogen that colonizes the blood stream of humans and rodents to an extremely high density. A T cell-independent IgM response is essential and sufficient for controlling B. hermsii bacteremia. Mice deficient in Bruton's tyrosine kinase (Btk), despite their known defect in BCR signaling, generated B. hermsii-specific IgM and resolved bacteremia, suggesting that an alternative activation or costimulatory pathway remained functional for T cell-independent B cells in Btk(-/-) mice. B. hermsii contains putative ligands for TLRs, and we found that mice deficient in TLR1, TLR2, or the TLR adaptor MyD88 generated anti-B. hermsii IgM with delayed kinetics and suffered more severe episodes of bacteremia. In striking contrast to the anti-B. hermsii IgM response in mice deficient only in Btk, mice deficient in both Btk and MyD88 were entirely incapable of generating B. hermsii-specific Ab or resolving bacteremia. The response to a T cell-dependent model Ag was unaffected in Btk(-/-) x MyD88(-/-) mice. These results suggest that MyD88 specifically promotes T cell-independent BCR signaling and that, in the absence of Btk, this TLR-mediated stimulation is a required component of this signal.