Toll-like receptor signal adaptor protein MyD88 is required for sustained endotoxin-induced acute hypoferremic response in mice

Hypoferremia, associated with immune system activation, involves a marked reduction in the levels of circulating iron, coupled with iron sequestration within macrophages. Toll-like receptor (TLR) signaling plays an important role in the development of the hypoferremic response, but how downstream signaling events affect genes involved in iron metabolism is incompletely understood. We investigated the involvement of MyD88-dependent (MyD88) and MyD88-independent (TRIF) TLR signaling in the development of hypoferremia. Using MyD88-deficient and TRIF-deficient mice, we show that MyD88 and TRIF signaling pathways are critical for up-regulation by lipopolysaccharide (LPS) of the iron regulator hepcidin. In addition, MyD88 signaling is required for the induction of lipocalin 2 secretion and iron sequestration in the spleen. Activation of TLR4 and TLR3 signaling through LPS and polyinosinic:polycytidylic acid [poly(I:C)] treatments resulted in rapid down-regulation of HFE protein [encoded by the hemochromatosis gene (Hfe)] and ferroportin [encoded by solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1)] expression in the spleen, independent of MyD88 or TRIF signaling and proinflammatory cytokine production. However, lack of MyD88 signaling significantly impaired the hypoferremic response triggered by LPS, indicating that ferroportin and HFE protein down-regulation alone are insufficient to maintain hypoferremia. The extent of the hypoferremic response was found to be limited by initial, basal iron levels. Together, these results suggest that targeting specific TLR signaling pathways by affecting the function of adaptor molecules may provide new strategies to counteract iron sequestration within macrophages during inflammation.     

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.     

MyD88 and TRIF synergistic interaction is required for TH1‐cell polarization with a synthetic TLR4 agonist adjuvant

Glucopyranosyl lipid adjuvant‐stable emulsion (GLA‐SE) is a synthetic adjuvant TLR4 agonist that promotes potent poly‐functional T_H1 responses. Different TLR4 agonists may preferentially signal via MyD88 or TIR‐domain‐containing adapter inducing IFN‐beta (TRIF) to exert adjuvant effects; however, the contribution of MyD88 and TRIF signaling to the induction of polyclonal T_H1 responses by TLR4 agonist adjuvants has not been studied in vivo. To determine whether GLA‐SE preferentially signals through MyD88 or TRIF, we evaluated the immune response against a candidate tuberculosis (TB) vaccine Ag following immunization of mice lacking either signaling adapter compared with that of wild‐type mice. We find that both MyD88 and TRIF are necessary for GLA‐SE to induce a poly‐functional T_H1 immune response characterized by CD4⁺ T cells producing IFN‐γ, TNF, and IL‐2, as well as IgG2c class switching, when paired with the TB vaccine Ag ID93. Accordingly, the protective efficacy of ID93/GLA‐SE immunization against aerosolized Mycobacterium tuberculosis was lost when either signaling molecule was ablated. We demonstrate that MyD88 and TRIF must be expressed in the same cell for the in vivo T_H1‐skewing adjuvant activity, indicating that these two signaling pathways cooperate on an intracellular level. Thus engagement of both the MyD88 and TRIF signaling pathways are essential for the effective adjuvant activity of this TLR4 agonist.     

1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine Suppresses Toll-Like Receptor 4 Dimerization Induced by Lipopolysaccharide

Toll-like receptor 4 (TLR4) recognizes LPS and triggers the activation of the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter, inducing interferon-β (TRIF)-dependent major downstream signaling pathways. Previously, we presented biochemical evidence that 1-[4-Fluoro-2-(2-nitrovinyl)phenyl]pyrrolidine (FPP), which was synthesized in our laboratory, inhibits NF-κB activation induced by LPS. Here, we investigated whether FPP modulates the TLR4 downstream signaling pathways and what anti-inflammatory target in TLR4 signaling is regulated by FPP. FPP inhibited LPS-induced NF-κB activation by targeting TLR4 dimerization. These results suggest that FPP can modulate the TLR4 signaling pathway at the receptor level to decrease inflammatory gene expression.     

A variety of microbial components induce tolerance to lipopolysaccharide by differentially affecting MyD88-dependent and -independent pathways

Exposure of macrophages to lipopolysaccharide (LPS) induces a hypo-responsive state to a second challenge with LPS that is termed LPS tolerance. LPS tolerance is also induced by pre-exposure to lipopeptides and lipoteichoic acid, which trigger Toll-like receptor (TLR) 2-mediated signaling. LPS signaling involves at least two pathways: a MyD88-dependent cascade that is essential for production of inflammatory cytokines and a MyD88-independent cascade that mediates the expression of IFN-inducible genes. We analyzed the induction of LPS tolerance by several microbial components in mouse peritoneal macrophages. Pre-exposure to LPS led to impaired activation of both the pathways. In contrast, mycoplasmal lipopeptides did not affect the MyD88-independent pathway, but impaired the MyD88-dependent signaling by inhibiting LPS-mediated activation of IL-1 receptor-associated kinase (IRAK) 1. The induction of LPS tolerance by recently identified TLR ligands was analyzed. Pretreatment with double-stranded RNA, which triggers the activation of TLR3, led to defective activation of the MyD88-independent, but not the MyD88-dependent, pathway. Imidazoquinoline compounds, which are recognized by TLR7, had no effect on the MyD88-independent pathway, but inhibited LPS-induced activation of MyD88-dependent signaling through down-regulation of IRAK1 expression. Thus, each microbial component induced LPS tolerance in macrophages.     

Toll‐like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin‐specific allergic airway disease: role of MyD88 adaptor molecule and interleukin‐12/interferon‐γ axis

Background Epidemiological and experimental data suggest that bacterial lipopolysaccharides (LPS) can either protect from or exacerbate allergic asthma. Lipopolysaccharides trigger immune responses through toll‐like receptor 4 (TLR4) that in turn activates two major signalling pathways via either MyD88 or TRIF adaptor proteins. The LPS is a pro‐Type 1 T helper cells (Th1) adjuvant while aluminium hydroxide (alum) is a strong Type 2 T helper cells (Th2) adjuvant, but the effect of the mixing of both adjuvants on the development of lung allergy has not been investigated. Objective We determined whether natural (LPS) or synthetic (ER‐803022) TLR4 agonists adsorbed onto alum adjuvant affect allergen sensitization and development of airway allergic disease. To dissect LPS‐induced molecular pathways, we used TLR4‐, MyD88‐, TRIF‐, or IL‐12/IFN‐γ‐deficient mice. Methods Mice were sensitized with subcutaneous injections of ovalbumin (OVA) with or without TLR4 agonists co‐adsorbed onto alum and challenged with intranasally with OVA. The development of allergic lung disease was evaluated 24 h after last OVA challenge. Results Sensitization with OVA plus LPS co‐adsorbed onto alum impaired in dose‐dependent manner OVA‐induced Th2‐mediated allergic responses such as airway eosinophilia, type‐2 cytokines secretion, airway hyper‐reactivity, mucus hyper production and serum levels of IgE or IgG1 anaphylactic antibodies. Although the levels of IgG2a, Th1‐affiliated isotype increased, investigation into the lung‐specific effects revealed that LPS did not induce a Th1 pattern of inflammation. Lipopolysaccharides impaired the development of Th2 immunity, signaling via TLR4 and MyD88 molecules and via the IL‐12/IFN‐γ axis, but not through TRIF pathway. Moreover, the synthetic TLR4 agonists that proved to have a less systemic inflammatory response than LPS also protected against allergic asthma development. Conclusion Toll‐like receptor 4 agonists co‐adsorbed with allergen onto alum down‐modulate allergic lung disease and prevent the development of polarized T cell‐mediated airway inflammation.     

SARM inhibits both TRIF‐ and MyD88‐mediated AP‐1 activation

SARM (sterile α‐ and armadillo‐motif‐containing protein), the fifth identified TIR (Toll–interleukin 1 receptor (IL‐1R)) domain‐containing adaptors in humans, downregulates NF‐κB and IRF3 (interferon‐regulatory factor 3)‐mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR‐domain‐containing adaptor protein inducing IFN‐β)‐dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF‐ and MyD88‐mediated, as well as basal MAPK activity, were repressed, indicating that SARM‐mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP‐1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N‐terminal and sterile alpha motif domains in the autoregulation of SARM activity.     

Rapid CD4+ T‐cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9

Toll‐like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as‐MyD88 or (TRIF TIR‐domain‐containing adapter‐inducing interferon‐β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin‐specific T‐cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR‐signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin‐specific CD4⁺ T‐cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin‐specific T‐cell responses.     

Educating CD4 T cells with vaccine adjuvants: lessons from lipopolysaccharide

Toll-like receptor (TLR) adjuvants are capable of driving T cell immunity. The TLR4 agonist LPS activates antigen-presenting cells through myeloid differentiation primary response gene 88 (MyD88) and TIR domain-containing adaptor inducing interferon-beta (TRIF)-dependent signaling pathways, initiating CD4 T helper cell clonal expansion and differentiation. Lipopolysaccharide (LPS) supports the development of diverse T helper (Th) lineages depending on the tissue microenvironment. For instance, peripheral immunization with LPS drives Th1 priming in lymphoid tissue and Th17 priming in the gut. This could be due to commensal bacteria inducing Th17-stabilizing cytokines within the intestinal lamina propria. Here, we detail how the response to LPS stimulates CD4 T cell priming in lymphoid tissue and the intestinal mucosa. How this knowledge might be exploited to target specific features of T cell immunity by vaccine adjuvants is also considered.     

Downregulation of Paralemmin-3 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury in Rats by Regulating Inflammatory Response and Inhibiting Formation of TLR4/MyD88 and TLR4/TRIF Complexes

Previous studies have demonstrated paralemmin-3 (PALM3) participates in Toll-like receptor (TLR) signaling. This study investigated the effect of PALM3 knockdown on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its underlying mechanisms. We constructed a recombinant adenoviral vector containing short hairpin RNA for PALM3 to knockdown PALM3 expression. A transgene-free adenoviral vector was used as a negative control. The ALI rat model was established by LPS peritoneal injection at 48-h post-transfection. Results showed that downregulation of PALM3 improved the survival rate, attenuated lung pathological changes, alleviated pulmonary edema, lung vascular leakage and neutrophil infiltration, inhibited the production of proinflammatory cytokines and activation of nuclear factor κB and interferon β regulatory factor 3, and promoted the secretion of anti-inflammatory cytokine interleukin-10 and expression of suppressor of cytokine signaling-3 in the ALI rat model. However, PALM3 knockdown had no effect on TLR4, myeloid differentiation factor 88 (MyD88), and Toll-interleukin-1 receptor domain-containing adaptor inducing interferon β (TRIF) expression. Moreover, PALM3 knockdown reduced the interaction of TLR4 with MyD88 or TRIF induced by LPS in rat lungs. Therefore, the downregulation of PALM3 protected rats from LPS-induced ALI and its mechanisms were partially associated with the modulation of inflammatory responses and inhibition of TLR4/MyD88 and TLR4/TRIF complex formation.     

Toll-like receptor-mediated tyrosine phosphorylation of paxillin via MyD88-dependent and -independent pathways

Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity. A proximal signaling event of TLR is the direct binding of an adaptor protein MyD88 to TLR and recruitment of the IL-1R-associated kinase (IRAK). In the present study, we examined the effect of several TLR ligands on protein tyrosine phosphorylation in rat macrophages. Macrophage-activating lipopeptide-2 kDa (MALP2) and lipoarabinomannan were used as activators of TLR2, while lipopolysaccharides (LPS) and lipoteichoic acid were used as TLR4 ligands. All these ligands induced tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) and its substrate paxillin, an integrin-associated focal adhesion adaptor protein, in the macrophages. PP2, an inhibitor of Src family tyrosine kinases, prevented the TLR-induced phosphorylation of paxillin and Pyk2 without affecting TLR-induced IRAK activation. MALP2 failed to induce paxillin phosphorylation in the macrophages from MyD88-knockout mice. In contrast, the effect of LPS weakened, but was still observed even in the MyD88-deficient cells. Thus, TLR regulate the function of paxillin in an Src family-dependent mechanism through both MyD88-dependent and MyD88-independent pathways.     

The CD20 homolog Ms4a8a integrates pro‐ and anti‐inflammatory signals in novel M2‐like macrophages and is expressed in parasite infection

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor‐associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a⁺ macrophages in M2‐associated infectious pathologies. In late‐stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll‐like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL‐4). LPS/dexamethasone/IL‐4‐induced Ms4a8a⁺ BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF‐κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL‐4‐stimulated Ms4a8a⁺ BMDMs and in peritoneal macrophages from late‐stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2‐like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.     

Toll-like receptor 4 is a sensor for autophagy associated with innate immunity

Autophagy has recently been shown to be an important component of the innate immune response. The signaling pathways leading to activation of autophagy in innate immunity are not known. Here we showed that Toll-like receptor 4 (TLR4) served as a previously unrecognized environmental sensor for autophagy. Autophagy was induced by lipopolysaccharide (LPS) in primary human macrophages and in the murine macrophage RAW264.7 cell line. We defined a new molecular pathway in which LPS-induced autophagy was regulated through a Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF)-dependent, myeloid differentiation factor 88 (MyD88)-independent TLR4 signaling pathway. Receptor-interacting protein (RIP1) and p38 mitogen-activated protein kinase were downstream components of this pathway. This signaling pathway did not affect cell viability, indicating that it is distinct from the autophagic death signaling pathway. We further showed that LPS-induced autophagy could enhance mycobacterial colocalization with the autophagosomes. This study links two ancient processes, autophagy and innate immunity, together through a shared signaling pathway.     

TIR domain-containing adaptors define the specificity of TLR signaling

The concept that Toll-like receptors (TLRs) recognize specific molecular patterns in various pathogens has been established. In signal transduction via TLRs, MyD88, which harbors a Toll/IL-1 receptor (TIR)-domain and a death domain, has been shown to link between TLRs and MyD88-dependent downstream events leading to proinflammatory cytokine production and splenocyte proliferation. However, recent studies using MyD88-deficient mice have revealed that some TLRs possess a MyD88-independent pathway, which is represented by interferon (IFN)-beta production induced by LPS stimulation. This indicates that additional signaling molecules other than MyD88 exist in the TLR signaling pathway. Indeed, two additional TIR domain-containing adaptors, TIRAP/Mal and TRIF, have recently been identified. Both define the specific biological responses of each TLR.     

Nonredundant roles of TIRAP and MyD88 in airway response to endotoxin, independent of TRIF, IL-1 and IL-18 pathways

Inhaled endotoxins induce an acute inflammatory response in the airways mediated through Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). However, the relative roles of the TLR4 adaptor proteins TIRAP and TRIF and of the MyD88-dependent IL-1 and IL-18 receptor pathways in this response are unclear. Here, we demonstrate that endotoxin-induced acute bronchoconstriction, vascular damage resulting in protein leak, Th1 cytokine and chemokine secretion and neutrophil recruitment in the airways are abrogated in mice deficient for either TIRAP or MyD88, but not in TRIF deficient mice. The contribution of other TLR-independent, MyD88-dependent signaling pathways was investigated in IL-1R1, IL-18R and caspase-1 (ICE)-deficient mice, which displayed normal airway responses to endotoxin. In conclusion, the TLR4-mediated, bronchoconstriction and acute inflammatory lung pathology to inhaled endotoxin critically depend on the expression of both adaptor proteins, TIRAP and MyD88, suggesting cooperative roles, while TRIF, IL-1R1, IL-18R signaling pathways are dispensable.     

B cell‐intrinsic MyD88 signaling controls IFN‐γ‐mediated early IgG2c class switching in mice in response to a particulate adjuvant

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR‐dependent and ‐independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR‐independent MOAs of adjuvants is poorly understood. Here, using the T‐dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class‐switch recombination (CSR) in mice. Using cell‐type‐specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell‐intrinsic MyD88 signaling. Notably, IFN‐γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B‐cell intrinsic MyD88 is required for IFN‐γ production. Moreover, IFN‐γ receptor (IFNγR) deficiency abolished sHZ‐induced IgG2c production, while recombinant IFN‐γ administration successfully rescued IgG2c CSR impairment in mice lacking B‐cell intrinsic MyD88. Together, our results show that B cell‐intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.     

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.