MyD88 but not TRIF is essential for osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide, and IL-1alpha

Myeloid differentiation factor 88 (MyD88) plays essential roles in the signaling of the Toll/interleukin (IL)-1 receptor family. Toll-IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF)-mediated signals are involved in lipopolysaccharide (LPS)-induced MyD88-independent pathways. Using MyD88-deficient (MyD88-/-) mice and TRIF-deficient (TRIF-/-) mice, we examined roles of MyD88 and TRIF in osteoclast differentiation and function. LPS, diacyl lipopeptide, and IL-1alpha stimulated osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells obtained from TRIF-/- mice, but not MyD88-/- mice. These factors stimulated receptor activator of nuclear factor-kappaB ligand mRNA expression in TRIF-/- osteoblasts, but not MyD88-/- osteoblasts. LPS stimulated IL-6 production in TRIF-/- osteoblasts, but not TRIF-/- macrophages. LPS and IL-1alpha enhanced the survival of TRIF-/- osteoclasts, but not MyD88-/- osteoclasts. Diacyl lipopeptide did not support the survival of osteoclasts because of the lack of Toll-like receptor (TLR)6 in osteoclasts. Macrophages expressed both TRIF and TRIF-related adaptor molecule (TRAM) mRNA, whereas osteoblasts and osteoclasts expressed only TRIF mRNA. Bone histomorphometry showed that MyD88-/- mice exhibited osteopenia with reduced bone resorption and formation. These results suggest that the MyD88-mediated signal is essential for the osteoclastogenesis and function induced by IL-1 and TLR ligands, and that MyD88 is physiologically involved in bone turnover.     

TIR domain--containing adaptors regulate TLR-mediated signaling pathways

Recognition of pathogens by Toll-like receptors (TLRs) triggers innate immune responses via signaling pathways mediated by several Toll/IL-1R (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 that is responsible for type I interferon production in response to double-stranded RNA and LPS, respectively. TIRAP specifically participates in the MyD88-dependent pathways shared by TLR2 and TLR4, and TRAM is essential for the TLR4-mediated MyD88-independent pathway. Thus, TIR domain-containing adaptors play an important role in the TLR mediated signaling pathways.     

Role of MyD88 and TLR9 in the innate immune response elicited by serotype 5 adenoviral vectors

A replication-incompetent adenoviral (Ad) vector is generating interest for both gene therapy and immunotherapy. A major limitation of the use of Ad vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage; however, the precise mechanism of the innate immune response remains to be clarified. Here, we show that serotype 5 human Ad vectors elicit innate immune responses through a myeloid differentiating factor 88 (MyD88)/Toll-like receptor (TLR)-9-dependent and/or -independent manner according to cell type. After stimulation with Ad vectors, the production of interleukin (IL)-6 and IL-12 was significantly decreased in MyD88- or TLR9-deficient dendritic cells (DCs), compared with wild-type DCs. In addition, the surface expression of maturation marker proteins, such as CD40, CD80, CD86, and MHC class II, in MyD88- or TLR9-deficient granulocyte-macrophage colony-stimulating factor (GM-CSF)-DCs was similar to that in wild-type DCs. On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors. We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3, TLR7, and TLR9, between DCs and macrophages. The intravenous injection of luciferase-expressing Ad vectors into MyD88- or TLR9-deficient mice resulted in almost comparable levels of IL-6 and IL-12 production and luciferase expression with wild-type mice. These results suggest that Ad vectors can activate innate immunity via MyD88/TLR9-dependent and -independent mechanisms.     

The Fas-associated death domain protein suppresses activation of NF-kappa B by LPS and IL-1 beta

Activation of NF-kappa B by bacterial LPS promotes the upregulation of proinflammatory cytokines that contribute to the pathogenesis of Gram-negative septic shock. LPS activation of NF-kappa B is dependent upon the interaction of two death domain-containing (DD-containing) proteins, MyD88 and IL-1 receptor-associated kinase IRAK. Another DD-containing protein, Fas-associated death domain (FADD), also binds MyD88 through respective DD-DD interactions. Although FADD has been classically described as a proapoptotic signaling molecule, several reports have implicated a role for FADD in mediating NF-kappa B activation. In the present report, we investigated whether FADD could mediate LPS activation of NF-kappa B. Overexpression of FADD blocked LPS-induced NF-kappa B activation, whereas absence of FADD enhanced activation of NF-kappa B by LPS. Further, LPS-induced expression of two NF-kappa B-dependent gene products, IL-6 and KC, was enhanced in FADD(-/-) mouse embryo fibroblasts (MEFs) compared with wild-type. This increase in NF-kappa B activity correlated with enhanced I kappa B degradation. FADD(-/-) MEFs were also resistant to NF-kappa B activation induced by IL-1 beta. Finally, reconstitution of full-length FADD in the FADD(-/-) MEFs completely reversed the enhanced activation of NF-kappa B elicited by either LPS or IL-1 beta. Together, these data indicate that FADD negatively regulates LPS- and IL-1 beta-induced NF-kappa B activation and that this regulation occurs upstream of I kappa B degradation.     

Leukotriene B4 amplifies NF-κB activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-κB and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B4 (LTB4) are pivotal components of host defense and inflammatory responses. However, the role of LTB4 in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1β and IL-18) are reduced in mice lacking either 5-LO or the LTB4 receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-κB. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-κB through Stat1-dependent expression of MyD88.     

Microbiota-induced IL-1β, but not IL-6, is critical for the development of steady-state TH17 cells in the intestine

T(H)17 cells are a lineage of CD4(+) T cells that are critical for host defense and autoimmunity by expressing the cytokines IL-17A, IL-17F, and IL-22. A feature of T(H)17 cells at steady state is their ubiquitous presence in the lamina propria of the small intestine. The induction of these steady-state intestinal T(H)17 (sT(H)17) cells is dependent on the presence of the microbiota. However, the signaling pathway linking the microbiota to the development of intestinal sT(H)17 cells remains unclear. In this study, we show that IL-1β, but not IL-6, is induced by the presence of the microbiota in intestinal macrophages and is required for the induction of sT(H)17 cells. In the absence of IL-1β-IL-1R or MyD88 signaling, there is a selective reduction in the frequency of intestinal sT(H)17 cells and impaired production of IL-17 and IL-22. Myeloid differentiation factor 88-deficient (MyD88(-/-)) and germ-free (GF) mice, but not IL-1R(-/-) mice, exhibit impairment in IL-1β induction. Microbiota-induced IL-1β acts directly on IL-1R-expressing T cells to drive the generation of sT(H)17 cells. Furthermore, administration of IL-1β into GF mice induces the development of retinoic acid receptor-related orphan receptor γt-expressing sT(H)17 cells in the small intestine, but not in the spleen. Thus, commensal-induced IL-1β production is a critical step for sT(H)17 differentiation in the intestine, which may have therapeutic implications for T(H)17-mediated pathologies.     

Early interleukin 12 production by macrophages in response to mycobacterial infection depends on interferon gamma and tumor necrosis factor alpha

Interleukin 12 (IL-12) produced by macrophages immediately after infection is considered essential for activation of a protective immune response against intracellular pathogens. In the murine Mycobacterium bovis Bacillus Calmette-Guerin (BCG) model we assessed whether early IL- 12 production by macrophages depends on other cytokines. In vitro, murine bone marrow-derived macrophages produced IL-12 after infection with viable M. bovis BCG or stimulation with LPS, however, priming with recombinant interferon gamma (rIFN-gamma) was necessary. In addition, IL-12 production by these macrophages was blocked by specific anti- tumor necrosis factor alpha (TNF-alpha) antiserum. Macrophages from gene deletion mutant mice lacking either the IFN-gamma receptor or the TNF receptor 1 (p55) failed to produce IL-12 in vitro after stimulation with rIFN-gamma and mycobacterial infection. In vivo, IL-12 production was induced in spleens of immunocompetent mice early during M. bovis BCG infection but not in those of mutant mice lacking the receptors for IFN-gamma or TNF. Our results show that IL-12 production by macrophages in response to mycobacterial infection depends on IFN-gamma and TNF. Hence, IL-12 is not the first cytokine produced in mycobacterial infections.     

LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF

Toll-IL-1-resistance (TIR) domain-containing adaptor-inducing IFN-beta (TRIF)-related adaptor molecule (TRAM) is the fourth TIR domain-containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-kappaB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-alpha/beta, regulated on activation, normal T cell expressed and secreted (RANTES), and gamma interferon-inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.     

Tumor development in murine ulcerative colitis depends on MyD88 signaling of colonic F4/80+CD11b(high)Gr1(low) macrophages

Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13-producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11b(high)Gr1(low) M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate-induced colitis, F4/80+CD11b(high)Gr1(intermediate) macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88-deficient (Myd88-/-) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88-/- mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11b(high)Gr1(intermediate) macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.     

Fatal Mycobacterium tuberculosis infection despite adaptive immune response in the absence of MyD88

Toll-like receptors (TLRs) such as TLR2 and TLR4 have been implicated in host response to mycobacterial infection. Here, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88) were infected with Mycobacterium tuberculosis (MTB). While primary MyD88(-/-) macrophages and DCs are defective in TNF, IL-12, and NO production in response to mycobacterial stimulation, the upregulation of costimulatory molecules CD40 and CD86 is unaffected. Aerogenic infection of MyD88(-/-) mice with MTB is lethal within 4 weeks with 2 log(10) higher CFU in the lung; high pulmonary levels of cytokines and chemokines; and acute, necrotic pneumonia, despite a normal T cell response with IFN-gamma production to mycobacterial antigens upon ex vivo restimulation. Vaccination with Mycobacterium bovis bacillus Calmette-Guerin conferred a substantial protection in MyD88(-/-) mice from acute MTB infection. These data demonstrate that MyD88 signaling is dispensable to raise an acquired immune response to MTB. Nonetheless, this acquired immune response is not sufficient to compensate for the profound innate immune defect and the inability of MyD88(-/-) mice to control MTB infection.