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416 VOLUME 12 NUMBER 5 MAY 2011 nature immunology
A rt i c l e s
The ability of the innate immune system to recognize and eliminate invading microbial pathogens has been largely attributed to Toll-like receptors (TLRs) and TLR-triggered immune response. TLRs, the key pattern-recognition receptors expressed on antigen-presenting cells (APCs) such as macrophages and dendritic cells (DCs), have important roles in the initiation of innate immune responses as well as the subsequent induction of adaptive immune responses1. After the recognition of pathogen-associated molecule patterns, TLRs initiate shared and distinct signaling pathways by recruiting various combina-tions of four Toll–interleukin 1 (IL-1) receptor domain–containing adapters proteins: MyD88, TIRAP (Mal), TRIF and TRAM. These signaling pathways activate the transcription factors NF-κB and AP-1, which are common to all TLRs, leading to the production of inflammatory cytokines and chemokines. TLR3, TLR4, TLR7, TLR8 and TLR9 also activate the transcription factors IRF3 and/or IRF7, leading to the production of type I interferon2–4. Full activation of TLRs is essential for initiation of the innate immune response and enhancement of adaptive immunity to eliminate invading pathogens; however, TLR signaling is well regulated, positively and negatively, to prevent inappropriate activation or overactivation, which may cause autoinflammatory disorders5. So far, various signaling molecules have been shown to be involved in the tight regulation of the TLR pathway to maintain the immunological balance4,6. For example, the kinases MEKK3 (ref. 7) and CaMKII (ref. 8) can be activated by TLR ligands and then enhance TLR-triggered innate immune responses. However, the identification of cofactors and their underlying mechanisms for the initiation and full activation of TLR responses remain to be fully elucidated.
Major histocompatibility complex (MHC) class II molecules (encoded by H2) are expressed mainly by professional APCs, includ-ing DCs, macrophages and B cells. MHC class II molecules, which are heterodimers composed of an α-chain and a β-chain, are type I integral membrane proteins with short cytoplasmic domains and four large extracellular domains9. Their main function is to present peptides processed from extracellular proteins to CD4+ helper T cells and to direct the processes of positive and negative selection, shaping the repertoire during T cell maturation and lineage commitment10,11. In addition to that classical function, cell surface MHC class II mol-ecules can function as receptors to mediate reverse signal transduc-tion after ligation with agonist antibodies, T cell antigen receptors or CD4 molecules. Engagement of cell surface MHC class II may regulate cell adhesion, cytokine production and the expression of costimulatory molecules12–15 and may also induce the apoptosis, proliferation or differentiation of B cells16,17. Engagement of MHC class II mainly activates two distinct signal pathways. One increases cAMP and subsequently induces the translocation of protein kinase C to the nucleus18,19. Another increases activity of the Src family tyrosine kinase Lyn and the non-Src family tyrosine kinase Syk. These activated tyrosine kinases mediate activation of phospholipase C-γ, leading to the production of inositol-(1,4,5)-trisphosphate and diacylglycerol, which mediate calcium mobilization and activation of protein kinase C20,21.
MHC class II molecules can contribute to the responsiveness of cells to microbial components, and pathogens have developed strategies to downregulate expression of MHC class II molecules on APCs and thereby evade immunological surveillance22,23. For example,
1National Key Laboratory of Medical Immunology & Institute of Immunology, Second Military Medical University, Shanghai, China. 2Institute of Immunology, Zhejiang University School of Medicine, Hangzhou, China. 3Chinese Academy of Medical Sciences, Beijing, China. Correspondence should be addressed to X.C. ([email protected]).
Received 25 October 2010; accepted 28 February 2011; published online 27 March 2011; doi:10.1038/ni.2015
Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase BtkXingguang Liu1, Zhenzhen Zhan1, Dong Li2, Li Xu1, Feng Ma2, Peng Zhang1, Hangping Yao2 & Xuetao Cao1,3
The molecular mechanisms involved in the full activation of innate immunity achieved through Toll-like receptors (TLRs) remain to be fully elucidated. In addition to their classical antigen-presenting function, major histocompatibility complex (MHC) class II molecules might mediate reverse signaling. Here we report that deficiency in MHC class II attenuated the TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and dendritic cells, which protected mice from endotoxin shock. Intracellular MHC class II molecules interacted with the tyrosine kinase Btk via the costimulatory molecule CD40 and maintained Btk activation, but cell surface MHC class II molecules did not. Then, Btk interacted with the adaptor molecules MyD88 and TRIF and thereby promoted TLR signaling. Therefore, intracellular MHC class II molecules can act as adaptors, promoting full activation of TLR-triggered innate immune responses.
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deficiency in MHC class II results in less production of lipopoly-saccharide (LPS)-induced tumor necrosis factor (TNF) by human peripheral blood monocytes and mouse macrophages24,25. However, the detailed mechanisms by which MHC class II molecules are involved in TLR-triggered innate immune responses remain uncharacterized.
MHC class II molecules and TLRs are both expressed mainly on APCs; therefore, we sough to determine whether MHC class II mol-ecules have another, nonclassical function and somehow intersect with the TLR signaling pathway. Here we found that MHC class II–deficient mice were more resistant to endotoxin shock induced by either lethal challenge with LPS or infection with Gram-negative bacteria, with less production of proinflammatory cytokines and type I interferon in vivo. Deficiency in MHC class II attenuated the pro-duction of proinflammatory cytokines and type I interferon triggered by TLR4, TLR3 or TLR9 in macrophages and DCs. Furthermore, intracellular MHC class II molecules interacted with the tyrosine kinase Btk via the costimulatory molecule CD40 in endosomes and maintained activation of Btk, but cell surface MHC class II molecules did not. Activated Btk interacted with MyD88 and TRIF, promoting the activation of MyD88-dependent and TRIF-dependent pathways. Therefore, intracellular MHC class II molecules are needed to pro-mote the full activation of TLR signaling.
RESULTSH2 deficiency protects mice from LPS and bacterial challengeMHC class II–deficient (H2−/−) mice (with a 78.8-kilobase deletion in H2) and wild-type (H2+/+) littermate mice had similar numbers of splenic macrophages and DCs, as well as peritoneal macrophages and bone marrow–derived macrophages and DCs (Supplementary Fig. 1a,b). Furthermore there were no substantial differences between H2−/− and H2+/+ mice in the expression of CD40, CD80 or CD86 on splenic DCs or immature or mature bone marrow–derived DCs (Supplementary Fig. 1c). Therefore, H2−/− mice had normal mye-loid development and macrophage differentiation. To investigate the role of MHC class II molecules in the TLR-triggered innate immune response, we challenged H2−/− mice with the TLR ligands LPS, CpG ODN or poly(I:C). H2−/− mice produced significantly less TNF, IL-6 and interferon-β (IFN-β) than H2+/+ mice did in response to challenge with LPS, CpG ODN or poly(I:C) (Fig. 1a–c). Accordingly, H2−/− mice had prolonged survival relative to that of H2+/+ mice after lethal
challenge with LPS (Fig. 1d). H2−/− mice were also more resistant to lethal challenge with high-dose poly(I:C) (data not shown). Given that H2−/− mice have considerably fewer CD4+ T cells in thymus, spleen and lymph nodes, we further explored the effects of the lack of CD4+ T cells on the resistance of H2−/− mice to sepsis. We transplanted bone marrow cells from H2+/+ or H2−/− mice into lethally irradi-ated wild-type mice. The reconstituted H2−/− mixed–bone marrow chimeras had numbers of CD4+ T cells in spleen and lymph nodes similar to those in H2+/+ chimeras but had no expression of MHC class II in macrophages or DCs (data not shown). The LPS-induced in vivo production of TNF, IL-6 and IFN-β was much lower in the reconstituted H2−/− chimeras than in H2+/+ chimeras (Fig. 1e), which therefore excluded the possibility that the lack of CD4+ T cells was involved in the resistance of H2−/− mice to sepsis. These data demon-strate that H2−/− mice showed impaired TLR-triggered inflammatory innate responses and were more resistant to endotoxin shock, which indicates that MHC class II molecules have an important role in the full activation of TLR-triggered immune responses.
To assess the role of MHC class II molecules in the host innate response to infection with intact Gram-negative bacteria, we injected H2−/− mice intraperitoneally with Escherichia coli serotype 0111:B4, the most frequent cause of bacterial sepsis in humans. The production of TNF and IL-6 in the serum of H2−/− mice after injection of E. coli was significantly less than that in H2+/+ mice (Fig. 2a,b). The survival of H2−/− mice after lethal challenge with E. coli was also prolonged (Fig. 2c). These data indicate that deficiency in MHC class II attenuates the inflammatory innate response of host to Gram-negative bacteria and protects mice from lethal challenge by Gram-negative bacteria.
Impaired cytokine production in TLR-triggered H2−/− APCsNext we assessed whether deficiency in MHC class II attenu-ated the production of proinflammatory cytokines and type I
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Figure 1 Deficiency in MHC class II protects mice from challenge with TLR ligands. (a–c) Enzyme-linked immunosorbent assay (ELISA) of TNF (a), IL-6 (b) and IFN-β (c) in the serum of H2−/− or H2+/+ mice (n = 5 per genotype) 2 h after intraperitoneal administration of PBS or LPS, CpG-ODN (CpG) or poly(I:C) (at a dose of 15, 20 or 20 mg per kg body weight, respectively). *P < 0.01 (Student’s t-test). (d) Survival of H2−/− mice and H2+/+ mice (n = 10 per genotype) given intraperitoneal injection of LPS (15 mg per kg body weight). P < 0.01 (Wilcoxon test). (e) ELISA of TNF, IL-6 and IFN-β in serum from wild-type mice lethally irradiated and given intravenous transplantation of 1 × 107 bone marrow cells from H2−/− or H2+/+ mice 3 weeks before challenge with PBS or LPS, assessed 2 h after challenge. *P < 0.01 (Student’s t-test). Data are from three independent experiments (mean ± s.e.m.).
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Figure 2 Deficiency in MHC class II protects mice from sepsis induced by live E. coli. (a,b) ELISA of TNF (a) and IL-6 (b) in serum from H2−/− or H2+/+ mice (n = 3 per genotype) 4 h after intraperitoneal infection with E. coli 0111:B4 (1 × 107 colony-forming units per mouse). *P < 0.01 (Student’s t-test). (c) Survival of mice (n = 10 per genotype) treated as described in a,b. P < 0.01 (Wilcoxon test). Data are from three independent experiments (mean ± s.e.m.).
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interferon in TLR-triggered macrophages and DCs in vitro. H2−/− peritoneal macrophages had lower expression of TNF, IL-6, IFN-α and IFN-β mRNA and protein than did macrophages from H2+/+ mice in response to LPS, CpG-ODN or poly(I:C) (Fig. 3a and Supplementary Fig. 2). Similarly, we also detected less production of TNF, IL-6 and IFN-β in H2−/− DCs (Fig. 3a). H2−/− peritoneal macrophages responded normally to the phorbol ester PMA, to IL-1β and to MDP (the ligand for the intracellular bacteria sensor Nod2) and produced amounts of TNF and IL-6 similar to those produced by H2+/+ macrophages (Supplementary Fig. 3a), which indicated that deficiency in MHC class II selectively impaired the activation of TLR signaling. We also did a ‘rescue’ experiment by transfecting expression vectors encoding MHC class II α-chain and β-chain into H2−/− peritoneal macrophages and found that overexpression of both MHC class II α-chain and β-chain restored the production of TNF, IL-6 and IFN-β induced by LPS, CpG-ODN or poly(I:C), whereas overexpression of either α-chain or β-chain alone did not (Fig. 3b,c); this suggested that both the α-chain and β-chain are required for full activation of TLR-triggered macro-phages. In addition, there was no substantial difference between H2−/− and H2+/+ mice in the expression of TLR4, TLR3 or TLR9 protein and mRNA in peritoneal macrophages and bone marrow–derived DCs (Supplementary Fig. 3b,c). Overexpression of MHC class II α-chain and β-chain in H2−/− peritoneal macrophages did not affect the expression of TLR4, TLR3 or TLR9 (Supplementary Fig. 4). Thus, MHC class II expression did not affect the expres-sion pattern of TLRs, which excluded the possibility that the
attenuation of TLR responses achieved by deficiency in MHC class II was due simply to lower expression of TLRs.
We further observed the effect of knockdown of MHC class II on cytokine production by TLR-activated macrophages. The endog-enous expression of total or cell surface MHC class II in peritoneal macrophages was diminished considerably by MHC class II–specific small interfering RNA (siRNA; Fig. 3d). MHC class II–specific siRNA resulted in significantly less production of TNF, IL-6 and IFN-β in peritoneal macrophages stimulated with LPS, CpG-ODN or poly(I:C) (Fig. 3e). To further confirm that the impaired TLR-triggered inflam-matory response in vivo was due to deficiency in MHC class II in myeloid cells, we adoptively transferred H2+/+ or H2−/− bone marrow–derived macrophages into wild-type mice depleted of endogenous macrophages by pretreatment with clodronate liposomes. Mice reconstituted with H2−/− macrophages produced less proinflamma-tory cytokines and IFN-β in response to LPS, CpG-ODN or poly(I:C) challenge than did mice reconstituted with H2+/+ macrophages (Fig. 3f). Therefore, deficiency in MHC class II attenuated the TLR-triggered production of proinflammatory cytokines and type I interferon in APCs, including macrophages and DCs.
Impaired TLR signaling in H2−/− macrophagesWe further investigated the effect of deficiency in MHC class II on TLR-activated downstream signal pathways in macrophages. We observed impaired phosphorylation of the kinases Erk, Jnk and p38 and inhibitor IκBα in LPS-stimulated H2−/− peritoneal macrophages (Fig. 4a). Deficiency in MHC class II resulted in less LPS-induced
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Figure 3 Deficiency in MHC class II attenuates TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and DCs. (a) ELISA of cytokines in supernatants of H2−/− or H2+/+ macrophages (top row) or DCs (bottom row) left unstimulated (Med) or stimulated for 6 h with LPS (100 ng/ml), CpG ODN (CpG; 0.3 µM) or poly(I:C) (10 µg/ml). (b,c) ELISA of cytokines in supernatants of H2−/− or H2+/+ macrophages given mock transfection (Mock) or transfected with vectors for the expression of MHC class II α-chain and β-chain (α+β-chain) or MHC class II α-chain alone (α-chain) or β-chain alone (β-chain) and, 36 h later, stimulated for 6 h with LPS (b), or CpG ODN or poly(I:C) (c). (d) Immunoblot analysis of the expression of MHC class II β-chain (MHCIIβ) and β-actin in lysates (left) and flow cytometry analysis of the surface expression of MHC class II (MHCII; right) of macrophages 48 h after transfection with control siRNA (Ctrl (left) or solid line with no shading (right)) or siRNA specific for MHC class II β-chain (siRNA (left) or solid line with gray shading (right)). Dotted line (right), isotype-matched control antibody. (e) ELISA of cytokines in supernatants of macrophages transfected as in d and, 48 h later, left unstimulated or stimulated for 6 h with LPS, CpG ODN or poly(I:C). (f) ELISA of cytokines in serum from wild-type mice first depleted of endogenous macrophages and then transplanted with 1 × 107 H2−/− or H2+/+ bone marrow–derived cells 6 h before challenge with LPS, CpG ODN or poly(I:C), measured 2 h after challenge. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from three independent experiments (a–c,e–f; mean ± s.e.m.) or are representative of three independent experiments with similar results (d).
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phosphorylation and nuclear translocation of IRF3 (Fig. 4a,b). We obtained similar results with H2−/− peritoneal macrophages stim-ulated with CpG ODN or poly(I:C) (Supplementary Fig. 5). We further evaluated the effect of deficiency in MHC class II on the transactivation of NF-κB, AP-1, IRF3 and IRF7. Transactivation of reporters for NF-κB and AP-1 was lower in H2−/− peritoneal macro-phages stimulated with LPS, CpG ODN or poly(I:C) (Fig. 4c). Deficiency in MHC class II also impaired the transactivation of an IRF3 reporter induced by LPS or poly(I:C) and the transactivation of an IRF7 reporter induced by CpG ODN (Fig. 4c). To assess the func-tional integrity of intracellular signal pathway in H2−/− macrophages, we monitored the activation of mitogen-activated protein kinases and NF-κB induced by TNF. Phosphorylation of Jnk, p38 and IκBα in TNF-stimulated H2−/− peritoneal macrophages was similar to that in TNF-stimulated H2+/+ macrophages (Supplementary Fig. 6), which indicated that deficiency in MHC class II selectively impaired TLR-triggered activation of mitogen-activated protein kinases and NF-κB. Immunoprecipitation showed that the interaction of MyD88 with the kinase IRAK1 or of TRIF with the kinase TBK1 was much lower in H2−/− macrophages stimulated with LPS than in H2+/+ macrophages stimulated with LPS (Fig. 4d,e). In vitro kinase assays showed that the activation of IRAK1, TAK1 and TBK1 induced by LPS, CpG ODN or poly(I:C) was impaired in H2−/− macrophages relative to that in H2+/+ macrophages (Fig. 4f). Collectively, these data suggest that MHC class II molecules promote TLR-triggered production of proinflammatory cytokines and type I interferon by enhancing the activation of both MyD88-dependent and TRIF-dependent pathways in macrophages.
MHC class II molecules maintain TLR-triggered Btk activationNext we explored which signal molecules mediate the nonclassical function of MHC class II molecules in promoting intracellular TLR signaling. Given that activation of tyrosine kinases is involved in TLR
signaling, we screened the activation status of various tyrosine kinases in TLR-triggered H2−/− and H2+/+ macrophages. Many such kinases showed similar activation in H2−/− and H2+/+ macrophages stimulated with LPS, except Btk, a member of the Btk-Tec family of cytoplasmic tyrosine kinases26. Activation of Btk was associated with phosphoryla-tion of two tyrosine residues: Tyr550 and Tyr222. Tyr550 in the activa-tion loop is transphosphorylated, leading to autophosphorylation at Tyr222, which is necessary for full activation27. TLR ligand-induced phosphorylation of Btk was much lower in H2−/− macrophages than in H2+/+ macrophages (Fig. 5a,b), which indicated that MHC class II molecules may increase TLR-triggered activation of Btk. We found no substantial difference between H2−/− and H2+/+ macrophages in PMA-triggered Btk activation (Supplementary Fig. 7a), which suggested that deficiency in MHC class II selectively impairs TLR- triggered Btk activation. Overexpression of MHC class II α-chain and β-chain in wild-type macrophages did not induce Btk activation (data not shown), whereas overexpression of MHC class II α-chain and β-chain in H2−/− peritoneal macrophages did restore the activation of Btk triggered by LPS, CpG-ODN or poly(I:C) (Supplementary Fig. 7b,c). These data indicate that MHC class II molecules act in synergy with TLR signaling to maintain Btk activation.
To investigate the role of Btk in TLR signaling, we examined the effect of Btk deficiency on TLR-triggered production of proinflam-matory cytokines and type I interferons in macrophages. Btk−/− peritoneal macrophages produced significantly less TNF, IL-6 and IFN-β than Btk+/+ macrophages did in response to LPS, CpG-ODN or poly(I:C) (Fig. 5c). We further observed the effect of knockdown of Btk on the production of cytokines in TLR-activated macrophages. Btk-specific siRNA substantially downregulated endogenous expres-sion of Btk (Supplementary Fig. 8a), which led to much lower pro-duction of TNF, IL-6 and IFN-β in macrophages stimulated with LPS, CpG-ODN or poly(I:C) (Supplementary Fig. 8b–d). In addition, LFM-A13, an inhibitor of Btk, also resulted in much less production
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Figure 4 Deficiency in MHC class II impairs the MyD88-dependent and TRIF-dependent activation of mitogen-activated protein kinases, NF-κB, IRF3 and IRF7 in TLR-triggered macrophages. (a) Immunoblot analysis of phosphorylated (p-) or total protein in lysates of H2−/− or H2+/+ macrophages stimulated for 0–60 min (above lanes) with LPS (100 ng/ml). (b) Immunoblot analysis of IRF3 among nuclear proteins from macrophages stimulated with LPS; lamin A serves as a loading control. (c) Luciferase activity in lysates of H2−/− or H2+/+ macrophages transfected with luciferase reporter plasmids for NF-κB, AP-1, IRF3 or IRF7 (vertical axes) and, 36 h later, left unstimulated or stimulated for 4 h with LPS (100 ng/ml), CpG ODN (0.3 µM) or poly(I:C) (10 µg/ml); results are presented relative to the activity in unstimulated H2+/+ macrophages, set as 1. (d,e) Immunoblot analysis (IB) of IRAK1 and MyD88 (d) or TBK1 and TRIF (e) immunoprecipitated (IP) with anti-MyD88 (d) or anti-TRIF (e) from lysates of H2−/− or H2+/+ macrophages stimulated for 0–90 min (above lanes) with LPS; immunoglobulin G (IgG) serves as an immunoprecipitation control. (f) In vitro kinase assay of IRAK1, TAK1 and TBK1 in lysates of H2−/− or H2+/+ macrophages left stimulated (Med) or stimulated for 30 min with LPS, CpG ODN or poly(I:C), assayed with the substrates MBP (for IRAK1), MKK4 (for TAK1) or recombinant IRF3 (for TBK1). *P < 0.01 (Student’s t-test). Data are from one experiment representative of three independent experiments with similar results (a,b,d,e; mean ± s.d. of four samples in c) or are from three independent experiments (f; mean ± s.e.m.).
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of TNF, IL-6 and IFN-β in macrophages stimulated with LPS, CpG-ODN or poly(I:C) (Supplementary Fig. 9). These data suggest that Btk is required for full activation of TLR signaling.
Given the positive role of Btk in TLR-triggered cytokine produc-tion and the lower activation of Btk in TLR-triggered H2−/− macro-phages, we sought to determine whether Btk contributes to MHC class II–mediated full activation of TLR signaling. We transfected H2−/− and H2+/+ macrophages with plasmid encoding constitutively active Btk, with substitution of lysine for glutamic acid at position 41 (Btk(E41K)), and found that overexpression of Btk(E41K) potently enhanced the production of TNF, IL-6 and IFN-β induced by LPS, CpG ODN or poly(I:C) in H2+/+ macrophages relative to that in mock-transfected control cells (Fig. 5d). Furthermore, overexpres-sion of Btk(E41K) restored the impaired production of inflammatory cytokines and type I interferon in H2−/− macrophages activated with LPS, CpG ODN or poly(I:C) (Fig. 5d). Together these data suggest that MHC class II molecules facilitate TLR-triggered inflammatory responses by enhancing Btk activation.
Intracellular MHC class II molecules bind Btk via CD40We further investigated the mechanisms underlying the involve-ment of MHC class II molecules in the activation of TLR signaling. First we sought to determine whether MHC class II molecules inter-acted directly with TLRs. Immunoprecipitation with antibody to MHC class II, TLR4, TLR9 or TLR3 showed that MHC class II mol-ecules did not interact with TLR4, TLR9 or TLR3 (Supplementary Fig. 10). So far, there has been no report to our knowledge showing that MHC class II molecules can recognize TLR ligands; therefore, we predicted that MHC class II molecules may not form a complex with the TLR as a cofactor to promote TLR signaling. MHC class II molecules are also abundant in the intracellular endosomal compart-ment9,10, whereas after stimulation by their respective ligands, TLR3 and TLR9 located on the endoplasmic reticulum membrane and TLR4 on the plasma membrane translocate into endosome. In the endosome, TLRs initiate signals by a MyD88- or TRIF-dependent pathway1. Thus, we investigated whether deficiency in MHC class II disrupted endosomal trafficking of TLR4, TLR3 or TLR9 in TLR-triggered macrophages. Confocal microscopy showed that the translocation of TLR4, TLR3 or TLR9 into endosomes in H2−/− macrophages was similar to that in H2+/+ macrophages, after stimu-lation with TLR ligands (Supplementary Fig. 11). The similar subcellular distribution of intracellular MHC class II molecules and TLR4, TLR3 and TLR9 inspired us to explore whether intracellular
MHC class II molecules interact with some intermediate proteins and thereby integrate into the TLR signaling pathway.
Given that the impaired activation of Btk in TLR-triggered H2−/− macrophages contributed to the lower production of inflammatory cytokines and type I interferon, we sought to determine whether MHC class II molecules interact with Btk. Immunoprecipitation showed that MHC class II molecules interacted with Btk (Fig. 6a). Specifically, intracellular MHC class II molecules associated with Btk, but plasma membrane MHC class II molecules did not. As MHC class II molecules have only short cytoplasmic domains, MHC class II might not directly interact with Btk; therefore, some other molecules might mediate the interaction of MHC class II with Btk. We immuno-precipitated proteins from lysates of LPS-stimulated macrophages with antibody to MHC class II and then used reverse-phase nano-spray liquid chromatography–tandem mass spectrometry to identify possible MHC class II–associated proteins, which might be involved in the association and activation of Btk, in the immunoprecipitates. Among the several proteins we detected (data not shown), CD40 attracted our attention because CD40 has been found to mediate Btk activation after stimulation of human B lymphocytes with its ligand, CD40L28. Further immunoprecipitation confirmed the finding that intracellular MHC class II molecules associated with CD40, but those at the plasma membrane did not (Fig. 6b). Confocal microscopy also showed that intracellular MHC class II localized together with CD40 and Btk in the endosomes of macrophages stimulated with LPS for 15 min (Fig. 6c–f), which indicated that intracellular but not plasma membrane MHC class II forms a complex with CD40 and Btk after TLR activation.
Binding of Btk with CD40 is required for full TLR responseImmunoprecipitation with antibody to Btk (anti-Btk) also showed that Btk interacted with CD40 and MHC class II molecules (Supplementary Fig. 12a). To determine which domain of Btk was required for the interaction of Btk with CD40, we constructed mutants of Btk with deletion of various domains and transfected the mutants into Btk-deficient macrophages to observe the restoration of LPS-induced cytokine production in the Btk-deficient macro-phages. Overexpression of mutant Btk with deletion of the pleck-strin homology domain or the kinase domain was unable to restore LPS-induced TNF production (Supplementary Fig. 12b). We trans-fected those two Btk mutants or wild-type Btk into macrophages, followed by immunoprecipitation. We found that mutant Btk with deletion of the pleckstrin homology domain did not interact with
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CD40 (Supplementary Fig. 12c). Therefore the pleckstrin homology domain of Btk is required for the interaction of Btk with CD40.
As described above (Fig. 5 and Supplementary Figs. 8 and 9), Btk activation was required for full activation of TLR signaling. We sought further to confirm that it was CD40 that mediated the interaction of MHC class II and Btk required for the TLR response. Immunoprecipitation of proteins from lysates of TLR-triggered CD40-deficient macrophages showed that MHC class II molecules did not interact with Btk without CD40 (Supplementary Fig. 13), which suggested that MHC class II molecules interact with Btk via CD40 in TLR responses. Btk activation triggered by LPS, CpG ODN or poly(I:C) was also impaired in Cd40−/− macrophages (Supplementary Fig. 14). Furthermore, Cd40−/− macrophages produced less proinflammatory
cytokines and IFN-β than did Cd40+/+ macrophages in response to stimulation with LPS, CpG ODN or poly(I:C) (Fig. 6g). These data indicate that CD40-mediated interaction of intracellular MHC class II molecules with Btk is involved in the positive regulation of TLR-triggered innate response.
Btk enhances TLR signaling by binding MyD88 and TRIFWe sought to elucidate the underlying molecular mechanisms by which the greater Btk activation contributed to the enhancement of TLR-triggered innate immune response by intracellular MHC class II. Given that signaling through TLR4, TLR9 and TLR3 was down-regulated by deficiency in MHC class II, the common adapters MyD88 and TRIF might be the potential targets of Btk and might be
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more activated in the presence of MHC class II. Immunoprecipitation with anti-Btk showed that Btk precipitated together with MyD88 and TRIF in H2+/+ macrophages after LPS stimulation and that this coimmunoprecipitation was diminished in H2−/− macrophages (Fig. 7a and Supplementary Fig. 15). We transfected hemaggluti-nin-tagged Btk together with Flag-tagged MyD88 or Flag-tagged TRIF into HEK293 human embryonic kidney cells; coimmunopre-cipitation showed that Btk interacted with either MyD88 or TRIF (Fig. 7b). We further examined the effect of Btk on the activation of a reporter for NF-κB or IRF3. Overexpression of constitutively active Btk(E41K) enhanced the MyD88- or TRIF-induced activation of NF-κB and the TRIF-induced activation of IRF3 (Fig. 7c). These data indicate that MHC class II molecules contribute to the mainte-nance of TLR-triggered Btk activation and subsequently enhance the interaction of Btk with MyD88 and TRIF, which promotes the acti-vation of MyD88-dependent and TRIF-dependent signal pathways, finally leading to full activation of TLR-triggered innate responses (Supplementary Fig. 16).
DISCUSSIONIt is well known that MHC class II molecules have a crucial role in the development and function of the immune system. In addition to the classical function of MHC class II molecules in presenting antigen to CD4+ T cells, MHC class II molecules can activate various cellular functions in immune or non-immune cells when crosslinked by antibody or superantigen12–14. These nonclassical functions are accomplished by MHC class II molecules at the cell surface acting as signal-transduction receptors. However, so far there has been no insight into any nonclassical functions of intracellular MHC class II molecules. Given reports that the expression of MHC class II can affect the response of macrophages to LPS24,25, we speculated that MHC class II molecules may be involved in the activation of TLR signaling. Here we have provided evidence that deficiency in MHC class II impaired TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and DCs, and this protected mice from lethal challenge with TLR ligands and live Gram-negative bacteria. A lower abundance of activated Btk in TLR-triggered H2−/− macrophages led to less interaction of Btk with MyD88 and TRIF, which attenuated the activation of MyD88- and TRIF-dependent pathways; this suggested that MHC class II molecules are required for full activation of TLR-triggered innate responses. Therefore, we have demonstrated a nonclassical function of MHC class II molecules in the TLR-triggered innate immune response.
Some reports have suggested a role for Btk in TLR signaling. Btk is phosphorylated in LPS-stimulated human monocytes and can interact with multiple components of TLR pathways, including TLR4, TLR6, TLR8, TLR9, MyD88, Mal and IRAK129. Btk phosphorylates Mal, which resulting in degradation of Mal30,31. Studies of peripheral blood monocytes from patients with Btk-deficient X-linked agammaglob-ulinemia and macrophages from Btk-mutant mice with X-linked immunodeficiency have indicated that Btk-dependent signaling is involved in the LPS-induced production of TNF and IL-1β32,33. However, it remained unclear whether Btk affects the production of cytokines by macrophages in response to other TLR ligands or whether it activates an altered signal pathway. Here we have shown that Btk interacted with TRIF and promoted TRIF-dependent activation of IRF3 and NF-κB, leading to enhanced TLR3- and TLR4-triggered production of type I interferons and proinflammatory cytokines. In addition, the constitutively active mutant Btk(E41K) also enhanced MyD88-triggered activation of NF-κB, AP-1 and IRF7. Given those findings and the observations that Btk interacted with TLRs, MyD88,
Mal and IRAK1, we conclude that Btk is necessary but is not essential for the full activation of MyD88- and TRIF-dependent pathways by interacting with multiple components in TLR signaling. As deficiency in MHC class II impairs Btk activation, H2−/− mice and cells (macro-phages and DCs) derived from them had lower but not completely abolished cytokine production and less death in response to challenge with TLR ligands. The data showing that overexpression of the con-stitutively active mutant Btk(E41k) corrected the lower abundance of proinflammatory cytokines and type I interferons in TLR-triggered H2−/− macrophages indicate that Btk activation has a pivotal role in MHC class II–mediated full activation of TLR signaling.
Reverse signaling mediated by MHC class II at the cell surface is involved in many cellular processes of B cells and DCs. The short cytosolic domain of MHC class II molecules seems inconsistent with these complex signal-transduction pathways, which suggests that the presence of membrane-associated signaling components that might provide this functionality. Indeed, a variety of cell surface molecules have been reported to immunoprecipitate together with and/or couple with MHC class II molecules. These MHC class II–associated molecules belong to various families, including the immunoglobulin superfamily (CD19), the tetraspanin family (CD37, CD53, CD81 and CD82), lectin (CD23) and the complement receptor family (CD21 and CD20)34–36. Furthermore, MHC class II molecules can associate with CD40 on human B cells37. Here we found that MHC class II molecules interacted with CD40 in the endosomes of TLR-activated macrophages. However, which region of MHC class II interacts with these molecules is still unclear. Several studies have shown that eight membrane-proximal amino acids of the cytoplasmic domain and transmembrane domain of MHC class II β-chain are required for distinct MHC class II–mediated signaling38,39, which suggests that these regions may also be required for the interaction of MHC class II with other molecules. Notably, we found that intracellular MHC class II molecules interacted with Btk after TLR ligation as early as 5 min after activation, but cell surface MHC class II molecules did not. This rapid interaction of intracellular MHC class II and Btk is consistent with rapid activation of the Btk and TLR signaling pathway. We further confirmed that MHC class II molecules formed a complex with CD40 and Btk in the endosomes of TLR-activated macrophages and that intracellular CD40 mediated the interaction of MHC class II and Btk. Although Btk has been reported to become activated after stimulation of human B lymphocytes with CD40L, the underlying mechanism is not clear. Thus, the mechanism by which the interaction of MHC class II molecules with CD40 maintains activation of Btk needs further investigation.
Coexpression of MHC class II (HLA-DR) in HEK293 cells over-expressing TLR2 or TLR4 results in much higher TLR2- or TLR4-triggered expression of human β-defensin40. Furthermore, in lysates of HEK293 cells overexpressing HLA-DR, radiolabeled recombinant TLR2 protein precipitates in vitro together with HLA-DR protein immunoprecipitated with anti-HLA-DR. So, TLR2 was proposed to associate with HLA-DR40. However, whether or not TLR2 and HLA-DR interact physically needs further investigation. In our study, we did not find direct interaction of cell surface MHC class II molecules with TLRs in macrophages, which suggests that MHC class II molecules may not form a complex with TLRs. In addition, we found that ligation of MHC class II by specific antibody did not induce the production of TNF, IL-6 or IFN-β in macrophages or DCs, which indicated that ligation of cell surface MHC class II alone did not induce the activation of signaling involved in the production of inflammatory cytokines in macrophages and DCs (data not shown). Instead, we found here that intracellular MHC class II molecules
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interacted with Btk via CD40 and subsequently maintained Btk acti-vation and thereby promoted TLR signaling through interaction of Btk with the adapters MyD88 and TRIF, but cell surface MHC class II molecules did not.
Clinical observations have shown that HLA-DR expression in peripheral blood monocytes is much lower in patients with septic shock than in normal subjects41,42. Such patients release much less TNF and IL-1β than do normal subjects in response to LPS. In addition, the ability of monocytes to express HLA-DR anti-gen correlates directly with the clinical course of trauma patients43 and septic patients44. However, treatment with IFN-γ restores HLA-DR expression and thus substantially enhances the induction of TNF by LPS in vitro in such situations44. The recovery of mono-cyte function results in clearance of sepsis and improved survival of the patients44. Thus, those clinical observations, together with our in vitro and in vivo data, demonstrate that MHC class II mol-ecules are required for full activation of macrophages in response to TLR ligands.
In conclusion, our study has demonstrated that intracellular MHC class II molecules interacted with Btk and maintained Btk activation after stimulation with TLR ligands. Activated Btk interacted with MyD88 and TRIF, promoting the activation of MyD88-dependent and TRIF-dependent pathway and thus leading to the enhanced produc-tion of proinflammatory cytokines and type I interferons. Therefore, intracellular MHC class II molecules are required for TLR-triggered full activation of macrophages and DCs. Our findings provide new insight into the regulation of TLR-triggered inflammatory responses and also indicate a previously unknown nonclassical role for MHC class II molecules in innate immunity.
METHODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/natureimmunology/.
Note: Supplementary information is available on the Nature Immunology website.
ACknowLeDgMentsWe thank P. Ma, M. Jin and Y. Li for technical assistance; and N. Li, H. An, T. Chen, S. Xu and C. Han for discussions. Supported by the National Key Basic Research Program of China (2007CB512403), National 115 Key Project (2008ZX10002-008, 2009ZX09503-023) and the National Natural Science Foundation of China (30721091).
AUtHoR ContRIBUtIonsX.C. and X.L. designed the experiments; X.L., Z.Z., D.L., L.X., F.M., P.Z. and H.Y. did the experiments; X.C. and X.L. analyzed data and wrote the paper; and X.C. was responsible for research supervision, coordination and strategy.
CoMPetIng FInAnCIAL InteRestsThe authors declare no competing financial interests.
Published online at http://www.nature.com/natureimmunology/. reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.
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nature immunologydoi:10.1038/ni.2015
ONLINE METHODSMice and reagents. Mice homozygous for deletion of all genes encoding clas-sical MHC class II (B6.129S-H2dlAb1-Ea; Mouse Genome Informatics acces-sion code, 003584), Btk-deficient mice (B6.129S-Btktm1Wk/J; Mouse Genome Informatics accession code, 002536) and Cd40-deficient mice (B6.129P2-Cd40tm1Kik/J; Mouse Genome Informatics accession code, 002928) were from Jackson Laboratories and were bred in specific pathogen–free condi-tions. Littermate mice 6 weeks of age were used (matched for body weight and sex). All animal experiments were in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Second Military Medical University, Shanghai. LPS (E. coli 0111:B4), CpG ODN and poly(I:C) have been described45. E. coli 0111:B4 was obtained from the China Center for Type Culture Collection. LFM-A13 was from Cayman Chemical. Recombinant MBP was from Upstate Biotechnology. Recombinant MKK4 was from Merck. Antibody to MHC class II (107610) was from BioLegend. Recombinant IRF3 and anti-TRIF (ab13810), anti-Btk (ab25971), anti-CD40 (ab13545), anti-TLR4 (ab22048), anti-TLR3 (ab62566), anti-TLR9 (ab52967) and antibody to Btk phosphorylated at Tyr222 (ab51210) or Tyr550 (ab52192) were from Abcam. Anti-IRAK1 (D51G7), anti-TBK1 (D1B4), anti-IRF3 (D83B9), anti-hemagglutinin (6E2), anti-EEA1 (2411), anti-Erk (9102), anti-Jnk (56G8), anti-p38 (9212) and antibodies to Erk phosphorylated at Thr202-Tyr204 (E10), to Jnk phosphorylated at Thr183-Tyr185 (G9), to p38 phosphorylated at Thr180-Tyr182 (9211), to IRF3 phosphorylated at Ser396(4D4G) or to IκBα phosphorylated at Ser32-Ser36 (5A5) were from Cell Signaling Technology. Anti-MyD88 (3244-100) was from BioVision. Anti–lamin A (133A2) and anti-β-actin (sc-130656) were from Santa Cruz. Anti-Flag (M2) was from Agilent Technologies.
Plasmid constructs. Recombinant vectors encoding mouse MHC class II α-chain (NM_010378.2) or β-chain (NM_207105.2), TRIF (BC094338), MyD88 (NM_010851) or Btk (NM_013482.2; GenBank accession numbers in parentheses) and mutants thereof were constructed by PCR-based ampli-fication from cDNA of mouse macrophages and then were subcloned into the pcDNA3.1 eukaryotic expression vector (Invitrogen) as described45. All constructs were confirmed by DNA sequencing. Luciferase reporter plasmids for NF-κB, AP-1, IRF3 and IRF7 have been described45,46.
Cell culture and transfection. Bone marrow–derived DCs from C57BL/6J mice were generated as described46. The HEK293 cell line (American Type Culture Collection) was transfected with JetPEI reagents (PolyPlus). Thioglycollate-elicited mouse peritoneal macrophages were prepared and cultured in endotoxin-free RPMI-1640 medium with 10% (vol/vol) FCS (Invitrogen) as described46 and were transfected by nucleofection with a Mouse Macrophage Nucleofector kit (Amaxa).
RNA-mediated interference. The sequences of siRNA targeting MHC class II β-chain and Btk were 5′-CCACACAGCTTATTAGGAA-3′ and 5′-GGAGTCTAGTGAAATGGAA-3′, respectively; the control siRNA sequence was 5′-TTCTCCGAACGTGTCACGT-3′. The siRNA duplexes were transfected into mouse peritoneal macrophages with INTERFERin reagent (Polyplus) according to a standard protocol.
Cytokine detection. TNF, IL-6 and IFN-β in supernatants and serum were measured with ELISA kits (R&D Systems).
RNA quantification. A LightCycler (Roche) and SYBR RT-PCR kit (Takara) were used for quantitative real-time RT-PCR analysis as described45. Data were normalized to β-actin expression.
Assay of luciferase reporter gene expression. HEK293 cells or mouse macro-phages were transfected with a mixture of the appropriate luciferase reporter plasmid, pRL-TK-renilla-luciferase plasmid and the appropriate additional constructs. The total amount of plasmid DNA was made equal by the addition of empty control vector. After 24 h or 36 h, cells were left untreated or were treated with LPS, CpG ODN or poly(I:C). Luciferase activity was measured
with a Dual-Luciferase Reporter Assay System according to the manufacturer’s instructions (Promega). Data were normalized for transfection efficiency by the division of firefly luciferase activity with that of renilla luciferase.
Immunoprecipitation and immunoblot analysis. Cells were lysed with cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor ‘cocktail’. Protein concentrations in the extracts were measured by BCA assay (Pierce). Immunoprecipitation and immunoblot analysis were done as described45,46.
Nanospray liquid chromatography–tandem mass spectrometry. Macrophages (3 × 108) were stimulated for 15 min with LPS and then were lysed for immunoprecipitation with antibody to MHC class II. Proteins were eluted and digested, followed by analysis by reverse-phase nanospray liquid chromatography–tandem mass spectrometry. The spectra from tandem mass spectrometry were automatically used for searching against the nonredundant International Protein Index mouse protein database (version 3.72) with the Bioworks browser (rev.3.1).
In vitro kinase assay. Proteins in total cell extracts (100 µg) were immuno-precipitated with the appropriate antibody, and kinase activity was measured as described45.
Confocal microscopy. Macrophages plated on glass coverslips in six-well plates were left unstimulated or stimulated with LPS, then were labeled with antibody to MHC class II, anti-CD40, anti-Btk, anti-TLR4, anti-TLR3, anti-TLR9 or anti-EEA1 (endosome marker). Cells were viewed with a Leica TCS SP2 confocal laser microscope.
Establishment of endotoxin shock model and bacterial sepsis model. The endotoxin shock mouse model was established by intraperitoneal injection of LPS (15 mg per kg body weight) as described45. E. coli 0111:B4 in midlogarith-mic growth were collected and concentrations were measured by counting of viable bacteria on agar plates. For injection, bacteria were washed twice with nonpyrogenic PBS. Mice were injected intraperitoneally with 0.5 ml bacteria suspension (1 × 107 colony-forming units)47.
Bone marrow transplantation. Bone marrow cells (1 × 107) from H2+/+ or H2−/− mice were transplanted into lethally irradiated wild-type C57BL/6J mice (cumulative dose, 10 Gy) by injection into the tail vein. After 3 weeks, CD4+ T cells in spleen and lymph nodes were counted and expression of MHC class II on macrophages and DCs was analyzed by flow cytometry.
Macrophage reconstitution. Bone marrow cells from H2+/+ or H2−/− mice were cultured for 7 d in mouse macrophage colony-stimulating factor (50 ng/ml; PeproTech) for the preparation of bone marrow–derived macro-phages. Clodronate liposomes (Sigma) were injected intraperitoneally into wild-type mice (50 mg in 200 µl per mouse) for the depletion of endogenous macrophages. Then, 2 d later, H2+/+ or H2−/− bone marrow–derived macrophages (1 × 107) were transplanted (by injection into the tail vein) into the mice depleted of macrophages, followed 6 h later by challenge with LPS48.
Statistical analysis. The statistical significance of comparisons between two groups was determined with Student’s t-test. The statistical significance of survival curves were estimated according to the method of Kaplan and Meier, and curves were compared with the generalized Wilcoxon test. P values of less than 0.05 were considered statistically significant.
45. Wang, C. et al. The E3 ubiquitin ligase Nrdp1 ‘preferentially’ promotes TLR-mediated production of type I interferon. Nat. Immunol. 10, 744–752 (2009).
46. An, H. et al. Phosphatase SHP-1 promotes TLR- and RIG-I-activated production of type I interferon by inhibiting the kinase IRAK1. Nat. Immunol. 9, 542–550 (2008).
47. Haziot, A. et al. Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice. Immunity 4, 407–414 (1996).
48. Han, C. et al. Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b. Nat. Immunol. 11, 734–742 (2010).
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