Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk

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1 Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk Xingguang Liu 1, Zhenzhen Zhan 1, Dong Li, Li Xu 1, Feng Ma, Peng Zhang 1, Hangping Yao & Xuetao Cao 1,3 11 Nature America, Inc. All rights reserved. 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 CD 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. 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 responses 1. After the recognition of pathogen-associated molecule patterns, TLRs initiate shared and distinct signaling pathways by recruiting various combinations 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, TLR, TLR7, TLR8 and TLR9 also activate the transcription factors IRF3 and/or IRF7, leading to the production of type I interferon. 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 disorders 5. So far, various signaling molecules have been shown to be involved in the tight regulation of the TLR pathway to maintain the immunological balance,. 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 H) are expressed mainly by professional APCs, including 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 domains 9. Their main function is to present peptides processed from extracellular proteins to CD + helper T cells and to direct the processes of positive and negative selection, shaping the repertoire during T cell maturation and lineage commitment 1,11. In addition to that classical function, cell surface MHC class II molecules can function as receptors to mediate reverse signal transduction after ligation with agonist antibodies, T cell antigen receptors or CD molecules. Engagement of cell surface MHC class II may regulate cell adhesion, cytokine production and the expression of costimulatory molecules 1 15 and may also induce the apoptosis, proliferation or differentiation of B cells 1,17. 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 nucleus 18,19. 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,,5)-trisphosphate and diacylglycerol, which mediate calcium mobilization and activation of protein kinase C,1. 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 surveillance,3. For example, 1 National Key Laboratory of ical Immunology & Institute of Immunology, Second Military ical University, Shanghai, China. Institute of Immunology, Zhejiang University School of icine, Hangzhou, China. 3 Chinese Academy of ical Sciences, Beijing, China. Correspondence should be addressed to X.C. (caoxt@immunol.org). Received 5 October 1; accepted 8 February 11; published online 7 March 11; doi:1.138/ni.15 1 VOLUME 1 NUMBER 5 MAY 11 nature immunology

2 11 Nature America, Inc. All rights reserved. a c e 5. H /.. H / PBS Poly(I:C) H / 1 H / 8 PBS Poly(I:C) PBS PBS PBS Poly(I:C) Time after challenge (h) chimeras H / chimeras deficiency in MHC class II results in less production of lipopolysaccharide ()-induced tumor necrosis factor (TNF) by human peripheral blood monocytes and mouse macrophages,5. 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 molecules 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 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 production of proinflammatory cytokines and type I interferon triggered by TLR, TLR3 or TLR9 in macrophages and DCs. Furthermore, intracellular MHC class II molecules interacted with the tyrosine kinase Btk via the costimulatory molecule CD 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 promote the full activation of TLR signaling. RESULTS H deficiency protects mice from and bacterial challenge MHC class II deficient (H / ) mice (with a 78.8-kilobase deletion in H) and wild-type ( ) 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 H / and mice in the expression of CD, CD8 or CD8 on splenic DCs or immature or mature bone marrow derived DCs (Supplementary Fig. 1c). Therefore, H / mice had normal myeloid development and macrophage differentiation. To investigate the role of MHC class II molecules in the TLR-triggered innate immune response, we challenged H / mice with the TLR ligands, ODN or poly(i:c). H / mice produced significantly less TNF, IL- and interferon-β (IFN-β) than mice did in response to challenge with, ODN or poly(i:c) (Fig. 1a c). Accordingly, H / mice had prolonged survival relative to that of mice after lethal b d Survival (%) PBS 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- (b) and IFN-β (c) in the serum of H / or mice (n = 5 per genotype) h after intraperitoneal administration of PBS or, -ODN () or poly(i:c) (at a dose of 15, or mg per kg body weight, respectively). P <.1 (Student s t-test). (d) Survival of H / mice and mice (n = 1 per genotype) given intraperitoneal injection of (15 mg per kg body weight). P <.1 (Wilcoxon test). (e) ELISA of TNF, IL- and IFN-β in serum from wild-type mice lethally irradiated and given intravenous transplantation of bone marrow cells from H / or mice 3 weeks before challenge with PBS or, assessed h after challenge. P <.1 (Student s t-test). Data are from three independent experiments (mean ± s.e.m.). challenge with (Fig. 1d). H / mice were also more resistant to lethal challenge with high-dose poly(i:c) (data not shown). Given that H / mice have considerably fewer CD + T cells in thymus, spleen and lymph nodes, we further explored the effects of the lack of CD + T cells on the resistance of H / mice to sepsis. We transplanted bone marrow cells from or H / mice into lethally irradiated wild-type mice. The reconstituted H / mixed bone marrow chimeras had numbers of CD + T cells in spleen and lymph nodes similar to those in chimeras but had no expression of MHC class II in macrophages or DCs (data not shown). The -induced in vivo production of TNF, IL- and IFN-β was much lower in the reconstituted H / chimeras than in chimeras (Fig. 1e), which therefore excluded the possibility that the lack of CD + T cells was involved in the resistance of H / mice to sepsis. These data demonstrate that H / 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 H / mice intraperitoneally with Escherichia coli serotype 111:B, the most frequent cause of bacterial sepsis in humans. The production of TNF and IL- in the serum of H / mice after injection of E. coli was significantly less than that in mice (Fig. a,b). The survival of H / mice after lethal challenge with E. coli was also prolonged (Fig. c). 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 H / APCs Next we assessed whether deficiency in MHC class II attenuated the production of proinflammatory cytokines and type I a b c 3 1 +/+ 1 H / 3 H / 8 H H / PBS 1 E. coli PBS E. coli Survival (%) Time after E. coli challenge (h) Figure Deficiency in MHC class II protects mice from sepsis induced by live E. coli. (a,b) ELISA of TNF (a) and IL- (b) in serum from H / or mice (n = 3 per genotype) h after intraperitoneal infection with E. coli 111:B (1 1 7 colony-forming units per mouse). P <.1 (Student s t-test). (c) Survival of mice (n = 1 per genotype) treated as described in a,b. P <.1 (Wilcoxon test). Data are from three independent experiments (mean ± s.e.m.). nature immunology VOLUME 1 NUMBER 5 MAY 11 17

3 11 Nature America, Inc. All rights reserved. a Macrophage d DC Counts interferon in TLR-triggered macrophages and DCs in vitro. H / peritoneal macrophages had lower expression of TNF, IL-, IFN-α and IFN-β mrna and protein than did macrophages from mice in response to, -ODN or poly(i:c) (Fig. 3a and Supplementary Fig. ). Similarly, we also detected less production of TNF, IL- and IFN-β in H / DCs (Fig. 3a). H / peritoneal macrophages responded normally to the phorbol ester PMA, to IL-1β and to MDP (the ligand for the intracellular bacteria sensor Nod) and produced amounts of TNF and IL- similar to those produced by 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 H / peritoneal macrophages and found that overexpression of both MHC class II α-chain and β-chain restored the production of TNF, IL- and IFN-β induced by, -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 macrophages. In addition, there was no substantial difference between H / and mice in the expression of TLR, 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 H / peritoneal macrophages did not affect the expression of TLR, TLR3 or TLR9 (Supplementary Fig. ). Thus, MHC class II expression did not affect the expression pattern of TLRs, which excluded the possibility that the e f b. 1. H / MHCIIβ β-actin. 1.. Ctrl sirna MHCII 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 H / or macrophages (top row) or DCs (bottom row) left unstimulated () or stimulated for h with (1 ng/ml), ODN (;.3 µm) or poly(i:c) (1 µg/ml). (b,c) ELISA of cytokines in supernatants of H / or macrophages given mock transfection () or transfected with vectors for the expression of MHC class II α-chain and β-chain () or MHC class II α-chain 1 8 c α-chain β-chain PBS α-chain β-chain 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 endogenous 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- and IFN-β in peritoneal macrophages stimulated with, -ODN or poly(i:c) (Fig. 3e). To further confirm that the impaired TLR-triggered inflammatory response in vivo was due to deficiency in MHC class II in myeloid cells, we adoptively transferred or H / bone marrow derived macrophages into wild-type mice depleted of endogenous macrophages by pretreatment with clodronate liposomes. Mice reconstituted with H / macrophages produced less proinflammatory cytokines and IFN-β in response to, -ODN or poly(i:c) challenge than did mice reconstituted with 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 H / macrophages We 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 -stimulated H / peritoneal macrophages (Fig. a). Deficiency in MHC class II resulted in less -induced PBS α-chain β-chain PBS H / H / H / alone (α-chain) or β-chain alone (β-chain) and, 3 h later, stimulated for h with (b), or 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 8 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, 8 h later, left unstimulated or stimulated for h with, ODN or poly(i:c). (f) ELISA of cytokines in serum from wild-type mice first depleted of endogenous macrophages and then transplanted with H / or bone marrow derived cells h before challenge with, ODN or poly(i:c), measured h after challenge. P <.5 and P <.1 (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) Ctrl sirna H / 18 VOLUME 1 NUMBER 5 MAY 11 nature immunology

4 11 Nature America, Inc. All rights reserved. a H b c / (min) p-erk Erk p-jnk Jnk p-p38 p38 p-lκbα p-irf3 IRF3 NF-κB activation (fold) d e f (min) IP: MyD IP: IgG + IB: IRAK1 IB: MyD88 H / H / (min) Nuclear IRF3 Lamin A phosphorylation and nuclear translocation of IRF3 (Fig. a,b). We obtained similar results with H / peritoneal macrophages stimulated with 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 H / peritoneal macrophages stimulated with, ODN or poly(i:c) (Fig. c). Deficiency in MHC class II also impaired the transactivation of an IRF3 reporter induced by or poly(i:c) and the transactivation of an IRF7 reporter induced by ODN (Fig. c). To assess the functional integrity of intracellular signal pathway in H / 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 H / peritoneal macrophages was similar to that in TNF-stimulated macrophages (Supplementary Fig. ), which indicated that deficiency in MHC class II selectively impaired TLRtriggered 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 H / macrophages stimulated with than in macrophages stimulated with (Fig. d,e). In vitro kinase assays showed that the activation of IRAK1, TAK1 and TBK1 induced by, ODN or poly(i:c) was impaired in H / macrophages relative to that in macrophages (Fig. f). 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 activation Next 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 (min) IP: TRIF IP: IgG + IB: TBK1 IB: TRIF H / Poly(I:C) IRAK1 activity (1 3 c.p.m.) AP-1 activation (fold) 1 8 TAK1 activity (1 3 c.p.m.) Poly(I:C) IRF3 activation (fold) signaling, we screened the activation status of various tyrosine kinases in TLR-triggered H / and macrophages. Many such kinases showed similar activation in H / and macrophages stimulated with, except Btk, a member of the Btk-Tec family of cytoplasmic tyrosine kinases. Activation of Btk was associated with phosphorylation of two tyrosine residues: Tyr55 and Tyr. Tyr55 in the activation loop is transphosphorylated, leading to autophosphorylation at Tyr, which is necessary for full activation 7. TLR ligand-induced phosphorylation of Btk was much lower in H / macrophages than in macrophages (Fig. 5a,b), which indicated that MHC class II molecules may increase TLR-triggered activation of Btk. We found no substantial difference between H / and macrophages in PMA-triggered Btk activation (Supplementary Fig. 7a), which suggested that deficiency in MHC class II selectively impairs TLRtriggered 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 H / peritoneal macrophages did restore the activation of Btk triggered by, -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 proinflammatory cytokines and type I interferons in macrophages. Btk / peritoneal macrophages produced significantly less TNF, IL- and IFN-β than Btk +/+ macrophages did in response to, -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 expression of Btk (Supplementary Fig. 8a), which led to much lower production of TNF, IL- and IFN-β in macrophages stimulated with, -ODN or poly(i:c) (Supplementary Fig. 8b d). In addition, LFM-A13, an inhibitor of Btk, also resulted in much less production 8 Poly(I:C) Figure 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 H / or macrophages stimulated for min (above lanes) with (1 ng/ml). (b) Immunoblot analysis of IRF3 among nuclear proteins from macrophages stimulated with ; lamin A serves as a loading control. (c) Luciferase activity in lysates of H / or macrophages transfected with luciferase reporter plasmids for NF-κB, AP-1, IRF3 or IRF7 (vertical axes) and, 3 h later, left unstimulated or stimulated for h with (1 ng/ml), ODN (.3 µm) or poly(i:c) (1 µg/ml); results are presented relative to the activity in unstimulated 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 H / or macrophages stimulated for 9 min (above lanes) with ; immunoglobulin G (IgG) serves as an immunoprecipitation control. (f) In vitro kinase assay of IRAK1, TAK1 and TBK1 in lysates of H / or macrophages left stimulated () or stimulated for 3 min with, ODN or poly(i:c), assayed with the substrates MBP (for IRAK1), MKK (for TAK1) or recombinant IRF3 (for TBK1). P <.1 (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.). Poly(I:C) TBK1 activity (1 3 c.p.m.) IRF7 activation (fold) Poly(I:C) H / H / nature immunology VOLUME 1 NUMBER 5 MAY 11 19

5 a b c (min) p-btk(y55) p-btk(y) Btk H / H / p-btk(y55) p-btk(y) Figure 5 MHC class II molecules promote TLR-triggered inflammatory innate responses by maintaining Btk activation. (a,b) Immunoblot analysis of Btk phosphorylated at Tyr55 (p-btk(y55)) or Tyr (p-btk(y)) or total Btk in lysates of H / or peritoneal macrophages left unstimulated or stimulated for min with Btk d H / E1K E1K E1K E1K E1K E1K E1K E1K E1K (1 ng/ml; a) or for 3 min with ODN (.3 µm) or poly(i:c) (1 µg/ml; b). (c) ELISA of TNF, IL- and IFN-β in supernatants of Btk +/+ or Btk / peritoneal macrophages left unstimulated or stimulated for h with, ODN or poly(i:c). (d) ELISA of TNF, IL- and IFN-β in supernatants of H / or peritoneal macrophages mock-transfected or transfected with constitutively active Btk(E1K) and, 8 h later, stimulated for h with, ODN or poly(i:c). P <.5 and P <.1 (Student s t-test). Data are representative of three independent experiments with similar results (a,b) or are from three independent experiments (c,d; mean ± s.e.m.).... Btk +/+ Btk / 11 Nature America, Inc. All rights reserved. of TNF, IL- and IFN-β in macrophages stimulated with, -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 production and the lower activation of Btk in TLR-triggered H / macrophages, we sought to determine whether Btk contributes to MHC class II mediated full activation of TLR signaling. We transfected H / and macrophages with plasmid encoding constitutively active Btk, with substitution of lysine for glutamic acid at position 1 (Btk(E1K)), and found that overexpression of Btk(E1K) potently enhanced the production of TNF, IL- and IFN-β induced by, ODN or poly(i:c) in macrophages relative to that in mock-transfected control cells (Fig. 5d). Furthermore, overexpression of Btk(E1K) restored the impaired production of inflammatory cytokines and type I interferon in H / macrophages activated with, 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 CD We further investigated the mechanisms underlying the involvement of MHC class II molecules in the activation of TLR signaling. First we sought to determine whether MHC class II molecules interacted directly with TLRs. Immunoprecipitation with antibody to MHC class II, TLR, TLR9 or TLR3 showed that MHC class II molecules did not interact with TLR, TLR9 or TLR3 (Supplementary Fig. 1). 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 compartment 9,1, whereas after stimulation by their respective ligands, TLR3 and TLR9 located on the endoplasmic reticulum membrane and TLR on the plasma membrane translocate into endosome. In the endosome, TLRs initiate signals by a MyD88- or TRIF-dependent pathway 1. Thus, we investigated whether deficiency in MHC class II disrupted endosomal trafficking of TLR, TLR3 or TLR9 in TLRtriggered macrophages. Confocal microscopy showed that the translocation of TLR, TLR3 or TLR9 into endosomes in H / macrophages was similar to that in macrophages, after stimulation with TLR ligands (Supplementary Fig. 11). The similar subcellular distribution of intracellular MHC class II molecules and TLR, 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 H / 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. a). 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 immunoprecipitated proteins from lysates of -stimulated macrophages with antibody to MHC class II and then used reverse-phase nanospray 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), CD attracted our attention because CD has been found to mediate Btk activation after stimulation of human B lymphocytes with its ligand, CDL 8. Further immunoprecipitation confirmed the finding that intracellular MHC class II molecules associated with CD, but those at the plasma membrane did not (Fig. b). Confocal microscopy also showed that intracellular MHC class II localized together with CD and Btk in the endosomes of macrophages stimulated with for 15 min (Fig. c f), which indicated that intracellular but not plasma membrane MHC class II forms a complex with CD and Btk after TLR activation. Binding of Btk with CD is required for full TLR response Immunoprecipitation with antibody to Btk (anti-btk) also showed that Btk interacted with CD and MHC class II molecules (Supplementary Fig. 1a). To determine which domain of Btk was required for the interaction of Btk with CD, we constructed mutants of Btk with deletion of various domains and transfected the mutants into Btk-deficient macrophages to observe the restoration of -induced cytokine production in the Btk-deficient macrophages. Overexpression of mutant Btk with deletion of the pleckstrin homology domain or the kinase domain was unable to restore -induced TNF production (Supplementary Fig. 1b). We transfected 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 VOLUME 1 NUMBER 5 MAY 11 nature immunology

6 a Plasma Plasma b c d (min) IP: MHCll IP: IgG IB: Btk IB: MHCll Cytoplasmic 15 membrane (min) 15 e IP: MHCll IP: IgG IB: CD IB: MHCll Cytoplasmic membrane MHCll CD Merge Btk CD Merge min 15 min MHCll Btk Merge min min f MHCll EEA1 Merge 15 min 15 min 15 min 11 Nature America, Inc. All rights reserved. Figure Intracellular MHC class II molecules interact with CD and Btk. (a,b) Immunoblot analysis of Btk (a), CD (b) or MHC class II (a,b) immunoprecipitated with antibody to MHC class II from cytoplasmic and plasma membrane proteins in lysates of peritoneal macrophages stimulated for 15 min with. Immunoglobulin G serves as an immunoprecipitation control. (c f) Confocal microscopy of macrophages left unstimulated ( min) or stimulated for 15 min with CD (Supplementary Fig. 1c). Therefore the pleckstrin homology domain of Btk is required for the interaction of Btk with CD. 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 CD that mediated the interaction of MHC class II and Btk required for the TLR response. Immunoprecipitation of proteins from lysates of TLR-triggered CD-deficient macrophages showed that MHC class II molecules did not interact with Btk without CD (Supplementary Fig. 13), which suggested that MHC class II molecules interact with Btk via CD in TLR responses. Btk activation triggered by, ODN or poly(i:c) was also impaired in Cd / macrophages (Supplementary Fig. 1). Furthermore, Cd / macrophages produced less proinflammatory g cytokines and IFN-β than did Cd +/+ macrophages in response to stimulation with, ODN or poly(i:c) (Fig. g). These data indicate that CD-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 TRIF We 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 TLR, TLR9 and TLR3 was downregulated by deficiency in MHC class II, the common adapters MyD88 and TRIF might be the potential targets of Btk and might be... (1 ng/ml), then labeled with antibodies to the appropriate molecules (above images). Original magnification, 3. (g) ELISA of TNF, IL- and IFN-β in supernatants of Cd +/+ or Cd / peritoneal macrophages left unstimulated or stimulated for h with (1 ng/ml), ODN (.3 µm) or poly(i:c) (1 µg/ml). P <.1 (Student s t-test). Data are representative of three independent experiments with similar results (a f) or are from three independent experiments (g; mean ± s.e.m.). Cd +/+ Cd / a b + + c H / (min) IP: Btk IP: IgG + IB: MyD88 IB: TRIF IB: Btk HA-Btk Flag-MyD88 Flag-TRIF IP: HA IB: Flag IB: HA TCL: Flag NF-κB activation (fold) 3 1 NF-κB activation (fold) 3 1 Ctrl MyD Ctrl TRIF MyD88+E1K TRIF+E1K IRF3 activation (fold) 1 8 Ctrl TRIF TRIF+E1K Figure 7 Activated Btk interacts with MyD88 and TRIF, promoting the activation of MyD88-dependent and TRIF-dependent pathways. (a) Immunoblot analysis of MyD88, TRIF or Btk immunoprecipitated with anti-btk from lysates of H / or macrophages stimulated for 9 min with. (b) Immunoblot analysis of HEK93 cells 8 h after cotransfection of Flag-tagged MyD88 or Flag-tagged TRIF plus hemagglutinin (HA)-tagged Btk, followed by immunoprecipitation with anti-hemagglutinin. TCL, immunoblot analysis of total cell lysates with anti-flag. (c) Luciferase assay of the activation of NF-κB or IRF3 in lysates of HEK93 cells h after transfection of luciferase reporter plasmid for NF-κB or IRF3, plus empty vector control (Ctrl) or plasmid expressing MyD88 or TRIF either alone (MyD88 or TRIF) or together with plasmid expressing Btk(E1K) (MyD88+E1K or TRIF+E1K; dose, horizontal axis); results were normalized to renilla luciferase activity and are presented relative to the activity in cells transfected with empty vector control, set as 1. P <.5 and P <.1 (Student s t-test). Data are from one experiment representative of three independent experiments with similar results (mean ± s.d. of six samples in c). nature immunology VOLUME 1 NUMBER 5 MAY 11 1

7 11 Nature America, Inc. All rights reserved. more activated in the presence of MHC class II. Immunoprecipitation with anti-btk showed that Btk precipitated together with MyD88 and TRIF in macrophages after stimulation and that this coimmunoprecipitation was diminished in H / macrophages (Fig. 7a and Supplementary Fig. 15). We transfected hemagglutinin-tagged Btk together with Flag-tagged MyD88 or Flag-tagged TRIF into HEK93 human embryonic kidney cells; coimmunoprecipitation 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(E1K) 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 maintenance of TLR-triggered Btk activation and subsequently enhance the interaction of Btk with MyD88 and TRIF, which promotes the activation of MyD88-dependent and TRIF-dependent signal pathways, finally leading to full activation of TLR-triggered innate responses (Supplementary Fig. 1). DISCUSSION It 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 CD + T cells, MHC class II molecules can activate various cellular functions in immune or non-immune cells when crosslinked by antibody or superantigen 1 1. 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,5, 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 H / 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 -stimulated human monocytes and can interact with multiple components of TLR pathways, including TLR, TLR, TLR8, TLR9, MyD88, Mal and IRAK1 9. Btk phosphorylates Mal, which resulting in degradation of Mal 3,31. Studies of peripheral blood monocytes from patients with Btk-deficient X-linked agammaglobulinemia and macrophages from Btk-mutant mice with X-linked immunodeficiency have indicated that Btk-dependent signaling is involved in the -induced production of TNF and IL-1β 3,33. 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 TLR-triggered production of type I interferons and proinflammatory cytokines. In addition, the constitutively active mutant Btk(E1K) 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, H / mice and cells (macrophages 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 constitutively active mutant Btk(E1k) corrected the lower abundance of proinflammatory cytokines and type I interferons in TLR-triggered H / 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 CD8), lectin (CD3) and the complement receptor family (CD1 and CD) 3 3. Furthermore, MHC class II molecules can associate with CD on human B cells 37. Here we found that MHC class II molecules interacted with CD 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 signaling 38,39, 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 CD and Btk in the endosomes of TLR-activated macrophages and that intracellular CD mediated the interaction of MHC class II and Btk. Although Btk has been reported to become activated after stimulation of human B lymphocytes with CDL, the underlying mechanism is not clear. Thus, the mechanism by which the interaction of MHC class II molecules with CD maintains activation of Btk needs further investigation. Coexpression of MHC class II (HLA-DR) in HEK93 cells overexpressing TLR or TLR results in much higher TLR- or TLR- triggered expression of human β-defensin. Furthermore, in lysates of HEK93 cells overexpressing HLA-DR, radiolabeled recombinant TLR protein precipitates in vitro together with HLA-DR protein immunoprecipitated with anti-hla-dr. So, TLR was proposed to associate with HLA-DR. However, whether or not TLR 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- 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 VOLUME 1 NUMBER 5 MAY 11 nature immunology

8 11 Nature America, Inc. All rights reserved. interacted with Btk via CD and subsequently maintained Btk activation 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 subjects 1,. Such patients release much less TNF and IL-1β than do normal subjects in response to. In addition, the ability of monocytes to express HLA-DR antigen correlates directly with the clinical course of trauma patients 3 and septic patients. However, treatment with IFN-γ restores HLA- DR expression and thus substantially enhances the induction of TNF by in vitro in such situations. The recovery of monocyte function results in clearance of sepsis and improved survival of the patients. Thus, those clinical observations, together with our in vitro and in vivo data, demonstrate that MHC class II molecules 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 production 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. Methods Methods and any associated references are available in the online version of the paper at Note: Supplementary information is available on the Nature Immunology website. Acknowledgments We 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 (7CB513), National 115 Key Project (8ZX1-8, 9ZX953-3) and the National Natural Science Foundation of China (37191). AUTHOR CONTRIBUTIONS X.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 INTERESTS The authors declare no competing financial interests. Published online at Reprints and permissions information is available online at reprintsandpermissions/. 1. Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 1, (3).. Barton, G.M. & zhitov, R. Toll-like receptor signaling pathways. Science 3, (3). 3. O Neill, L.A. & Bowie, A.G. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat. Rev. Immunol. 7, (7).. Kawai, T. & Akira, S. TLR signaling. Semin. Immunol. 19, 3 (7). 5. Marshak-Rothstein, A. & Rifkin, I.R. Immunologically active autoantigens: the role of toll-like receptors in the development of chronic inflammatory disease. Annu. Rev. Immunol. 5, 19 1 (7).. Liew, F.Y., Xu, D., Brint, E.K. & O Neill, L.A. Negative regulation of Toll-like receptor-mediated immune responses. Nat. Rev. Immunol. 5, 58 (5). 7. Huang, Q. et al. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3. Nat. Immunol. 5, (3). 8. Liu, X. et al. CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages. Blood 11, (8). 9. Schafer, P.H., Pierce, S.K. & Jardetzky, T.S. The structure of MHC class II: a role for dimer of dimers. Semin. Immunol. 7, (1995). 1. McDevitt, H.O. Discovering the role of the major histocompatibility complex in the immune response. Annu. Rev. Immunol. 18, 1 17 (). 11. Al-Daccak, R., Mooney, N. & Charron, D. MHC class II signaling in antigenpresenting cells. Curr. Opin. Immunol. 1, (). 1. Mourad, W., Geha, R.S. & Chatila, T. Engagement of major histocompatibility complex class II molecules induces sustained, lymphocyte functionassociated molecule 1-dependent cell adhesion. J. Exp.. 17, (199). 13. Spertini, F., Chatila, T. & Geha, R.S. Signals delivered via MHC class II molecules synergize with signals delivered via TCR/CD3 to cause proliferation and cytokine gene expression in T cells. J. Immunol. 19, 5 7 (199). 1. Hauschildt, S., Bessler, W.G. & Scheipers, P. Engagement of major histocompatibility complex class II molecules leads to nitrite production in bone marrow derived macrophages. Eur. J. Immunol. 3, (1993). 15. Trede, N.S., Geha, R.S. & Chatila, T. Transcriptional activation of IL-1β and tumor necrosis factor α genes by MHC class II ligands. J. Immunol. 1, (1991). 1. Cambier, J.C. & Lehmann, K.R. Ia-mediated signal transduction leads to proliferation of primed B lymphocytes. J. Exp.. 17, (1989). 17. Drenou, B. et al. Caspase-independent pathway of MHC class II antigenmediated apoptosis of human B lymphocytes. J. Immunol. 13, (1999). 18. Bishop, G. Requirements of class II-mediated B cell differentiation for class II cross-linking and cyclic AMP. J. Immunol. 17, (1991). 19. Cambier, J.C. et al. Ia binding ligand and camp stimulate translocation of PKC in B lymphocytes. Nature 37, 9 3 (1987).. Mooney, N.A., Grillot-Courvalin, C., Hivroz, C., Ju, L.Y. & Charron, D. Early Biochemical events after MHC class II-mediated signaling on human B lymphocytes. J. Immunol. 15, 7 7 (199). 1. Lane, P.J.L., McConnell, F.M., Schieven, G.L., Clark, E.A. & Ledbetter, J.A. The role of class II molecules in human B cell activation: association with phosphatidyl inositol turnover, protein tyrosine phosphorylation, and proliferation. J. Immunol. 1, (199).. Neumann, J., Eis-Hübinger, A.M. & Koch, N. Herpes simplex virus type 1 targets the MHC class II processing pathway for immune evasion. J. Immunol. 171, (3). 3. Lapaque, N. et al. Salmonella regulates polyubiquitination and surface expression of MHC class II antigens. Proc. Natl. Acad. Sci. USA 1, (9).. Piani, A. et al. Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness. Eur. J. Immunol. 3, (). 5. Beharka, A.A., Armstrong, J.W. & Chapes, S.K. Macrophage cell lines derived from major histocompatibility complex II-negative mice. In Vitro Cell. Dev. Biol. Anim. 3, (1998).. de Weers, M. et al. B-cell antigen receptor stimulation activates the human Bruton s tyrosine kinase, which is deficient in X-linked agammaglobulinemia. J. Biol. Chem. 9, (199). 7. Wahl, M.I. et al. Phosphorylation of two regulatory tyrosine residues in the activation of Bruton s tyrosine kinase via alternative receptors. Proc. Natl. Acad. Sci. USA 9, (1997). 8. Brunner, C., Avots, A., Kreth, H.W., Serfling, E. & Schuster, V. Bruton s tyrosine kinase is activated upon CD stimulation in human B lymphocytes. Immunobiology, 3 (). 9. Jefferies, C.A. et al. Bruton s tyrosine kinase is a Toll/interleukin-1 receptor domainbinding protein that participates in nuclear factor κb activation by Toll-like receptor. J. Biol. Chem. 78, 58 (3). 3. Gray, P. et al. MyD88 adapter-like (Mal) is phosphorylated by Bruton s tyrosine kinase during TLR and TLR signal transduction. J. Biol. Chem. 81, (). 31. Mansell, A. et al. Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation. Nat. Immunol. 7, (). 3. Horwood, N.J. et al. Bruton s tyrosine kinase is required for lipopolysaccharideinduced tumor necrosis factor-α production. J. Exp.. 197, (3). 33. Mukhopadhyay, S. et al. Macrophage effector functions controlled by Bruton s tyrosine kinase are more crucial than the cytokine balance of T cell responses for microfilarial clearance. J. Immunol. 18, (). 3. Bonnefoy, J.Y. et al. The low-affinity receptor for IgE (CD3) on B lymphocytes is spatially associated with HLA-DR antigens. J. Exp.. 17, 57 7 (1988). 35. Bradbury, L.E. et al. The CD19/CD1 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 molecules. J. Immunol. 19, (199). 3. Léveillé, C., AL-Daccak, R. & Mourad, W. CD is physically and functionally coupled to MHC class II and CD on human B cell lines. Eur. J. Immunol. 9, 5 7 (1999). nature immunology VOLUME 1 NUMBER 5 MAY 11 3