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

Xingguang 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 -presenting function, major 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 and type I 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.

The ability of the innate to recognize and eliminate Major histocompatibility complex (MHC) class II molecules invading microbial pathogens has been largely attributed to Toll-like (encoded by H2) are expressed mainly by professional APCs, includ- receptors (TLRs) and TLR-triggered . TLRs, the ing DCs, macrophages and B cells. MHC class II molecules, which key pattern-recognition receptors expressed on antigen-presenting are heterodimers composed of an α-chain and a β-chain, are type I cells (APCs) such as macrophages and dendritic cells (DCs), have integral membrane proteins with short cytoplasmic domains and important roles in the initiation of innate immune responses as well four large extracellular domains9. Their main function is to present as the subsequent induction of adaptive immune responses1. After the peptides processed from extracellular proteins to CD4+ helper T cells recognition of pathogen-associated molecule patterns, TLRs initiate and to direct the processes of positive and negative selection, shaping Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature shared and distinct signaling pathways by recruiting various combina- the repertoire during maturation and lineage commitment10,11. tions of four Toll–interleukin 1 (IL-1) domain–containing In addition to that classical function, cell surface MHC class II mol­ © 20 adapters proteins: MyD88, TIRAP (Mal), TRIF and TRAM. These ecules can function as receptors to mediate reverse signal transduc- signaling pathways activate the transcription factors NF-κB and tion after ligation with agonist , T cell antigen receptors AP-1, which are common to all TLRs, leading to the production of or CD4 molecules. Engagement of cell surface MHC class II may inflammatory cytokines and chemokines. TLR3, TLR4, TLR7, TLR8 regulate cell adhesion, production and the expression of and TLR9 also activate the transcription factors IRF3 and/or IRF7, costimulatory molecules12–15 and may also induce the apoptosis, leading to the production of type I interferon2–4. Full activation of proliferation or differentiation of B cells16,17. Engagement of MHC TLRs is essential for initiation of the innate immune response and class II mainly activates two distinct signal pathways. One increases enhancement of adaptive immunity to eliminate invading pathogens; cAMP and subsequently induces the translocation of protein kinase C however, TLR signaling is well regulated, positively and negatively, to to the nucleus18,19. Another increases activity of the Src family prevent inappropriate activation or overactivation, which may cause tyrosine kinase Lyn and the non-Src family tyrosine kinase Syk. autoinflammatory disorders5. So far, various signaling molecules have These activated tyrosine mediate activation of phospholipase been shown to be involved in the tight regulation of the TLR pathway C-γ, leading to the production of inositol-(1,4,5)-trisphosphate and to maintain the immunological balance4,6. For example, the kinases ­diacylglycerol, which mediate calcium mobilization and activation MEKK3 (ref. 7) and CaMKII (ref. 8) can be activated by TLR ligands of protein kinase C20,21. and then enhance TLR-triggered innate immune responses. However, MHC class II molecules can contribute to the responsiveness of cells the identification of cofactors and their underlying mechanisms for to microbial components, and pathogens have developed strategies the initiation and full activation of TLR responses remain to be to downregulate expression of MHC class II molecules on APCs fully elucidated. 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

416 VOLUME 12 NUMBER 5 MAY 2011 nature immunology A rt i c l e s

Figure 1 Deficiency in MHC class II protects mice from challenge with a 5.0 * H2+/+ b H2+/+ –/– 4.0 –/– TLR ligands. (a–c) Enzyme-linked immunosorbent assay (ELISA) of 4.0 H2 * H2 TNF (a), IL-6 (b) and IFN- (c) in the serum of H2−/− or H2+/+ mice (n = 5 * 3.0 β 3.0 * * per genotype) 2 h after intraperitoneal administration of PBS or LPS, 2.0 2.0 CpG-ODN (CpG) or poly(I:C) (at a dose of 15, 20 or 20 mg per kg body * −/− TNF (ng/ml) 1.0 IL-6 (ng/ml) 1.0 weight, respectively). *P < 0.01 (Student’s t-test). (d) Survival of H2 mice and H2+/+ mice (n = 10 per genotype) given intraperitoneal injection 0 0 PBS LPS CpG Poly(I:C) PBS LPS CpG Poly(I:C) 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 1.6 H2 * H2+/+ c –/– d 100 7 H2 H2–/– and given intravenous transplantation of 1 × 10 bone marrow cells from 1.2 80 H2−/− or H2+/+ mice 3 weeks before challenge with PBS or LPS, assessed * 60 2 h after challenge. *P < 0.01 (Student’s t-test). Data are from three

(ng/ml) 0.8 40 β independent experiments (mean ± s.e.m.).

* Survival (%) 20

IFN- 0.4 0 0 12 24 36 48 60 72 0 −/− PBS LPS CpG Poly(I:C) Time after LPS challenge (h) challenge with LPS (Fig. 1d). H2 mice were also more resistant to e H2+/+ chimeras lethal challenge with high-dose poly(I:C) (data not shown). Given that H2–/– chimeras −/− + 6.0 H2 mice have considerably fewer CD4 T cells in , spleen * 4.0 * 1.2 * 3.0 + 4.0 0.8 and lymph nodes, we further explored the effects of the lack of CD4 2.0 (ng/ml) −/− 2.0 1.0 β 0.4 T cells on the resistance of H2 mice to sepsis. We transplanted

IL-6 (ng/ml) +/+ −/− TNF (ng/ml)

0 0 IFN- 0 bone marrow cells from H2 or H2 mice into lethally irradi- PBS LPS PBS LPS PBS LPS ated wild-type mice. The reconstituted H2−/− mixed–bone marrow chimeras had numbers of CD4+ T cells in spleen and lymph nodes ­deficiency in MHC class II results in less production of lipopoly­ similar to those in H2+/+ chimeras but had no expression of MHC saccharide (LPS)-induced tumor necrosis factor (TNF) by human class II in macrophages or DCs (data not shown). The LPS-induced peripheral blood and mouse macrophages24,25. However, in vivo production of TNF, IL-6 and IFN-β was much lower in the the detailed mechanisms by which MHC class II molecules are involved reconstituted H2−/− chimeras than in H2+/+ chimeras (Fig. 1e), which in TLR-triggered innate immune responses remain uncharacterized. therefore excluded the possibility that the lack of CD4+ T cells was MHC class II molecules and TLRs are both expressed mainly on involved in the resistance of H2−/− mice to sepsis. These data demon- APCs; therefore, we sough to determine whether MHC class II mol­ strate that H2−/− mice showed impaired TLR-triggered inflammatory ecules have another, nonclassical function and somehow intersect innate responses and were more resistant to endotoxin shock, which with the TLR signaling pathway. Here we found that MHC class indicates that MHC class II molecules have an important role in the II–deficient mice were more resistant to endotoxin shock induced full activation of TLR-triggered immune responses. by either lethal challenge with LPS or infection with Gram-negative To assess the role of MHC class II molecules in the host innate bacteria, with less production of proinflammatory cytokines and type response to infection with intact Gram-negative bacteria, we injected I interferon in vivo. Deficiency in MHC class II attenuated the pro- H2−/− mice intraperitoneally with Escherichia coli 0111:B4, duction of proinflammatory cytokines and type I interferon triggered the most frequent cause of bacterial sepsis in humans. The production by TLR4, TLR3 or TLR9 in macrophages and DCs. Furthermore, of TNF and IL-6 in the serum of H2−/− mice after injection of E. coli Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature intracellular MHC class II molecules interacted with the tyrosine was significantly less than that in H2+/+ mice (Fig. 2a,b). The survival kinase Btk via the costimulatory molecule CD40 in endosomes and of H2−/− mice after lethal challenge with E. coli was also prolonged © 20 maintained activation of Btk, but cell surface MHC class II molecules (Fig. 2c). These data indicate that deficiency in MHC class II attenuates did not. Activated Btk interacted with MyD88 and TRIF, promoting the inflammatory innate response of host to Gram-negative bacteria the activation of MyD88-dependent and TRIF-dependent pathways. and protects mice from lethal challenge by Gram-negative bacteria. Therefore, intracellular MHC class II molecules are needed to pro- mote the full activation of TLR signaling. Impaired cytokine production in TLR-triggered H2−/− APCs Next we assessed whether deficiency in MHC class II attenu- RESULTS ated the production of proinflammatory cytokines and type I H2 deficiency protects mice from LPS and bacterial challenge −/− MHC class II–deficient (H2 ) mice (with a 78.8-kilobase deletion a b * c +/+ +/+ 4 +/+ +/+ in H2) and wild-type (H2 ) littermate mice had similar numbers 3 H2 * H2 100 H2 H2–/– H2–/– H2–/– of splenic macrophages and DCs, as well as peritoneal macrophages 3 80 and bone marrow–derived macrophages and DCs (Supplementary 2 60 Fig. 1a,b). Furthermore there were no substantial differences between 2 40

−/− +/+ 1 Survival (%) H2 and H2 mice in the expression of CD40, CD80 or CD86 IL-6 (ng/ml) 20 TNF (ng/ml) 1 on splenic DCs or immature or mature bone marrow–derived DCs 0 −/− 0 0 0 12 24 36 48 60 72 (Supplementary Fig. 1c). Therefore, H2 mice had normal mye- PBS E. coli PBS E. coli Time after E. coli challenge (h) loid development and macrophage differentiation. To investigate the role of MHC class II molecules in the TLR-triggered innate immune 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 response, we challenged H2 mice with the TLR ligands LPS, CpG +/+ −/− or H2 mice (n = 3 per genotype) 4 h after intraperitoneal infection ODN or poly(I:C). H2 mice produced significantly less TNF, IL-6 with E. coli 0111:B4 (1 × 107 colony-forming units per mouse). +/+ and interferon-β (IFN-β) than H2 mice did in response to challenge *P < 0.01 (Student’s t-test). (c) Survival of mice (n = 10 per genotype) −/− with LPS, CpG ODN or poly(I:C) (Fig. 1a–c). Accordingly, H2 treated as described in a,b. P < 0.01 (Wilcoxon test). Data are from mice had prolonged survival relative to that of H2+/+ mice after lethal three independent experiments (mean ± s.e.m.).

nature immunology VOLUME 12 NUMBER 5 MAY 2011 417 A rt i c l e s

LPS LPS LPS a +/+ b 3.0 ** 2.0 ** 1.0 H2 4.0 2.5 0.6 ** ** ** H2+/+ ** 0.8 –/– ** ** ** –/– ** 1.5 ** ** H2 3.0 ** ** ** 2.0 H2 2.0 ** 0.6 1.5 0.4

(ng/ml) ** Macrophage 1.0 2.0 (ng/ml)

β 0.4 1.0 ** 1.0 β 0.2 0.5 0.2 1.0 0.5 IL-6 (ng/ml) IL-6 (ng/ml) TNF (ng/ml) TNF (ng/ml) IFN- 0 0 0 0 0 IFN- 0

Med LPS CpG Med LPSCpG Med LPS CpG Mock-chain-chain -chain Mock -chain-chain -chain Mock -chain -chain -chain β β β β β β Poly(l:C) Poly(l:C) Poly(l:C) + α + α + α α α α CpG Poly(l:C) CpG Poly(l:C) CpG Poly(l:C) ** +/+ c 2.0 1.5 0.6 ** H2 3.0 2.0 0.8 +/+ ** ** –/– H2 1.5 ** H2 ** ** ** ** ** –/– 1.0 ** 0.4 ** 2.0 1.5 0.6 H2

DC (ng/ml) ** 1.0 ** 1.0 (ng/ml) 0.4 β 0.5 0.2 β ** 0.5 1.0 0.5 0.2 IL-6 (ng/ml) TNF (ng/ml) IL-6 (ng/ml) TNF (ng/ml) IFN- 0 0 0 0 0 IFN- 0

Med LPSCpG Med LPSCpG Med LPSCpG Mock -chain Mock -chain Mock -chain Mock-chain Mock-chain Mock -chain β β β β β β Poly(l:C) Poly(l:C) Poly(l:C) + + + + + + α α α α α α Ctrl d 100 e 4.0 ** 2.5 ** 0.8 ** Ctrl siRNA 80 3.0 ** 2.0 ** ** 0.6 ** siRNA 60 ** 1.5 MHCIIβ 2.0 (ng/ml) 0.4 ** 40 1.0 β 1.0 0.5 0.2 β- Counts 20 IL-6 (ng/ml) TNF (ng/ml) 0 0 0 IFN- 0 100101102103 MHCII Med LPS CpG Med LPS CpG Med LPS CpG Figure 3 Deficiency in MHC class II Poly(l:C) Poly(l:C) Poly(l:C) attenuates TLR-triggered production +/+ f 5.0 4.0 1.2 H2 * * –/– of proinflammatory cytokines and type I interferon in macrophages and 4.0 3.0 * * H2 −/− +/+ 3.0 * * 0.8 DCs. (a) ELISA of cytokines in supernatants of H2 or H2 macrophages 2.0 * (ng/ml) 2.0 * β 0.4 * (top row) or DCs (bottom row) left unstimulated (Med) or stimulated for 1.0 1.0 IL-6 (ng/ml) TNF (ng/ml)

0 IFN- 0 6 h with LPS (100 ng/ml), CpG ODN (CpG; 0.3 µM) or poly(I:C) (10 µg/ml). 0 (b,c) ELISA of cytokines in supernatants of H2−/− or H2+/+ macrophages PBS LPS CpG PBS LPS CpG PBS LPS CpG given mock transfection (Mock) or transfected with vectors for the expression Poly(l:C) Poly(l:C) Poly(l:C) 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 . (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).

interferon in TLR-triggered macrophages and DCs in vitro. H2−/− attenuation of TLR responses achieved by deficiency in MHC peritoneal macrophages had lower expression of TNF, IL-6, IFN-α class II was due simply to lower expression of TLRs. Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature and IFN-β mRNA and protein than did macrophages from H2+/+ We further observed the effect of knockdown of MHC class II on mice in response to LPS, CpG-ODN or poly(I:C) (Fig. 3a and cytokine production by TLR-activated macrophages. The endog- © 20 Supplementary Fig. 2). Similarly, we also detected less production enous expression of total or cell surface MHC class II in peritoneal of TNF, IL-6 and IFN-β in H2−/− DCs (Fig. 3a). H2−/− peritoneal macrophages was diminished considerably by MHC class II–specific macrophages responded normally to the phorbol ester PMA, to small interfering RNA (siRNA; Fig. 3d). MHC class II–specific siRNA IL-1β and to MDP (the ligand for the intracellular bacteria sensor resulted in significantly less production of TNF, IL-6 and IFN-β in Nod2) and produced amounts of TNF and IL-6 similar to those peritoneal macrophages stimulated with LPS, CpG-ODN or poly(I:C) produced by H2+/+ macrophages (Supplementary Fig. 3a), which (Fig. 3e). To further confirm that the impaired TLR-triggered inflam- indicated that deficiency in MHC class II selectively impaired the matory response in vivo was due to deficiency in MHC class II in activation of TLR signaling. We also did a ‘rescue’ experiment by myeloid cells, we adoptively transferred H2+/+ or H2−/− bone marrow– transfecting expression vectors encoding MHC class II α-chain derived macrophages into wild-type mice depleted of endogenous and β-chain into H2−/− peritoneal macrophages and found that macrophages by pretreatment with clodronate liposomes. Mice overexpression of both MHC class II α-chain and β-chain restored reconstituted with H2−/− macrophages produced less proinflamma- the production of TNF, IL-6 and IFN-β induced by LPS, CpG-ODN tory cytokines and IFN-β in response to LPS, CpG-ODN or poly(I:C) or poly(I:C), whereas overexpression of either α-chain or β-chain challenge than did mice reconstituted with H2+/+ macrophages alone did not (Fig. 3b,c); this suggested that both the α-chain and (Fig. 3f). Therefore, deficiency in MHC class II attenuated the β-chain are required for full activation of TLR-triggered macro- TLR-triggered production of proinflammatory cytokines and type I phages. In addition, there was no substantial difference between interferon in APCs, including macrophages and DCs. H2−/− and H2+/+ mice in the expression of TLR4, TLR3 or TLR9 protein and mRNA in peritoneal macrophages and bone marrow– Impaired TLR signaling in H2−/− macrophages derived DCs (Supplementary Fig. 3b,c). Overexpression of MHC We further investigated the effect of deficiency in MHC class II on class II α-chain and β-chain in H2−/− peritoneal macrophages did TLR-activated downstream signal pathways in macrophages. We not affect the expression of TLR4, TLR3 or TLR9 (Supplementary observed impaired phosphorylation of the kinases Erk, Jnk and p38 Fig. 4). Thus, MHC class II expression did not affect the expres- and inhibitor IκBα in LPS-stimulated H2−/− peritoneal macrophages sion pattern of TLRs, which excluded the possibility that the (Fig. 4a). Deficiency in MHC class II resulted in less LPS-induced

418 VOLUME 12 NUMBER 5 MAY 2011 nature immunology A rt i c l e s

a H2–/– H2+/+ b c 12 LPS (min) 0 15 30 45 60 0 15 30 45 60 * +/+ p-Erk 10 * 10 8 * H2 * –/– –/– +/+ 8 * 8 * 6 Erk H2 H2 * 6 * H2 6 6 4 * p-Jnk LPS (min) 0 30 60 90 0 30 60 90 (fold) 4 B activation (fold)

4 (fold) 4 κ Jnk Nuclear 2 (fold) 2 IRF3 2 2 NF- IRF3 activation 0 AP-1 activation 0 0 0 p-p38 Lamin A IRF7 activation p38 Med LPS Med Med LPS CpG Med LPS CpG CpG p-lκBα Poly(I:C) Poly(I:C) Poly(I:C) p-IRF3 IRF3

d –/– +/+ e –/– +/+ f * H2 H2 H2 H2 6 5 7 * * * 6 * LPS (min) 30 0 1530456090 0 153045 60 90 LPS (min) 30 0 1530456090 0 153045 6090 5 * 4 * 5 +/+ IP: MyD88 – + + + + + + + + + + + + IP: TRIF – + + + + + + + + + + + + 4 3 4 H2 3 –/– c.p.m.) c.p.m.) IP: IgG + – –––––– ––– –– IP: IgG + – –––––– –– ––– c.p.m.) 3 2 3 H2 3 3 2 2 IB: IRAK1 IB: TBK1 1 1 (10 1 (10 (10 TBK1 activity TAK1 activity IRAK1 activity 0 0 0 IB: MyD88 IB: TRIF

Med LPS CpG Med LPS CpG Med LPS Poly(I:C) Poly(I:C)

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.).

phosphorylation and nuclear translocation of IRF3 (Fig. 4a,b). We signaling, we screened the activation status of various tyrosine kinases obtained similar results with H2−/− peritoneal macrophages stim- in TLR-triggered H2−/− and H2+/+ macrophages. Many such kinases ulated with CpG ODN or poly(I:C) (Supplementary Fig. 5). We showed similar activation in H2−/− and H2+/+ macrophages stimulated further evaluated the effect of deficiency in MHC class II on the with LPS, except Btk, a member of the Btk-Tec family of cytoplasmic transactivation of NF-κB, AP-1, IRF3 and IRF7. Transactivation of tyrosine kinases26. Activation of Btk was associated with phosphoryla- reporters for NF-κB and AP-1 was lower in H2−/− peritoneal macro­ tion of two tyrosine residues: Tyr550 and Tyr222. Tyr550 in the activa- phages stimulated with LPS, CpG ODN or poly(I:C) (Fig. 4c). tion loop is transphosphorylated, leading to autophosphorylation at Deficiency in MHC class II also impaired the transactivation of an Tyr222, which is necessary for full activation27. TLR ligand-induced Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature IRF3 reporter induced by LPS or poly(I:C) and the transactivation of phosphorylation of Btk was much lower in H2−/− macrophages than an IRF7 reporter induced by CpG ODN (Fig. 4c). To assess the func- in H2+/+ macrophages (Fig. 5a,b), which indicated that MHC class II © 20 tional integrity of intracellular signal pathway in H2−/− macrophages, molecules may increase TLR-triggered activation of Btk. We found we monitored the activation of mitogen-activated protein kinases and no substantial difference between H2−/− and H2+/+ macrophages NF-κB induced by TNF. Phosphorylation of Jnk, p38 and IκBα in in PMA-triggered Btk activation (Supplementary Fig. 7a), which TNF-stimulated H2−/− peritoneal macrophages was similar to that in suggested that deficiency in MHC class II selectively impairs TLR- TNF-stimulated H2+/+ macrophages (Supplementary Fig. 6), which ­triggered Btk activation. Overexpression of MHC class II α-chain and indicated that deficiency in MHC class II selectively impaired TLR- β-chain in wild-type macrophages did not induce Btk activation (data triggered activation of mitogen-activated protein kinases and NF-κB. not shown), whereas overexpression of MHC class II α-chain and Immunoprecipitation showed that the interaction of MyD88 with the β-chain in H2−/− peritoneal macrophages did restore the activation kinase IRAK1 or of TRIF with the kinase TBK1 was much lower in of Btk triggered by LPS, CpG-ODN or poly(I:C) (Supplementary H2−/− macrophages stimulated with LPS than in H2+/+ macrophages Fig. 7b,c). These data indicate that MHC class II molecules act in stimulated with LPS (Fig. 4d,e). In vitro kinase assays showed that synergy with TLR signaling to maintain Btk activation. the activation of IRAK1, TAK1 and TBK1 induced by LPS, CpG To investigate the role of Btk in TLR signaling, we examined the ODN or poly(I:C) was impaired in H2−/− macrophages relative to effect of Btk deficiency on TLR-triggered production of proinflam- that in H2+/+ macrophages (Fig. 4f). Collectively, these data suggest matory cytokines and type I in macrophages. Btk−/− that MHC class II molecules promote TLR-triggered production of peritoneal macrophages produced significantly less TNF, IL-6 and proinflammatory cytokines and type I interferon by enhancing the IFN-β than Btk+/+ macrophages did in response to LPS, CpG-ODN activation of both MyD88-dependent and TRIF-dependent pathways or poly(I:C) (Fig. 5c). We further observed the effect of knockdown in macrophages. of Btk on the production of cytokines in TLR-activated macrophages. Btk-specific siRNA substantially downregulated endogenous expres- MHC class II molecules maintain TLR-triggered Btk activation sion of Btk (Supplementary Fig. 8a), which led to much lower pro- Next we explored which signal molecules mediate the nonclassical duction of TNF, IL-6 and IFN-β in macrophages stimulated with LPS, function of MHC class II molecules in promoting intracellular TLR CpG-ODN or poly(I:C) (Supplementary Fig. 8b–d). In addition, signaling. Given that activation of tyrosine kinases is involved in TLR LFM-A13, an inhibitor of Btk, also resulted in much less production

nature immunology VOLUME 12 NUMBER 5 MAY 2011 419 A rt i c l e s

a b –/– +/+ c H2 H2 3.0 ** 1.5 0.6 –/– +/+ ** ** H2 H2 ) ) Btk+/+ 2.0 ** ** ** –/– LPS (min) 0 5 15 3045 60 05 15 30 4560 G G ** 1.0 0.4 ** Btk MedCp Poly(l:CMedCp Poly(l:C (ng/ml)

p-Btk(Y550) β ** p-Btk(Y550) 1.0 0.5 0.2 TNF (ng/ml) IL-6 (ng/ml) p-Btk(Y222) p-Btk(Y222) IFN- 0 0 0 Btk Btk Med LPS CpG Poly(l:C) Med LPS CpG Poly(l:C) Med LPS CpG Poly(l:C) H2+/+ d * 3.0 H2–/– Figure 5 MHC class II molecules promote TLR-triggered 5.0 ** * 1.2 * 4.0 2.5 ** * * ** * * 2.0 ** ** * inflammatory innate responses by maintaining Btk 3.0 ** ** 0.8 ** 1.5 * (ng/ml)

activation. (a,b) Immunoblot analysis of Btk phosphorylated β 2.0 1.0 0.4 ** 1.0 IL-6 (ng/ml) at Tyr550 (p-Btk(Y550)) or Tyr222 (p-Btk(Y222)) or total TNF (ng/ml) 0.5 −/− +/+ 0 0 IFN- 0 Btk in lysates of H2 or H2 peritoneal macrophages Mock E41K Mock E41K Mock E41K Mock E41K Mock E41K Mock E41K Mock E41K Mock E41K Mock E41K left unstimulated or stimulated for 0–60 min with LPS LPS CpG Poly(l:C) LPS CpG Poly(l:C) LPS CpG Poly(l:C) (100 ng/ml; a) or for 30 min with CpG ODN (0.3 µM) or poly(I:C) (10 µg/ml; b). (c) ELISA of TNF, IL-6 and IFN-β in supernatants of Btk+/+ or Btk−/− peritoneal macrophages left unstimulated or stimulated for 6 h with LPS, CpG ODN or poly(I:C). (d) ELISA of TNF, IL-6 and IFN-β in supernatants of H2−/− or H2+/+ peritoneal macrophages mock-transfected or transfected with constitutively active Btk(E41K) and, 48 h later, stimulated for 6 h with LPS, CpG ODN or poly(I:C). *P < 0.05 and **P < 0.01 (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.).

of TNF, IL-6 and IFN-β in macrophages stimulated with LPS, MHC class II molecules interact with some intermediate proteins CpG-ODN or poly(I:C) (Supplementary Fig. 9). These data suggest and thereby integrate into the TLR signaling pathway. that Btk is required for full activation of TLR signaling. Given that the impaired activation of Btk in TLR-triggered H2−/− Given the positive role of Btk in TLR-triggered cytokine produc- macrophages contributed to the lower production of inflammatory tion and the lower activation of Btk in TLR-triggered H2−/− macro- cytokines and type I interferon, we sought to determine whether phages, we sought to determine whether Btk contributes to MHC MHC class II molecules interact with Btk. Immunoprecipitation class II–mediated full activation of TLR signaling. We transfected showed that MHC class II molecules interacted with Btk (Fig. 6a). H2−/− and H2+/+ macrophages with plasmid encoding constitutively Specifically, intracellular MHC class II molecules associated with Btk, active Btk, with substitution of lysine for glutamic acid at position 41 but plasma membrane MHC class II molecules did not. As MHC (Btk(E41K)), and found that overexpression of Btk(E41K) potently class II molecules have only short cytoplasmic domains, MHC class II enhanced the production of TNF, IL-6 and IFN-β induced by LPS, might not directly interact with Btk; therefore, some other molecules CpG ODN or poly(I:C) in H2+/+ macrophages relative to that in might mediate the interaction of MHC class II with Btk. We immuno­ mock-transfected control cells (Fig. 5d). Furthermore, overexpres- precipitated proteins from lysates of LPS-stimulated macrophages sion of Btk(E41K) restored the impaired production of inflammatory with antibody to MHC class II and then used reverse-phase nano- cytokines and type I interferon in H2−/− macrophages activated with spray liquid chromatography–tandem mass spectrometry to identify LPS, CpG ODN or poly(I:C) (Fig. 5d). Together these data suggest possible MHC class II–associated proteins, which might be involved that MHC class II molecules facilitate TLR-triggered inflammatory in the association and activation of Btk, in the immunoprecipitates. responses by enhancing Btk activation. Among the several proteins we detected (data not shown), CD40 Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature attracted our attention because CD40 has been found to mediate Btk Intracellular MHC class II molecules bind Btk via CD40 activation after stimulation of human B with its ligand, © 20 We further investigated the mechanisms underlying the involve- CD40L28. Further immunoprecipitation confirmed the finding that ment of MHC class II molecules in the activation of TLR signaling. intracellular MHC class II molecules associated with CD40, but those First we sought to determine whether MHC class II molecules inter- at the plasma membrane did not (Fig. 6b). Confocal microscopy also acted directly with TLRs. Immunoprecipitation with antibody to showed that intracellular MHC class II localized together with CD40 MHC class II, TLR4, TLR9 or TLR3 showed that MHC class II mol­ and Btk in the endosomes of macrophages stimulated with LPS for ecules did not interact with TLR4, TLR9 or TLR3 (Supplementary 15 min (Fig. 6c–f), which indicated that intracellular but not plasma Fig. 10). So far, there has been no report to our knowledge showing membrane MHC class II forms a complex with CD40 and Btk after that MHC class II molecules can recognize TLR ligands; therefore, TLR activation. 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 Binding of Btk with CD40 is required for full TLR response molecules are also abundant in the intracellular endosomal compart- Immunoprecipitation with antibody to Btk (anti-Btk) also showed ment9,10, whereas after stimulation by their respective ligands, TLR3 that Btk interacted with CD40 and MHC class II molecules and TLR9 located on the endoplasmic reticulum membrane and (Supplementary Fig. 12a). To determine which domain of Btk TLR4 on the plasma membrane translocate into endosome. In the was required for the interaction of Btk with CD40, we constructed endosome, TLRs initiate signals by a MyD88- or TRIF-dependent mutants of Btk with deletion of various domains and transfected the pathway1. Thus, we investigated whether deficiency in MHC class II mutants into Btk-deficient macrophages to observe the restoration disrupted endosomal trafficking of TLR4, TLR3 or TLR9 in TLR- of LPS-induced cytokine production in the Btk-deficient macro- triggered macrophages. Confocal microscopy showed that the phages. Overexpression of mutant Btk with deletion of the pleck- translocation of TLR4, TLR3 or TLR9 into endosomes in H2−/− strin homology domain or the kinase domain was unable to restore macrophages was similar to that in H2+/+ macrophages, after stimu­ LPS-induced TNF production (Supplementary Fig. 12b). We trans- lation with TLR ligands (Supplementary Fig. 11). The similar fected those two Btk mutants or wild-type Btk into macrophages, subcellular distribution of intracellular MHC class II molecules and followed by immunoprecipitation. We found that mutant Btk with TLR4, TLR3 and TLR9 inspired us to explore whether intracellular deletion of the pleckstrin homology domain did not interact with

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Plasma Plasma MHCll Btk Merge a Cytoplasmic membrane b Cytoplasmic membrane c LPS (min) 15 0 5 15 15 0 5 15 LPS (min) 15 0 5 15 15 0 5 15 IP: MHCll – + + + – + + + IP: MHCll – + + + – + + + LPS 0 min IP: IgG + –– – + –– – IP: IgG + –– – + –– – IB: Btk IB: CD40 IB: MHCll IB: MHCll LPS d MHCll CD40 Merge e Btk CD40 Merge 15 min

LPS LPS 0 min 0 min f MHCll EEA1 Merge

LPS LPS LPS 15 min 15 min 15 min

g * Cd40+/+ 3.0 * 1.5 * –/– Figure 6 Intracellular MHC class II molecules interact with 0.6 Cd40 * * * CD40 and Btk. (a,b) Immunoblot analysis of Btk (a), CD40 (b) 2.0 * 1.0 0.4 *

or MHC class II (a,b) immunoprecipitated with antibody to (ng/ml)

β * 1.0 0.5 0.2 IL-6 (ng/ml) MHC class II from cytoplasmic and plasma membrane proteins TNF (ng/ml) in lysates of peritoneal macrophages stimulated for 0–15 min 0 0 IFN- 0 with LPS. Immunoglobulin G serves as an immunoprecipitation Med LPS CpG Med LPS CpG Med LPS CpG control. (c–f) Confocal microscopy of macrophages left Poly(l:C) Poly(l:C) Poly(l:C) unstimulated (0 min) or stimulated for 15 min with LPS (100 ng/ml), then labeled with antibodies to the appropriate molecules (above images). Original magnification, ×630. (g) ELISA of TNF, IL-6 and IFN-β in supernatants of Cd40+/+ or Cd40−/− peritoneal macrophages left unstimulated or stimulated for 6 h with LPS (100 ng/ml), CpG ODN (0.3 µM) or poly(I:C) (10 µg/ml). *P < 0.01 (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.).

CD40 (Supplementary Fig. 12c). Therefore the pleckstrin homology cytokines and IFN-β than did Cd40+/+ macrophages in response domain of Btk is required for the interaction of Btk with CD40. to stimulation with LPS, CpG ODN or poly(I:C) (Fig. 6g). These As described above (Fig. 5 and Supplementary Figs. 8 and 9), data indicate that CD40-mediated interaction of intracellular MHC Btk activation was required for full activation of TLR signaling. class II molecules with Btk is involved in the positive regulation of We sought further to confirm that it was CD40 that mediated the TLR-triggered innate response. interaction of MHC class II and Btk required for the TLR response. Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature Immunoprecipitation of proteins from lysates of TLR-triggered Btk enhances TLR signaling by binding MyD88 and TRIF CD40-deficient macrophages showed that MHC class II molecules did We sought to elucidate the underlying molecular mechanisms by © 20 not interact with Btk without CD40 (Supplementary Fig. 13), which which the greater Btk activation contributed to the enhancement of suggested that MHC class II molecules interact with Btk via CD40 in TLR-triggered innate immune response by intracellular MHC class II. TLR responses. Btk activation triggered by LPS, CpG ODN or poly(I:C) Given that signaling through TLR4, TLR9 and TLR3 was down­ was also impaired in Cd40−/− macrophages (Supplementary Fig. 14). regulated by deficiency in MHC class II, the common adapters Furthermore, Cd40−/− macrophages produced less proinflammatory MyD88 and TRIF might be the potential targets of Btk and might be

Mock +– + – a –/– +/+ b c H2 H2 HA-Btk ––+ + LPS (min) 30 0 15 30 60 90 0 15 30 60 90 Flag-MyD88 ++ – – IP: Btk – + + + + + + + + + + Flag-TRIF –– + + 40 ** 40 IP: HA ++ + + * 12 ** IP: IgG + – – ––– –– –– – ** * IB: MyD88 30 30 * IB: Flag 8 20 20 IB: TRIF IB: HA 10 10 4 B activation (fold) B activation (fold) κ IB: Btk κ 0 0 0 TCL: Flag IRF3 activation (fold) NF- Ctrl MyD88 25 50 100 NF- Ctrl TRIF 25 50 100 Ctrl TRIF 25 50 100 MyD88+E41K TRIF+E41K TRIF+E41K

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 H2−/− or H2+/+ macrophages stimulated for 0–90 min with LPS. (b) Immunoblot analysis of HEK293 cells 48 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 HEK293 cells 24 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(E41K) (MyD88+E41K or TRIF+E41K; 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 < 0.05 and **P < 0.01 (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).

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more activated in the presence of MHC class II. Immunoprecipitation Mal and IRAK1, we conclude that Btk is necessary but is not essential with anti-Btk showed that Btk precipitated together with MyD88 for the full activation of MyD88- and TRIF-dependent pathways by and TRIF in H2+/+ macrophages after LPS stimulation and that interacting with multiple components in TLR signaling. As deficiency this coimmunoprecipitation was diminished in H2−/− macrophages in MHC class II impairs Btk activation, H2−/− mice and cells (macro­ (Fig. 7a and Supplementary Fig. 15). We transfected hemaggluti- phages and DCs) derived from them had lower but not completely nin-tagged Btk together with Flag-tagged MyD88 or Flag-tagged abolished cytokine production and less death in response to challenge TRIF into HEK293 human embryonic kidney cells; coimmunopre- with TLR ligands. The data showing that overexpression of the con- cipitation showed that Btk interacted with either MyD88 or TRIF stitutively active mutant Btk(E41k) corrected the lower abundance of (Fig. 7b). We further examined the effect of Btk on the activation proinflammatory cytokines and type I interferons in TLR-triggered of a reporter for NF-κB or IRF3. Overexpression of constitutively H2−/− macrophages indicate that Btk activation has a pivotal role in active Btk(E41K) enhanced the MyD88- or TRIF-induced activation MHC class II–mediated full activation of TLR signaling. of NF-κB and the TRIF-induced activation of IRF3 (Fig. 7c). These Reverse signaling mediated by MHC class II at the cell surface is data indicate that MHC class II molecules contribute to the mainte- involved in many cellular processes of B cells and DCs. The short nance of TLR-triggered Btk activation and subsequently enhance the cytosolic domain of MHC class II molecules seems inconsistent with interaction of Btk with MyD88 and TRIF, which promotes the acti- these complex signal-transduction pathways, which suggests that the vation of MyD88-dependent and TRIF-dependent signal pathways, presence of membrane-associated signaling components that might finally leading to full activation of TLR-triggered innate responses provide this functionality. Indeed, a variety of cell surface molecules (Supplementary Fig. 16). have been reported to immunoprecipitate together with and/or couple with MHC class II molecules. These MHC class II–associated DISCUSSION molecules belong to various families, including the immunoglobulin It is well known that MHC class II molecules have a crucial role in superfamily (CD19), the tetraspanin family (CD37, CD53, CD81 and the development and function of the immune system. In addition CD82), lectin (CD23) and the complement receptor family (CD21 to the classical function of MHC class II molecules in presenting and CD20)34–36. Furthermore, MHC class II molecules can associate antigen to CD4+ T cells, MHC class II molecules can activate various with CD40 on human B cells37. Here we found that MHC class II cellular functions in immune or non-immune cells when crosslinked molecules interacted with CD40 in the endosomes of TLR-activated by antibody or superantigen12–14. These nonclassical functions are macrophages. However, which region of MHC class II interacts with accomplished by MHC class II molecules at the cell surface acting these molecules is still unclear. Several studies have shown that eight as signal-transduction receptors. However, so far there has been no membrane-proximal amino acids of the cytoplasmic domain and insight into any nonclassical functions of intracellular MHC class II transmembrane domain of MHC class II β-chain are required for molecules. Given reports that the expression of MHC class II can affect distinct MHC class II–mediated signaling38,39, which suggests that the response of macrophages to LPS24,25, we speculated that MHC these regions may also be required for the interaction of MHC class II class II molecules may be involved in the activation of TLR signaling. with other molecules. Notably, we found that intracellular MHC Here we have provided evidence that deficiency in MHC class II class II molecules interacted with Btk after TLR ligation as early as impaired TLR-triggered production of proinflammatory cytokines 5 min after activation, but cell surface MHC class II molecules did and type I interferon in macrophages and DCs, and this protected not. This rapid interaction of intracellular MHC class II and Btk is mice from lethal challenge with TLR ligands and live Gram-negative consistent with rapid activation of the Btk and TLR signaling pathway. Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature bacteria. A lower abundance of activated Btk in TLR-triggered H2−/− We further confirmed that MHC class II molecules formed a complex macrophages led to less interaction of Btk with MyD88 and TRIF, with CD40 and Btk in the endosomes of TLR-activated macrophages © 20 which attenuated the activation of MyD88- and TRIF-dependent and that intracellular CD40 mediated the interaction of MHC class II pathways; this suggested that MHC class II molecules are required and Btk. Although Btk has been reported to become activated after for full activation of TLR-triggered innate responses. Therefore, we stimulation of human B lymphocytes with CD40L, the underlying have demonstrated a nonclassical function of MHC class II molecules mechanism is not clear. Thus, the mechanism by which the interaction in the TLR-triggered innate immune response. of MHC class II molecules with CD40 maintains activation of Btk Some reports have suggested a role for Btk in TLR signaling. Btk is needs further investigation. phosphorylated in LPS-stimulated human monocytes and can interact Coexpression of MHC class II (HLA-DR) in HEK293 cells over- with multiple components of TLR pathways, including TLR4, TLR6, expressing TLR2 or TLR4 results in much higher TLR2- or TLR4- TLR8, TLR9, MyD88, Mal and IRAK129. Btk phosphorylates Mal, triggered expression of human β-defensin40. Furthermore, in lysates which resulting in degradation of Mal30,31. Studies of peripheral blood of HEK293 cells overexpressing HLA-DR, radiolabeled recombinant monocytes from patients with Btk-deficient X-linked agammaglob- TLR2 protein precipitates in vitro together with HLA-DR protein ulinemia and macrophages from Btk-mutant mice with X-linked immunoprecipitated with anti-HLA-DR. So, TLR2 was proposed immunodeficiency have indicated that Btk-dependent signaling to associate with HLA-DR40. However, whether or not TLR2 and is involved in the LPS-induced production of TNF and IL-1β32,33. HLA-DR interact physically needs further investigation. In our However, it remained unclear whether Btk affects the production of study, we did not find direct interaction of cell surface MHC class II cytokines by macrophages in response to other TLR ligands or whether molecules with TLRs in macrophages, which suggests that MHC it activates an altered signal pathway. Here we have shown that Btk class II molecules may not form a complex with TLRs. In addition, interacted with TRIF and promoted TRIF-dependent activation of we found that ligation of MHC class II by specific antibody did not IRF3 and NF-κB, leading to enhanced TLR3- and TLR4-triggered induce the production of TNF, IL-6 or IFN-β in macrophages or DCs, production of type I interferons and proinflammatory cytokines. In which indicated that ligation of cell surface MHC class II alone did addition, the constitutively active mutant Btk(E41K) also enhanced not induce the activation of signaling involved in the production of MyD88-triggered activation of NF-κB, AP-1 and IRF7. Given those inflammatory cytokines in macrophages and DCs (data not shown). findings and the observations that Btk interacted with TLRs, MyD88, Instead, we found here that intracellular MHC class II molecules

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interacted with Btk via CD40 and subsequently maintained Btk acti- 7. Huang, Q. et al. Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3. Nat. Immunol. 5, 98–103 (2003). vation and thereby promoted TLR signaling through interaction of 8. Liu, X. et al. CaMKII promotes TLR-triggered proinflammatory cytokine and type I Btk with the adapters MyD88 and TRIF, but cell surface MHC class II interferon production by directly binding and activating TAK1 and IRF3 in molecules did not. macrophages. Blood 112, 4961–4970 (2008). 9. Schafer, P.H., Pierce, S.K. & Jardetzky, T.S. The structure of MHC class II: a role Clinical observations have shown that HLA-DR expression for dimer of dimers. Semin. Immunol. 7, 389–398 (1995). in peripheral blood monocytes is much lower in patients with 10. McDevitt, H.O. Discovering the role of the major histocompatibility complex in the 41,42 immune response. Annu. Rev. 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© 20 Basic Research Program of China (2007CB512403), National 115 Key Project tyrosine kinase, which is deficient in X-linked agammaglobulinemia.J. Biol. Chem. 269, (2008ZX10002-008, 2009ZX09503-023) and the National Natural Science 23857–23860 (1994). Foundation of China (30721091). 27. 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 94, AUTHOR CONTRIBUTIONS 11526–11533 (1997). 28. Brunner, C., Avots, A., Kreth, H.W., Serfling, E. & Schuster, V. Bruton’s tyrosine kinase X.C. and X.L. designed the experiments; X.L., Z.Z., D.L., L.X., F.M., P.Z. and is activated upon CD40 stimulation in human B lymphocytes. Immunobiology 206, H.Y. did the experiments; X.C. and X.L. analyzed data and wrote the paper; and 432–440 (2002). X.C. was responsible for research supervision, coordination and strategy. 29. Jefferies, C.A. et al. Bruton’s tyrosine kinase is a Toll/interleukin-1 receptor domain- binding protein that participates in nuclear factor κB activation by Toll-like receptor 4. COMPETING FINANCIAL INTERESTS J. Biol. Chem. 278, 26258–26264 (2003). The authors declare no competing financial interests. 30. Gray, P. et al. MyD88 adapter-like (Mal) is phosphorylated by Bruton’s tyrosine kinase during TLR2 and TLR4 signal transduction. J. Biol. Chem. 281, Published online at http://www.nature.com/natureimmunology/. 10489–10495 (2006). Reprints and permissions information is available online at http://npg.nature.com/ 31. Mansell, A. et al. Suppressor of cytokine signaling 1 negatively regulates Toll-like reprintsandpermissions/. receptor signaling by mediating Mal degradation. Nat. Immunol. 7, 148–155 (2006). 32. Horwood, N.J. et al. Bruton’s tyrosine kinase is required for lipopolysaccharide- induced tumor necrosis factor-α production. J. Exp. Med. 197, 1603–1611 1. Takeda, K., Kaisho, T. & Akira, S. Toll-like receptors. Annu. Rev. Immunol. 21, (2003). 335–376 (2003). 33. Mukhopadhyay, S. et al. Macrophage effector functions controlled by Bruton’s 2. Barton, G.M. & Medzhitov, R. Toll-like receptor signaling pathways. Science 300, tyrosine kinase are more crucial than the cytokine balance of T cell responses for 1524–1525 (2003). microfilarial clearance. J. Immunol. 168, 2914–2921 (2002). 3. O’Neill, L.A. & Bowie, A.G. The family of five: TIR-domain-containing adaptors in 34. Bonnefoy, J.Y. et al. The low-affinity receptor for IgE (CD23) on B lymphocytes Toll-like receptor signalling. Nat. Rev. Immunol. 7, 353–364 (2007). is spatially associated with HLA-DR antigens. J. Exp. Med. 167, 57–72 (1988). 4. Kawai, T. & Akira, S. TLR signaling. Semin. Immunol. 19, 24–32 (2007). 35. Bradbury, L.E. et al. The CD19/CD21 signal transducing complex of human 5. Marshak-Rothstein, A. & Rifkin, I.R. Immunologically active autoantigens: the role B lymphocytes includes the target of antiproliferative antibody-1 and Leu-13 of toll-like receptors in the development of chronic inflammatory disease. molecules. J. Immunol. 149, 2841–2850 (1992). Annu. Rev. Immunol. 25, 419–441 (2007). 36. Léveillé, C., AL-Daccak, R. & Mourad, W. CD20 is physically and functionally 6. Liew, F.Y., Xu, D., Brint, E.K. & O’Neill, L.A. Negative regulation of Toll-like coupled to MHC class II and CD40 on human B cell lines. Eur. J. Immunol. 29, receptor-mediated immune responses. Nat. Rev. Immunol. 5, 446–458 (2005). 65–74 (1999).

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37. Léveillé, C., Chandad, F., Al-Daccak, R. & Mourad, W. CD40 associates with the 41. Appel, S.H., Wellhausen, S.R., Montgomery, R., DeWeese, R.C. & Polk, H.C. Jr. MHC class II molecules on human B cells. Eur. J. Immunol. 29, 3516–3526 Experimental and clinical significance of endotoxin-dependent HLA-DR expression (1999). on monocytes. J. Surg. Res. 47, 39–44 (1989). 38. Harton, J.A., Van Hagen, A.E. & Bishop, G.A. The cytoplasmic and transmembrane 42. Astiz, M., Saha, D., Lustbader, D., Lin, R. & Rackow, E. response to domains of MHC class II β chains deliver distinct signals required for MHC class II- bacterial toxins, expression of cell surface receptors, and release of anti-inflammatory mediated B cell activation. Immunity 3, 349–358 (1995). cytokines during sepsis. J. Lab. Clin. Med. 128, 594–600 (1996). 39. Harton, J.A. & Bishop, G.A. Length and sequence requirements of the cytoplasmic 43. Hershman, M.J., Cheadle, W.G., Wellhausen, S.R., Davidson, P.F. & Polk, H.C. Jr. domain of the Aβ molecule for class II-mediated B cell signaling. J. Immunol. 151, Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma 5282–5289 (1993). patient. Br. J. Surg. 77, 204–207 (1990). 40. Frei, R. et al. MHC class II molecules enhance Toll-like receptor mediated innate 44. Docke, W.D. et al. Monocyte deactivation in septic patients: restoration by IFN-γ immune responses. PLoS ONE 5, e8808 (2010). treatment. Nat. Med. 3, 678–681 (1997). Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature © 20

424 VOLUME 12 NUMBER 5 MAY 2011 nature immunology ONLINE METHODS with a Dual-Luciferase Reporter Assay System according to the manufacturer’s Mice and reagents. Mice homozygous for deletion of all genes encoding clas- instructions (Promega). Data were normalized for transfection efficiency by sical MHC class II (B6.129S-H2dlAb1-Ea; Mouse Genome Informatics acces- the division of firefly luciferase activity with that of renilla luciferase. sion code, 003584), Btk-deficient mice (B6.129S-Btktm1Wk/J; Mouse Genome Informatics accession code, 002536) and Cd40-deficient mice (B6.129P2- Immunoprecipitation and immunoblot analysis. Cells were lysed with cell Cd40tm1Kik/J; Mouse Genome Informatics accession code, 002928) were lysis buffer (Cell Signaling Technology) supplemented with protease inhibitor from Jackson Laboratories and were bred in specific pathogen–free condi- ‘cocktail’. Protein concentrations in the extracts were measured by BCA tions. Littermate mice 6 weeks of age were used (matched for body weight assay (Pierce). Immunoprecipitation and immunoblot analysis were done and sex). All animal experiments were in accordance with the National as described45,46. 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 Nanospray liquid chromatography–tandem mass spectrometry. University, Shanghai. LPS (E. coli 0111:B4), CpG ODN and poly(I:C) have Macrophages (3 × 108) were stimulated for 15 min with LPS and then were been described45. E. coli 0111:B4 was obtained from the China Center for lysed for immunoprecipitation with antibody to MHC class II. Proteins were Type Culture Collection. LFM-A13 was from Cayman Chemical. Recombinant eluted and digested, followed by analysis by reverse-phase nanospray liquid MBP was from Upstate Biotechnology. Recombinant MKK4 was from Merck. chromatography–tandem mass spectrometry. The spectra from tandem mass Antibody to MHC class II (107610) was from BioLegend. Recombinant IRF3 spectrometry were automatically used for searching against the nonredundant and anti-TRIF (ab13810), anti-Btk (ab25971), anti-CD40 (ab13545), anti- International Protein Index mouse protein database (version 3.72) with the TLR4 (ab22048), anti-TLR3 (ab62566), anti-TLR9 (ab52967) and antibody Bioworks browser (rev.3.1). to Btk phosphorylated at Tyr222 (ab51210) or Tyr550 (ab52192) were from Abcam. Anti-IRAK1 (D51G7), anti-TBK1 (D1B4), anti-IRF3 (D83B9), anti- In vitro kinase assay. Proteins in total cell extracts (100 µg) were immuno- hemagglutinin (6E2), anti-EEA1 (2411), anti-Erk (9102), anti-Jnk (56G8), precipitated with the appropriate antibody, and kinase activity was measured anti-p38 (9212) and antibodies to Erk phosphorylated at Thr202-Tyr204 as described45. (E10), to Jnk phosphorylated at Thr183-Tyr185 (G9), to p38 phosphorylated at Thr180-Tyr182 (9211), to IRF3 phosphorylated at Ser396(4D4G) or to IκBα Confocal microscopy. Macrophages plated on glass coverslips in six-well phosphorylated at Ser32-Ser36 (5A5) were from Cell Signaling Technology. plates were left unstimulated or stimulated with LPS, then were labeled with Anti-MyD88 (3244-100) was from BioVision. Anti–lamin A (133A2) and antibody to MHC class II, anti-CD40, anti-Btk, anti-TLR4, anti-TLR3, anti- anti-β-actin (sc-130656) were from Santa Cruz. Anti-Flag (M2) was from TLR9 or anti-EEA1 (endosome marker). Cells were viewed with a Leica TCS Agilent Technologies. SP2 confocal laser microscope.

Plasmid constructs. Recombinant vectors encoding mouse MHC class II Establishment of endotoxin shock model and bacterial sepsis model. The α-chain (NM_010378.2) or β-chain (NM_207105.2), TRIF (BC094338), endotoxin shock mouse model was established by intraperitoneal injection of MyD88 (NM_010851) or Btk (NM_013482.2; GenBank accession numbers LPS (15 mg per kg body weight) as described45. E. coli 0111:B4 in midlogarith- in parentheses) and mutants thereof were constructed by PCR-based ampli- mic growth were collected and concentrations were measured by counting of fication from cDNA of mouse macrophages and then were subcloned into viable bacteria on agar plates. For injection, bacteria were washed twice with the pcDNA3.1 eukaryotic expression vector (Invitrogen) as described45. All nonpyrogenic PBS. Mice were injected intraperitoneally with 0.5 ml bacteria constructs were confirmed by DNA sequencing. Luciferase reporter plasmids suspension (1 × 107 colony-forming units)47. for NF-κB, AP-1, IRF3 and IRF7 have been described45,46. Bone marrow transplantation. Bone marrow cells (1 × 107) from H2+/+ or Cell culture and transfection. Bone marrow–derived DCs from C57BL/6J H2−/− mice were transplanted into lethally irradiated wild-type C57BL/6J mice were generated as described46. The HEK293 cell line (American mice (cumulative dose, 10 Gy) by injection into the tail vein. After 3 weeks, Nature America, Inc. All rights reserved. All rights Inc. America, 1 Nature Type Culture Collection) was transfected with JetPEI reagents (PolyPlus). CD4+ T cells in spleen and lymph nodes were counted and expression of MHC Thioglycollate-elicited mouse peritoneal macrophages were prepared and class II on macrophages and DCs was analyzed by flow cytometry. © 20 cultured in endotoxin-free RPMI-1640 medium with 10% (vol/vol) FCS (Invitrogen) as described46 and were transfected by nucleofection with a Macrophage reconstitution. Bone marrow cells from H2+/+ or H2−/− mice Mouse Macrophage Nucleofector kit (Amaxa). were cultured for 7 d in mouse macrophage colony-stimulating factor (50 ng/ml; PeproTech) for the preparation of bone marrow–derived macro- RNA-mediated interference. The sequences of siRNA targeting MHC phages. Clodronate liposomes (Sigma) were injected intraperitoneally into class II β-chain and Btk were 5′-CCACACAGCTTATTAGGAA-3′ and wild-type mice (50 mg in 200 µl per mouse) for the depletion of endogenous 5′-GGAGTCTAGTGAAATGGAA-3′, respectively; the control siRNA macrophages. Then, 2 d later, H2+/+ or H2−/− bone marrow–derived macrophages sequence was 5′-TTCTCCGAACGTGTCACGT-3′. The siRNA duplexes were (1 × 107) were transplanted (by injection into the tail vein) into the mice transfected into mouse peritoneal macrophages with INTERFERin reagent depleted of macrophages, followed 6 h later by challenge with LPS48. (Polyplus) according to a standard protocol. Statistical analysis. The statistical significance of comparisons between two Cytokine detection. TNF, IL-6 and IFN-β in supernatants and serum were groups was determined with Student’s t-test. The statistical significance of measured with ELISA kits (R&D Systems). 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 RNA quantification. A LightCycler (Roche) and SYBR RT-PCR kit (Takara) than 0.05 were considered statistically significant. were used for quantitative real-time RT-PCR analysis as described45. Data were normalized to β-actin expression. 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 Assay of luciferase reporter gene expression. HEK293 cells or mouse macro- type I interferon by inhibiting the kinase IRAK1. Nat. Immunol. 9, 542–550 phages were transfected with a mixture of the appropriate luciferase reporter (2008). plasmid, pRL-TK-renilla-luciferase plasmid and the appropriate additional 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). constructs. The total amount of plasmid DNA was made equal by the addition 48. Han, C. et al. Integrin CD11b negatively regulates TLR-triggered inflammatory of empty control vector. After 24 h or 36 h, cells were left untreated or were responses by activating Syk and promoting degradation of MyD88 and TRIF via treated with LPS, CpG ODN or poly(I:C). Luciferase activity was measured Cbl-b. Nat. Immunol. 11, 734–742 (2010).

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