SCIMP Is a Universal Toll‐Like Receptor Adaptor in Macrophages

SCIMP is a universal Toll-like receptor adaptor controlling selective cytokine outputs from macrophages Lin Luo*, James E. B. Curson, Liping Liu, Adam A. Wall, Neeraj Tuladhar, Richard M. Lucas, Matthew J. Sweet and Jennifer L. Stow Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, QLD 4072, Australia Summary sentence: The TLR adaptor SCIMP is used by multiple TLRs to generate cytokine specificity in macrophages *Correspondence should be addressed to: Dr Lin Luo Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia. Email: l.luo@imb.uq.edu.au Key words: SCIMP, Toll-like receptor, TRAP, inflammatory cytokines, macrophage, effectors This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/JLB.2MA0819-138RR. This article is protected by copyright. All rights reserved. Word counts: 4721 This article is protected by copyright. All rights reserved. ABSTRACT In innate immune cells, pathogens and danger signals activate Toll-like receptors (TLRs), unleashing potent and tailored inflammatory responses. Previously, we reported that an immune-specific transmembrane adaptor, SCIMP, interacts with TLR4 via direct binding to its cytoplasmic TIR domain. SCIMP scaffolds a Src family kinase, Lyn, for TLR4 phosphorylation and activation. Consequently, SCIMP is able to direct selective production of the pro-inflammatory cytokines IL-6 and IL-12p40 downstream of TLR4 in macrophages. Here we set out to investigate whether SCIMP also acts as an adaptor for other TLR family members. We report here that SCIMP is phosphorylated and activated in response to agonists of multiple TLRs, including TLR2, TLR3, TLR4 and TLR9. SCIMP also interacts with TLRs that are known to signal from both the cell surface and endosomal compartments. In so doing, this transmembrane adaptor presents Lyn, along with other effectors such as Grb2, Csk and SLP65, to multiple TLRs during cellular activation. CRISPR-mediated knockout or silencing of SCIMP in macrophages alters TLR signalling outputs and the production of IL-6 and IL- 12p40 downstream of multiple TLRs, and upon challenge with live bacteria. Furthermore, the selectivity in cytokine responses is preserved downstream of TLR3, with inducible expression of Il- 12p40 and IL-6, but not IFN, being SCIMP-dependent. SCIMP is thus a universal TLR adaptor for scaffolding the Lyn tyrosine kinase and its effectors to enable responses against a wide range of danger signals. This article is protected by copyright. All rights reserved. INTRODUCTION Macrophages are central to innate immune responses through their capacity to detect, interpret, and respond to danger signals presented by pathogens and/or other stimuli. Pattern recognition receptors, including the Toll-like receptors (TLRs), are activated by different pathogen-associated molecular patterns, and signalling from these receptors initiates the synthesis and secretion of inflammatory cytokines and other mediators (1). TLR4, the most widely studied member of the TLR family, senses and initiates responses to lipopolysaccharide (LPS) from Gram-negative bacteria (2). At the cell surface, TLR4 dimers recruit the cytoplasmic adaptors Mal (TIRAP) and MyD88, which bind directly through cognate Toll/IL-1 Receptor homology (TIR) domains. The TIR-containing adaptors TRIF and TRAM are subsequently recruited to TLR4 in endosomes (3). Other TLR family members signal from either the cell surface or from endosomal compartments, via one or more of the TIR-containing adaptors. TLR2 is a surface receptor that heterodimerises with TLR1 or TLR6 to recognize lipoproteins from Gram-positive bacteria, lipoarabinomannan from mycobacteria, and zymosan from yeast (4). Detection of intracellular viral and host dsRNAs by TLR3 (5), as well as unmethylated CpG-containing DNA by TLR9 (6), occurs in endosomal compartments. Many TLRs have surface- exposed tyrosine sites (e.g. Y674 in human TLR4) within the TIR domains of their cytoplasmic tails. Some of these residues are highly conserved in human and mouse and across many TLR family members, and are essential for TLR TIR-adaptor (MyD88/MAL or TRIF/TRAM) recruitment, signal transduction and cytokine production (7, 8). TLR2 (9, 10), TLR3 (11) and TLR4 (7, 12) are all known to be tyrosine phosphorylated in an agonist-induced manner. Despite the key role of tyrosine phosphorylation of these TLR TIR domains in signalling, only a limited number of studies have investigated the upstream kinases responsible for phosphorylation at these sites (13). In the case of TLR4, studies by us and others have implicated Lyn kinase as the responsible kinase (7, 12), although Syk is also reported to phosphorylate TLR4 (14). However, our knowledge of the identity of tyrosine kinase(s) acting on other TLR members, as well as the precise mechanisms involved, is still limited. This article is protected by copyright. All rights reserved. The transmembrane adaptor protein (TRAP) family of adaptors is typified by very short extracellular domains and longer cytoplasmic tails that serve as scaffolds for signalling kinases and effector proteins (including Lyn, Grb2, Csk and SLP65) (15, 16). pTRAPs are a sub-family of TRAPs that can be directed to specific membrane domains, such as lipid rafts or tetraspanin-enriched membranes (TEMs), by palmitoylation to enable exquisite signalling specificity from immune and non-immune receptors (17). The cell-type specific expression patterns of TRAPs and their capacity to recruit specific signalling components, such as tyrosine kinases and effector proteins, permits fine-tuning of cellular responses. Previously, we showed that SLP adaptor and CSK interacting membrane protein (SCIMP), a little known pTRAP family member, is an atypical adaptor for TLR4 that drives highly discriminate cytokine responses (IL-6 and IL-12p40, but not other inflammatory mediators examined) in macrophages, thus revealing an unrecognized level of regulation for innate immune signalling (12, 18). SCIMP is constitutively associated with the Lyn tyrosine kinase (19), and upon TLR4 activation, this adaptor presents Lyn to TLR4, resulting in tyrosine phosphorylation of this receptor (12). However, other SCIMP effectors may also be involved in TLR signalling. For example, Csk reportedly participates in signalling downstream of TLRs (20). SLP65 binds GRB2 through its proline-rich domain (21, 22). In B cells, SLP65 functions with GRB2 together to localize VAV3 to the membrane, where it can activate RAC1 and downstream signalling (23), but it is not well understood whether these effectors are involved in SCIMP-mediated TLR pathways. Here we investigate the role of the TLR adaptor SCIMP in driving cytokine production in macrophages upon TLR stimulation. Interestingly, we found that SCIMP is a common adaptor for multiple surface and endosomal TLRs, including TLR2, 3, 4, and 9 where it plays an important role in scaffolding the Lyn tyrosine kinase for production of specific proinflammatory cytokines during TLR-mediated macrophage activation. This article is protected by copyright. All rights reserved. MATERIALS AND METHODS: Plasmids, antibodies and reagents The mouse SCIMP full-length construct was amplified by PCR from cDNA and cloned into the pEF6/V5-His TOPO TA vector and confirmed by DNA sequencing. Primary rabbit antibodies recognizing SCIMP have previously been described (12). Antibodies to p-ERK1/2 (4370), p-Akt (9271), p-IκBα (2859), p-TBK1/NAK (5483), Csk (4980), GAPDH (2118), Grb2 (3972), Lyn (2796), SLP65 (12168), Myc (2276), p-Tyrosine (9411) and the p-Src family (Tyr416; 6943) were purchased from Cell Signalling Technology (Beverly, MA, USA). The anti-V5 (MCA1360) antibody were purchased from Bio-Rad Laboratories. The anti-TLR4 antibody targeting TLR4 amino acids 100-200 (ab22048), TLR3 antibody (ab62566) and TLR2 antibody (ab16894), used for immunoprecipitation and Western blotting, were from Abcam (Melbourne, VIC, Australia). HRP- conjugated goat anti-mouse and -rabbit antibodies (81-6520) were obtained from Zymed (San Francisco, CA, USA). Zymosan (Z4250) was purchased from Sigma Australia (Castle Hill, NSW, Australia). FSL-1 was purchased from Integrated Sciences (Chatswood, NSW, Australia). LPS, purified from Salmonella enterica serotype Minnesota Re 595, was purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). The CpG-containing oligonucleotide ODN-1688 (5’- TCCATGACGTTCCTGATGCT-3’) containing phosphorothioate linkages was purchased from GenScript (Piscataway, NJ, USA). The synthetic dsRNA poly I:C was purchased from Integrated Sciences (Chatswood, NSW, Australia). Pam3CSK4 was purchased from Life Research Pty Ltd (Scoresby, VIC, Australia). Zymosan, FSL-1, LPS, poly I:C, CpG DNA and Pam3CSK4 were used at 10 μg/ml, 100 μg/ml, 100 ng/ml, 10 µg/ml, 0.3 μM and 15 ng/mL, respectively, unless otherwise stated. All other chemicals and reagents were from Sigma-Aldrich (Castle Hill, NSW, Australia). Immunoprecipitation and immunoblotting This article is protected by copyright. All rights reserved. Immunoprecipitation and immunoblots were performed as described previously (24). Briefly, cells were lysed in lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 5% glycerol) and then passed successively through

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