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• W2904658243 abstract "Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease. Toll-like receptors (TLRs) are pathogen-recognition receptors that trigger the innate immune response. Recent reports have identified accessory proteins that provide essential support to TLR function through ligand delivery and receptor trafficking. Herein, we introduce leucine-rich repeats (LRRs) and calponin homology containing 4 (Lrch4) as a novel TLR accessory protein. Lrch4 is a membrane protein with nine LRRs in its predicted ectodomain. It is widely expressed across murine tissues and has two expression variants that are both regulated by lipopolysaccharide (LPS). Predictive modeling indicates that Lrch4 LRRs conform to the horseshoe-shaped structure typical of LRRs in pathogen-recognition receptors and that the best structural match in the protein database is to the variable lymphocyte receptor of the jawless vertebrate hagfish. Silencing Lrch4 attenuates cytokine induction by LPS and multiple other TLR ligands and dampens the in vivo innate immune response. Lrch4 promotes proper docking of LPS in lipid raft membrane microdomains. We provide evidence that this is through regulation of lipid rafts as Lrch4 silencing reduces cell surface gangliosides, a metric of raft abundance, as well as expression and surface display of CD14, a raft-resident LPS co-receptor. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response and a potential molecular target in inflammatory disease. Toll-like receptors (TLRs) 4The abbreviations used are:TLRToll-like receptorLPSlipopolysaccharideLrch4leucine-rich repeat and calponin homology containing protein 4LRRleucine-rich repeatMyD88myeloid differentiation primary response 88TMDtransmembrane domainTRIFToll/interleukin-1 receptor domain–containing adapter-inducing interferon-βIRFinterferon regulatory factorCHcalponin homologyAAamino acid(s)DAPI4′,6′-diamino-2-phenylindoleG-CSFgranulocyte–colony-stimulating factorILinterleukinHDAChistone deacetylaseTNFtumor necrosis factorqRT-PCRquantitative RT-PCR. are an evolutionarily conserved family of pattern recognition receptors that are thought to detect select pathogen- and damage-derived (i.e. host) molecules in part through leucine-rich repeat (LRR) motifs in the TLR ectodomain (1Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34 (21616434): 637-65010.1016/j.immuni.2011.05.006Abstract Full Text Full Text PDF PubMed Scopus (2603) Google Scholar). Ligation of TLRs, whether on the plasma membrane (TLR1, -2, -4, -5, and -6) or within endosomes (TLR3, -7, -8, and -9), triggers a complex series of signaling events through adaptor proteins and kinases, culminating in the activation of NF-κB and interferon regulatory factors (IRFs), master transcription factors that orchestrate the innate immune response via inducing pro-inflammatory cytokines and type I interferons. TLRs are pivotal for host defense but can also mediate inflammatory and autoimmune diseases (2Kawai T. Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors.Nat. Immunol. 2010; 11 (20404851): 373-38410.1038/ni.1863Crossref PubMed Scopus (6223) Google Scholar, 3Pasare C. Medzhitov R. Toll-like receptors and acquired immunity.Semin. Immunol. 2004; 16 (14751760): 23-2610.1016/j.smim.2003.10.006Crossref PubMed Scopus (168) Google Scholar); thus, an improved understanding of their molecular regulation is expected to enrich our insight into human disease pathogenesis and to reveal new therapeutic targets (4Hennessy E.J. Parker A.E. O'Neill L.A. Targeting Toll-like receptors: emerging therapeutics?.Nat. Rev. Drug Discov. 2010; 9 (20380038): 293-30710.1038/nrd3203Crossref PubMed Scopus (637) Google Scholar). Toll-like receptor lipopolysaccharide leucine-rich repeat and calponin homology containing protein 4 leucine-rich repeat myeloid differentiation primary response 88 transmembrane domain Toll/interleukin-1 receptor domain–containing adapter-inducing interferon-β interferon regulatory factor calponin homology amino acid(s) 4′,6′-diamino-2-phenylindole granulocyte–colony-stimulating factor interleukin histone deacetylase tumor necrosis factor quantitative RT-PCR. Recent literature has revealed a growing number of accessory proteins that play essential roles in supporting TLR function (5Lee C.C. Avalos A.M. Ploegh H.L. Accessory molecules for Toll-like receptors and their function.Nat. Rev. Immunol. 2012; 12 (22301850): 168-17910.1038/nri3151Crossref PubMed Scopus (313) Google Scholar). Thus, MD-2 assists TLR4 in binding of lipopolysaccharide (LPS) (6Park B.S. Song D.H. Kim H.M. Choi B.S. Lee H. Lee J.O. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex.Nature. 2009; 458 (19252480): 1191-119510.1038/nature07830Crossref PubMed Scopus (1606) Google Scholar), RP105 regulates TLR4 signaling in a cell type–dependent manner (7Blumenthal A. Kobayashi T. Pierini L.M. Banaei N. Ernst J.D. Miyake K. Ehrt S. RP105 facilitates macrophage activation by Mycobacterium tuberculosis lipoproteins.Cell Host Microbe. 2009; 5 (19154986): 35-4610.1016/j.chom.2008.12.002Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 8Divanovic S. Trompette A. Atabani S.F. Madan R. Golenbock D.T. Visintin A. Finberg R.W. Tarakhovsky A. Vogel S.N. Belkaid Y. Kurt-Jones E.A. Karp C.L. Negative regulation of Toll-like receptor 4 signaling by the Toll-like receptor homolog RP105.Nat. Immunol. 2005; 6 (15852007): 571-57810.1038/ni1198Crossref PubMed Scopus (317) Google Scholar), CD14 regulates ligand interactions for multiple TLRs (5Lee C.C. Avalos A.M. Ploegh H.L. Accessory molecules for Toll-like receptors and their function.Nat. Rev. Immunol. 2012; 12 (22301850): 168-17910.1038/nri3151Crossref PubMed Scopus (313) Google Scholar), and TRIL is thought to mediate ligand delivery to TLR3 and TLR4 (9Carpenter S. Carlson T. Dellacasagrande J. Garcia A. Gibbons S. Hertzog P. Lyons A. Lin L.L. Lynch M. Monie T. Murphy C. Seidl K.J. Wells C. Dunne A. O'Neill L.A. TRIL, a functional component of the TLR4 signaling complex, highly expressed in brain.J. Immunol. 2009; 183 (19710467): 3989-399510.4049/jimmunol.0901518Crossref PubMed Scopus (41) Google Scholar). However, additional regulators such as GRP94 and PRAT4A serve as chaperones for multiple TLRs via facilitating proper protein folding and maturation (10Yang Y. Liu B. Dai J. Srivastava P.K. Zammit D.J. Lefrançois L. Li Z. Heat shock protein gp96 is a master chaperone for toll-like receptors and is important in the innate function of macrophages.Immunity. 2007; 26 (17275357): 215-22610.1016/j.immuni.2006.12.005Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar, 11Takahashi K. Shibata T. Akashi-Takamura S. Kiyokawa T. Wakabayashi Y. Tanimura N. Kobayashi T. Matsumoto F. Fukui R. Kouro T. Nagai Y. Takatsu K. Saitoh S. Miyake K. A protein associated with Toll-like receptor (TLR) 4 (PRAT4A) is required for TLR-dependent immune responses.J. Exp. Med. 2007; 204 (17998391): 2963-297610.1084/jem.20071132Crossref PubMed Scopus (140) Google Scholar), whereas Unc93b1 interacts with multiple nucleic acid–sensing TLRs to mediate their delivery to endosomes (12Tabeta K. Hoebe K. Janssen E.M. Du X. Georgel P. Crozat K. Mudd S. Mann N. Sovath S. Goode J. Shamel L. Herskovits A.A. Portnoy D.A. Cooke M. Tarantino L.M. et al.The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9.Nat. Immunol. 2006; 7 (16415873, 10.1038/nrm1902, 10.1038/nrm1901): 156-16410.1038/ni1297Crossref PubMed Scopus (546) Google Scholar). Here, we introduce leucine-rich repeats and calponin homology containing protein 4 (Lrch4) as a novel accessory protein that regulates signaling by multiple TLRs. Lrch4 is predicted to be a single-pass transmembrane protein with approximately nine LRRs and a calponin homology (CH) motif in its ectodomain. It is widely expressed across murine tissues and is regulated by LPS. Lrch4 silencing attenuates cytokine induction by a wide array of TLR ligands in murine and human cells and also reduces inflammatory responses to LPS in vivo. Lrch4 co-precipitates with biotin-LPS from treated macrophages, suggesting interaction with LPS. Consistent with this, Lrch4 silencing reduces cell surface binding of LPS and alters the pattern of LPS deposition on the macrophage, reducing LPS localization to rafts. Taken together, we identify Lrch4 as a broad-spanning regulator of the innate immune response with potential as a therapeutic target in inflammatory disease. We recently identified Lrch4 in a proteomic screen as a protein increased in lipid raft microdomains of macrophages upon LPS exposure, suggestive of a potential role in TLR4 signaling (13Dhungana S. Merrick B.A. Tomer K.B. Fessler M.B. Quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated ERK activation in response to lipopolysaccharide.Mol. Cell. Proteomics. 2009; 8 (18815123): 201-21310.1074/mcp.M800286-MCP200Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Lrch4 (Gene ID: 231798) resides on chromosome 5 in the murine genome (7q22 in the human genome) and is predicted to have a 3,078-bp ORF (18 exons) that encodes a 680-amino acid (AA) protein (∼73 kDa) with an pI of ∼7.5. Sequence alignment using CLUSTAL (14Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. Thompson J.D. Gibson T.J. Higgins D.G. Clustal W and Clustal X version 2.0.Bioinformatics. 2007; 23 (17846036): 2947-294810.1093/bioinformatics/btm404Crossref PubMed Scopus (22593) Google Scholar) indicates a high degree of homology between murine Lrch4 and human LRCH4 (85.7%) and moderate homology between the human and zebrafish homologues (Fig. S1), suggesting significant evolutionary conservation. AA sequence-based prediction of conserved motifs (UniProt) in conjunction with transmembrane prediction (TMpred (15Hofmann K. TMBASE: a database of membrane spanning protein segments.Biol. Chem. Hoppe-Seyler. 1993; 374: 166Google Scholar) and Philius (16Reynolds S.M. Käll L. Riffle M.E. Bilmes J.A. Noble W.S. Transmembrane topology and signal peptide prediction using dynamic bayesian networks.PLoS Comput. Biol. 2008; 4 (18989393): e100021310.1371/journal.pcbi.1000213Crossref PubMed Scopus (183) Google Scholar)) indicates that Lrch4 has an ectodomain composed of a 19 AA N-terminal signal peptide followed by nine LRRs (each 21–23 AA in length), a central disordered region, and a CH motif; this is then followed by a transmembrane domain (TMD) and a short cytoplasmic tail (Fig. 1A). Recent revisions of the NCBI and ENSEMBL database suggest that Lrch4 has splice variants. The transcript support level is a method used by ENSEMBL to rate well-supported versus poorly supported transcript models. Three Lrch4 transcripts are identified at transcript support level 1 (i.e. all splice junctions of the transcript are supported by at least one nonsuspect mRNA). However, one of these (Lrch4-004) has incomplete 5′ and 3′ coding DNA sequences. We therefore focused on Lrch4-001 (referred to herein as variant 1) and Lrch4-002 (referred to as variant 2). Lrch4 variant 1 (680 AAs) is as described above, whereas variant 2 (649 AAs) is truncated C-terminal to the CH and omits the TMD, consistent with a soluble protein. Physiological expression of the predicted variants has not been verified to our knowledge. LRRs are 20–30-AA motifs that exist in thousands of proteins across phylogeny, typically appearing as tandem repeats that together form a solenoid-shaped domain thought to mediate protein–ligand and protein– binding (17Dolan J. Walshe K. Alsbury S. Hokamp K. O'Keeffe S. Okafuji T. Miller S.F. Tear G. Mitchell K.J. The extracellular leucine-rich repeat superfamily; a comparative survey and analysis of evolutionary relationships and expression patterns.BMC Genomics. 2007; 8 (17868438): 32010.1186/1471-2164-8-320Crossref PubMed Scopus (130) Google Scholar). Individual LRRs have been grouped based on sequence into seven categorical types, although the functional correlates of these categories remain unclear (18Ng A.C. Eisenberg J.M. Heath R.J. Huett A. Robinson C.M. Nau G.J. Xavier R.J. Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 4631-4638Crossref PubMed Scopus (146) Google Scholar). Alignment of LRR domains between murine Lrch4 and human LRCH4 suggests a very high degree of homology (Table S1), with manual inspection indicating that the majority of murine Lrch4 LRRs fall into the “plant-specific” LRR category, as was recently reported for human Lrch4 in a genome-wide survey of LRR proteins (18Ng A.C. Eisenberg J.M. Heath R.J. Huett A. Robinson C.M. Nau G.J. Xavier R.J. Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 4631-4638Crossref PubMed Scopus (146) Google Scholar). Using the threading algorithm pDomTHREADER (19Lobley A. Sadowski M.I. Jones D.T. pGenTHREADER and pDomTHREADER: new methods for improved protein fold recognition and superfamily discrimination.Bioinformatics. 2009; 25 (19429599): 1761-176710.1093/bioinformatics/btp302Crossref PubMed Scopus (223) Google Scholar), the best structural match in the protein database to the LRR domain of Lrch4 is a protein derived from the variable lymphocyte receptor of the jawless vertebrate, the hagfish. The LRR region matches well to known LRR structures, whereas there is less confidence that the N and C termini are modeled accurately. As shown in Fig. 1B, predictive modeling of Lrch4 (green) based on the hagfish protein (cyan) yields the horseshoe-shaped structure typical of LRRs. CH domains play a role in actin binding, although they may mediate additional interactions, especially when found singly rather than in tandem (20Sjöblom B. Ylänne J. Djinovic-Carugo K. Novel structural insights into F-actin-binding and novel functions of calponin homology domains.Curr. Opin. Struct. Biol. 2008; 18 (18952167): 702-70810.1016/j.sbi.2008.10.003Crossref PubMed Scopus (58) Google Scholar). A recent phylogenetic analysis indicated that the convergence of LRRs and CH domains in proteins (i.e. LRCH proteins) occurs rarely and only in animals, with just one protein (LRCH) in Drosophila melanogaster and four (Lrch1–4) in both Mus musculus and Homo sapiens (21Foussard H. Ferrer P. Valenti P. Polesello C. Carreno S. Payre F. LRCH proteins: a novel family of cytoskeletal regulators.PLoS One. 2010; 5 (20805893): e1225710.1371/journal.pone.0012257Crossref PubMed Scopus (15) Google Scholar). Interestingly, despite the very high degree of homology between murine Lrch4 and human LRCH4, sequence alignment reveals modest homology between murine Lrch4 and Lrch1 (35%), Lrch4 and Lrch2 (34%), and Lrch4 and Lrch3 (43%) (data not shown), suggesting divergent function among Lrch proteins. Expression profiling of transcripts for the four Lrch family members indicates that Lrch4 (detected using primers common to the two variants) is widely expressed across 14 murine tissues, with the most abundant expression in spleen, testes, thymus, intestine, and blood (Fig. 1C). As shown in Fig. 1D, immunoblotting of murine macrophage subcellular fractions for endogenous Lrch4 (using an antibody targeting a region common to variants 1 and 2) confirms that it is predominantly present in the membrane, with lesser expression in the cytoplasm and soluble nuclear fraction, and is detected as a single band in all fractions. Consistent results showing a single band in both RAW 264.7 and primary murine macrophage lysates were obtained with both a commercial anti-Lrch4 antibody and an anti-Lrch4 antibody raised by our laboratory. Given that only variant 1 has a predicted TMD, this finding suggests that, at the protein level, variant 1 is expressed much more highly than variant 2 within the cell. Microscopy of GFP-Lrch4 in HEK293 cells reveals staining that includes a cytoplasmic-type pattern, but formal analysis indicates a high degree of overlap with the plasma membrane stain CellMaskTM (Life Technologies) (22Gokhale N.A. Zaremba A. Janoshazi A.K. Weaver J.D. Shears S.B. PPIP5K1 modulates ligand competition between diphosphoinositol polyphosphates and PtdIns(3,4,5)P3 for polyphosphoinositide-binding domains.Biochem. J. 2013; 453 (23682967): 413-42610.1042/BJ20121528Crossref PubMed Scopus (48) Google Scholar), in particular in cells with low-medium forced expression, consistent with substantial localization to the plasma membrane (Fig. 1E). By contrast, the tGFP vector (control) displays a very different distribution, largely overlapping the nuclear DAPI stain (Fig. 1E). Based on its increase in rafts in response to LPS (13Dhungana S. Merrick B.A. Tomer K.B. Fessler M.B. Quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated ERK activation in response to lipopolysaccharide.Mol. Cell. Proteomics. 2009; 8 (18815123): 201-21310.1074/mcp.M800286-MCP200Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), its predicted receptor-like features, and its LRRs—a ligand-binding motif common to the ectodomain of all TLRs, as well as some TLR accessory proteins (CD14, RP105) (18Ng A.C. Eisenberg J.M. Heath R.J. Huett A. Robinson C.M. Nau G.J. Xavier R.J. Human leucine-rich repeat proteins: a genome-wide bioinformatic categorization and functional analysis in innate immunity.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 4631-4638Crossref PubMed Scopus (146) Google Scholar)—we hypothesized that Lrch4 regulates TLR4 activation by LPS. To address this, we generated two stable Lrch4 lentiviral shRNA knockdown RAW 264.7 macrophage lines in parallel with a scrambled (Scr) lentiviral shRNA control line. As shown in Fig. 2A, compared with the Scr line, both knockdown lines achieved ≥50% silencing of Lrch4 mRNA (using probes common to both variants) and protein. Although this degree of knockdown is somewhat modest, it is consistent with past reports using RNAi in macrophages and likely reflects the relatively refractory nature of macrophages to transfection/transduction (23Alper S. Laws R. Lackford B. Boyd W.A. Dunlap P. Freedman J.H. Schwartz D.A. Identification of innate immunity genes and pathways using a comparative genomics approach.Proc. Natl. Acad. Sci. U.S.A. 2008; 105 (18463287): 7016-702110.1073/pnas.0802405105Crossref PubMed Scopus (60) Google Scholar). Using variant-specific primers, we confirmed that both Lrch4 variants are expressed in the macrophage and that both are knocked down by Lrch4 shRNA (Fig. 2B). Of interest, LPS itself induced down-regulation of mRNA for Lrch4 variants 1 and 2 (Fig. 2, A and B). Confirming a role for Lrch4 in regulation of TLR4 signaling, both Lrch4 knockdown lines induced significantly less TNFα than the Scr line in response to two doses of LPS (Fig. 2C). A similar reduction in LPS-induced TNFα was observed in two Lrch4 disruption clones produced by CRISPR-Cas9 (Fig. 2D). Given that some accessory proteins are reported to regulate signaling by multiple TLRs (e.g. CD14, TRIL (5Lee C.C. Avalos A.M. Ploegh H.L. Accessory molecules for Toll-like receptors and their function.Nat. Rev. Immunol. 2012; 12 (22301850): 168-17910.1038/nri3151Crossref PubMed Scopus (313) Google Scholar)), we next tested a role for Lrch4 in additional TLR cascades. Lrch4 knockdown attenuated TNFα induction by the TLR1/2 ligand Pam3CSK4 (Fig. 2E), as well as by Pam2CSK4 (TLR2/6), imiquimod (TLR7), and ODN2395 (TLR9) (Fig. S2), indicating a broad-spanning role for Lrch4 in regulating plasmalemmal and endosomal TLRs. Induction of granulocyte–colony-stimulating factor (G-CSF) by both LPS and Pam3CSK4 was also reduced in Lrch4 knockdown cells, indicating that Lrch4 regulates multiple cytokines downstream of TLR2 and TLR4 (Fig. 2E). To test Lrch4 in human cells, we next silenced endogenous Lrch4 in HEK293 cells using an siRNA approach (Fig. 2F). HEK293 cells stably expressing either TLR4/MD-2/CD14 or TLR2 were transfected with Lrch4 siRNA or Scr siRNA and then exposed to LPS or Pam3CSK4, respectively. In preliminary experiments, we found that, in response to these stimuli, these cells produced very little TNFα (data not shown); thus, we surveyed for IL-8 protein production instead, as reported by others (24Trompette A. Divanovic S. Visintin A. Blanchard C. Hegde R.S. Madan R. Thorne P.S. Wills-Karp M. Gioannini T.L. Weiss J.P. Karp C.L. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein.Nature. 2009; 457 (19060881): 585-58810.1038/nature07548Crossref PubMed Scopus (593) Google Scholar). As shown in Fig. 2G, Lrch4 siRNA attenuated IL-8 induction by both ligands in HEK293 cells, providing further support that the shRNA results in macrophages are unlikely to reflect off-target effects. Confirming some selectivity for Lrch4 function, Lrch4 silencing did not affect induction of IL-8 by HEK293 cells in response to stimulation with TNFα (Fig. 2H). We next focused on Lrch4 function in the TLR4 pathway. Moving further “upstream” of cytokine protein expression, we confirmed that Lrch4 knockdown also attenuates transcript expression of TNFα and G-CSF in response to LPS (Fig. 3, A and B). This suggested that Lrch4 may regulate LPS induction of pro-inflammatory cytokines at or upstream of transcription. The TLR4 cascade bifurcates immediately downstream of the receptor into two signaling arms defined by alternate usage of the cytoplasmic adaptors, myeloid differentiation primary response 88 (MyD88), and Toll/interleukin-1 receptor domain–containing adapter-inducing interferon-β (TRIF) (1Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34 (21616434): 637-65010.1016/j.immuni.2011.05.006Abstract Full Text Full Text PDF PubMed Scopus (2603) Google Scholar). These so-called “MyD88-dependent” and “MyD88-independent” pathways lead to downstream induction of distinct cytokines, although it is thought that most cytokines likely have some input from both adaptor pathways (25Björkbacka H. Fitzgerald K.A. Huet F. Li X. Gregory J.A. Lee M.A. Ordija C.M. Dowley N.E. Golenbock D.T. Freeman M.W. The induction of macrophage gene expression by LPS predominantly utilizes Myd88-independent signaling cascades.Physiol. Genomics. 2004; 19 (15367722): 319-33010.1152/physiolgenomics.00128.2004Crossref PubMed Scopus (245) Google Scholar). Expression profiling of MyD88-dependent (Fig. 3C) and -independent (Fig. 3D) cytokines in LPS-stimulated macrophages after Lrch4 silencing indicated a role for Lrch4 in output from both adaptor arms, consistent with it regulating the proximal TLR4 pathway. However, differences in Lrch4 dependence were noted across cytokines, with some cytokines from both adaptor pathways showing dramatic reduction in Lrch4-silenced cells (e.g. IL-10, MCP-1), whereas other cytokines showed a more modest dependence on Lrch4 (e.g. IL-1β, IP-10). Taken together, these findings suggest that Lrch4 acts upstream of the MyD88/TRIF bifurcation in the TLR4 cascade. Moving further upstream in the TLR4 cascade to confirm more directly whether Lrch4 regulates early signaling responses to LPS, we next evaluated activation of the transcription factors NF-κB and IRF3. NF-κB is activated by both an early MyD88-dependent and late MyD88-independent (TRIF-dependent) pathway after LPS, whereas IRF3 activation is generally thought to be dependent upon the TRIF pathway (26O'Neill L.A. Bowie A.G. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling.Nat. Rev. Immunol. 2007; 7 (17457343): 353-36410.1038/nri2079Crossref PubMed Scopus (2011) Google Scholar). As shown in Fig. 4A, activation of NF-κB in macrophage nuclear isolates was attenuated in Lrch4-silenced cells at both 15 and 30 min after LPS. LPS-induced NF-κB luciferase was also reduced in Lrch4-silenced RAW 264.7 macrophages (Fig. 4B). Finally, PO4-IRF3 was also reduced in the nuclear fraction of Lrch4-silenced macrophages (Fig. 4C), indicating that Lrch4 is required for full LPS-induced activation of IRF3. Similar results for nuclear NF-κB activation, NF-κB luciferase, and PO4-IRF3 were obtained using cells with CRISPR-Cas9–mediated disruption of Lrch4 (Fig. 4, D–F). In addition to NF-κB and IRF3, LPS is well-known to lead to the rapid activation of MAPKs that, in turn, regulate multiple downstream functions including gene expression. Activation of these kinases is thought to be regulated by both MyD88 and TRIF (26O'Neill L.A. Bowie A.G. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling.Nat. Rev. Immunol. 2007; 7 (17457343): 353-36410.1038/nri2079Crossref PubMed Scopus (2011) Google Scholar). As shown in Fig. 4G, Lrch4-silenced macrophages displayed a marked reduction in LPS-induced p38 activation (phosphorylation). By contrast, JNK phosphorylation was reduced at 15 min but not at 30 min post-LPS, consistent with a delay in its activation by LPS in Lrch4-silenced cells. Taken together, these results indicate that Lrch4 regulates early signaling events in the proximal TLR4 pathway, with effects upon both MyD88 and TRIF arms, but with varying temporal effects on the MAPKs. Consistent with Lrch4 not exerting a global or indiscriminate effect upon TLR4 pathway output, we confirmed that neither silencing nor overexpression of Lrch4 altered cell surface display of TLR4 as assessed by flow cytometry (Fig. 5A and Fig. S3, A and B); nor did Lrch4 silencing or overexpression modify TLR4 gene expression (Fig. 5B). Similarly, Lrch4 knockdown had no effect on expression of the IL-6 receptor (Fig. S3C). Aiming to test whether Lrch4 could nonetheless impact ligand capture in the TLR4 pathway, we assessed cell-surface binding of LPS. Lrch4-silenced cells exhibited a significant, albeit modest reduction in overall surface binding of LPS (Fig. 5C). More remarkably, Lrch4-silenced cells showed reduced co-localization of LPS with the specific lipid raft marker cholera toxin subunit B (CtB; a ligand for the raft ganglioside GM1) (Fig. 5, D and E) (27Yvan-Charvet L. Welch C. Pagler T.A. Ranalletta M. Lamkanfi M. Han S. Ishibashi M. Li R. Wang N. Tall A.R. Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions.Circulation. 2008; 118 (18852364): 1837-184710.1161/CIRCULATIONAHA.108.793869Crossref PubMed Scopus (342) Google Scholar, 28Gale S.C. Gao L. Mikacenic C. Coyle S.M. Rafaels N. Murray Dudenkov T. Madenspacher J.H. Draper D.W. Ge W. Aloor J.J. Azzam K.M. Lai L. Blackshear P.J. Calvano S.E. Barnes K.C. et al.APOϵ4 is associated with enhanced in vivo innate immune responses in human subjects.J. Allergy Clin. Immunol. 2014; 134 (24655576): 127-13410.1016/j.jaci.2014.01.032Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), in conjunction with a much more punctate surface deposition of LPS as quantified by number of LPS foci per cell (Fig. 5F). Together, this suggests that Lrch4 is not just required for quantitative surface capture of LPS, but more specifically for successful delivery of LPS to lipid rafts, the site where CD14 and the TLR4 receptor cluster and are thought to interact (29Triantafilou M. Miyake K. Golenbock D.T. Triantafilou K. Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation.J. Cell Sci. 2002; 115 (12045230): 2603-2611Crossref PubMed Google Scholar). Testing more directly for a role of Lrch4 in LPS binding, we next performed a streptavidin bead pulldown after exposure of macrophages to biotin-LPS. As shown in Fig. 5G, Lrch4 was detected in the biotin-LPS pulldown (but not in cells treated with nonbiotinylated LPS, as expected), suggesting that it interacts either directly or indirectly with LPS. Lrch4 capture by biotin-LPS was effectively competed by nonbiotinylated LPS, consistent with a bona fide LPS interaction, whereas CD14 pulldown was not notably competed. This finding suggests either differing stoichiometry or avidity to" @default.
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