Promotion Of TLR-triggered Innate Immune Response By CLM3, CLM5 In Macrophages And The Underlying Mechanisms

CLM3 promotes TLR9-triggered production of proinflammatory cytokines in macrophages and the underlying mechanismsInnate immune system is the first line of host defense against pathogen infection. TLRs have emerged as the key sensors of microbial products, as they are expressed on sentinel cells in the immune system, most notably professional antigen-presenting cells (APC) including dendritic cells (DC) and macrophages, where they sense a range of chemicals produced by bacteria, viruses, fungi and protozoa. TLRs can be grouped into families according to the types of ligands they recognize. Lipid-based structures are recognized by TLR2 (in combination with TLR1 or TLR6 as heterodimers) and TLR4 (as a homodimer): the most studied examples of lipid-based recognition are bacterial or mycobacterial lipopeptides, or glycerophosphatidylinositol anchors from parasites, both of which are recognized by TLR2, and bacterial lipopolysaccharide (LPS), which is recognized by TLR4. Viral and/or bacterial nucleic acids are recognized by TLR3, TLR7, TLR8 and TLR9; the most characterized are the recognition of double stranded RNA (dsRNA) by TLR3 and recognition of CpG motifs in DNA by TLR9. Finally, TLR5 and TLR11 recognize proteins from pathogens (flagellin in the case of TLR5 and profilin in the case of TLR11. The events initiated inside cells when a given TLR is activated have been the subject of much investigation. Many biochemical details of these events have now been elucidated, including the description in the past years of novel adaptor proteins, protein kinases and transcription factors. However, the molecular mechanisms for the initiation and regulation of TLR response still remain to be fully understood now.In the TLR signaling, recognition of PAMPs by TLRs stimulates the recruitment of a set of intracellular TIR-domain- containing adaptors, including MyD88, TIRAP (also known as MAL), Trif (also known as TICAM1) and TRAM (also known as TICAM2) via TIR–TIR interactions. MyD88 is a universal adapter that activates inflammatory pathways, which is shared by all TLRs with the exception of TLR3. Recruitment of MyD88 leads to the activation of MAP kinases (MAPKs) (ERK, JNK, p38) and the transcription factor NF-κB to control the expression of inflammatory cytokine genes. TRIF is recruited to TLR3 and TLR4, and activates an alternative pathway (TRIF-dependent pathway) that culminates in the activation of NF-κB, MAPKs and the transcription factor IRF3. Activation of IRF3 is pivotal for induction of type I interferon (IFN), particularly IFN-β. The capacity not only to respond appropriately but also to self-regulate host responses to invading pathogens is critical to host immune system to mount an appropriate primary immune response against pathogens but avoid the pathogenesis of immune disorders. The continued focus of research on TLR signalling has provided a lot more information on pathways activated by TLRs, and new layers of complexity and regulation have been revealed, which present better understanding how TLR signaling is regulated and how to control TLR-mediated inflammatory diseases.Human CMRF35-A, previously isolated by recognition with the CMRF-35 monoclonal antibody, is a surface molecule with an IgV-like domain. It comprises a single extracellular Ig variable domain, a transmembrane domain and a cytoplasmic tail, located in chromosome 17. Anti-CMRF35 monoclonal antibody has been shown to be able to recognize other molecules which are homology of CMRF35, which located in chromosome 17 as cluster named CD300 family, and the mouse homology named as CMRF35-like molecules (CLM) family. The CLM family consists of nine members, which are encoded on mouse chromosome 11. CLM-1 to CLM-8 reside in close proximity in the ~250kb region of 11E2, whereas CLM-9 is separated from the other family members to be located in 11D. CLM family members have been proposed to play roles in the immune response, inflammatory and allergy. Firstly, CLM family members expressed on the surface of myeloid lineage cells. Secondly, the cross-linking with the active receptors such as CLM5,CLM7,CLM8 can promote neutrophilic leukocyte, mast cell, eosinophil, basophil degranulation. The cross-linking with inhibitory receptors such as CLM1 or CLM8 can inhibit inflammatory response or immune response. Thirdly, expression levels of CLM1/5, CLM4/8 are changed after stimulation with inflammatory factors. The investigations on the function and mechanism of CLM/CD300 family members will facilitate the development of new strategies to control inflammatory diseases, and also will contribute to the better understanding of immune recognition and immune regulation.CLM3 is a new member of CLM family, which has not been well investigated up to now. In this study, we cloned CLM3 full-length gene(accession No AY457049) from mouse abdominal cavity macrophages, which contains 738bp base, encoding 245 amino acids with a predicted molecular mass of 27 kD. The amino acid sequence begins with a hydrophobic signal peptide of 17 amino acids followed by an extracellular region composed of one IgV domains and a 22 amino acids transmember domain. CLM3 is located on mouse chromosome 11. CLM-1 to CLM-8 reside in close proximity in the region of 11E2. Comparative analysis indicated that CLM3 is closely related to some other CLM families, including CLM5 and CLM1 (85% identity with CLM3).Then we detected the expression patterns of CLM3 by RT-PCR. CLM3 mRNA was widely distributed in mouse tissues, including heart, spleen, liver, lymph nodes and testis, among which CLM3 is highest expressed in lymph nodes. RT-PCR analysis of cell lines and freshly isolated cells demonstrated that CLM3 mRNA was preferentially expressed in monocytes, and RAW264.7 macrophage cell lines.To explore the role of CLM3 in TLRs-triggered innate response in macrophages, we firstly examined whether the expression of CLM3 was changed in Raw264.7 cells after stimulated with TLR ligands. By using RT-PCR, we found that the mRNA expression of CLM3 was not changed after stimulation with different TLRs ligands during 24 hours.To identify the function of CLM3, we investigated the role of CLM3 in TLR-induced inflammatory response in macrophages. We found that CLM3 could promote CpG ODN-induced TNFαand IL6 production in macrophages. Mouse peritoneal macrophages were transfected with CLM3 siRNA. After 48h, the CLM3-silenced cells were treated with 0.3μM CpG ODN for 6 hrs. Then the concentrations of TNF-αand IL-6 in the supernatants were measured by ELISA. The productions of TNF-αand IL-6 were found to be decreased after silence of CLM3; Raw264.7 cells were transfected with CLM3 full-length cDNA. Then we found that overexpression of CLM3 promoted the production of TNF-αand IL-6 in macrophages in response to CpG ODN stimulation. These results indicate that CLM3 can promote TLR9-triggered proinfllamatory response in macrophages .To identify the mechanism of CLM3 involved in TLRs signaling, we cotransfected HEK293 with MyD88, IRAK1, TRAF6, TAK1, CLM3 plasmids and TNF-αluciferase reporter plasmid. After 24hrs of culture, luciferase activity was measured. Overexpression of CLM3 enhanced MyD88, IRAK1, TRAF6-induced but not TRIF, TAK1-induced TNF-αactivation, indicating that CLM3 promotes TLRs signaling through MyD88 pathway. Co-immunoprecipitation assays by cotransfection of HA-tagged MyD88, flag-tagged CLM3 plasmid in HEK293 cells, anti-flag antibody and anti-HA antibody proved that CLM3 could associate with MyD88, suggesting that CLM3 may influence TLR9 signalling via targeting IRAK1 and TRAF6 or TRAF6 and TAK1 complex formation.To analyze whether CLM3 could affect the activation of MAPK and NF-κB, CLM3 was transfected into RAW264.7 cells. After 24hrs of culture, the cells were treated with 0.3μM CpG ODN. The results showed that JNK and ERK phosphorylation and nuclear translocation of p65 subunit of NF-κB increased, compared with the negative control, indicating that CLM3 can enhance the TLR9-induced production of cytokines by activating MAPK and NF-κB pathway.In conclusion, we cloned CLM3 which belongs to CLM family. CLM3 is selectively expressed in lymph nodes and macrophages. CLM3 can promote TLR9-triggered production of inflammatory cytokines in macrophages. Co-immunoprecipitation experiments proved that CLM3 could promote TLRs signaling through association with MyD88 and enhance TLR9-induced activation of MAPK and NF-κB. We for the first time have provided the evidence that CLM3 promotes TLR9-triggered production of proinflammatory cytokines in macrophages though association with MyD88 and activation of MAPK, NF-κB pathways. Our findings provide new target and strategy to control inflammatory diseases. production of type I interferon and inflammatory cytokines in macrophages and the underlying mechanismsIn the immune response against viral invasion, RLHs (RIG-I-like Helicase) and TLRs are two major family of receptors which recognize RNA virus infection. To date, 11 human TLRs and 13 mouse TLRs have been identified. It has been shown that TLRs are activated by specific PAMPs and that the ability of specific TLRs to heterodimerize adds further to the diverse range of pathogens that may be recognized. For example, TLR1, -2, -4, -6, and -10 recognize lipids. TLR3, -7, -8, and -9, are endosomally localized, membrane-bound receptors which recognize virus RNA or DNA, so TLR3, -7, -8, -9 are named as anti-virus TLRs. After recognition of PAMPs, a cascade of intracellular signaling events is activated, which culminates in the induction of type I interferon (IFN) and proinflammatory cytokines such as tumor necrosis factor–α(TNF-α), interleukin (IL)-6, IL-1β.RIGI-like receptors (RLR), which are cytoplasmic RNA helicases, including retinoic acid-inducible gene-I (RIG-I), Mda5, and LGP2,Unlike membrane-bound TLRs, RLH reside in the cytoplasm and recognize RNA species produced in the cytoplasm. Whereas RIG-I and Mda5 contain a DExD/H box.RNA helicase domain and two caspase recruiting domain (CARD)-like domains required for eliciting downstream signaling pathways, but LGP2 lacks the CARD-like domains. RIG-I and Mda5 are able to interact with viral RNA, albeit with different ligand specificity, and elicit signaling leading to the transcription of type I IFN and inflammatory cytokines. RIG-I is essential for the recognition of a series of ssRNA viruses, which include paramyxoviruses, influenza virus, VSV, and Japanese encephalitis virus (JEV). Mda5 is required for the recognition of other RNA viruses, including picornaviruses such as EMCV, Mengo virus, and Theiler's virus. Furthermore, Mda5, but not RIG-I, participates in the recognition of polyI:C. LGP2 negatively or positively regulates RIG-I and Mda5 responses, depending on types of RNA viruses. Although the important roles of RLH and CLM5 promotes TLR- and RLH-triggered TLR in the recognition of PAMP and initiation of immune response against pathogens have been extensively studied, the molecular mechanisms by which RLH and TLR effectively activate innate immune response and how the process can be controlled at the proper level remain to be fully understood, so as to avoid the immunological disorders happened once the response is over-activated or out of control. New regulators for the RLH and TLR-triggered innate immune response attract much attention in recent years.CLM family member CLM5 is also designated as CD300d,DIgR1,MAIR-II,LMIR4. CLM-5 has a short cytoplasmic tail without any known motif sequences. In addition, the transmembrane domain of CLM5 possesses a negatively charged glutamic acid. These characteristics suggest that CLM5 may be an activating receptor interacting with a signaling motif-bearing adaptor molecule. It has been shown that CLM5 is expressed in the myeloid lineage cells such as dendritic cells (DC), macrophages, neutrophilic leukocytes but not in lymphocytes. When FLAG-CLM-5 on CLM-5/RAW264.7 cells was cross-linked with anti-FLAG mAb followed by the F(ab9)2 fragment of goat anti-mouse IgG, tyrosine phosphorylation of p38, ERK1/2 and JNK was significantly enhanced, and CLM-5 cross-linking also induced a fibroblastic morphological change in CLM-5/RAW264.7 cells similar to that observed when RAW264.7 cells were stimulated with LPS. Other FcRc-associating molecules, PIR-A, and osteoclast-associated receptor also enhance osteoclast formation upon cross-linking with antibodies. So, CLM-5 has been proposed to be involved in the differentiation of osteoclasts. However, up to now, there is no report about the role of CLM5 in the RLH and TLR-triggered innate immune response against virus infection and the underlying mechanisms.We cloned CLM5 full-length gene (accession No AY457051) from mouse primary abdominal macrophages, which contains 666bp base, encoding 222 amino acids with a predicted molecular mass of 25 kD. The amino acid sequence begins with a hydrophobic signal peptide of 17 amino acids followed by an extracellular region composed of one IgV domains and a 23 amino acids transmember domain. CLM5 is located on mouse chromosome 11, in which CLM-1 to CLM-8 reside in close proximity in the region of 11E2. CLM-5 has a short cytoplasmic tail without any known motif sequences indicating its active function. Comparative analysis indicated that CLM5 is closely related to some other CLM families, including CLM1 and CLM3 (91% identity with CLM1; 85% identity with CLM3).Then we detected the expression profile of CLM5 by RT-PCR. CLM5 mRNA was widely distributed in mouse tissues, including heart, spleen, liver, lymph nodes, brain and testis, with highest level in spleen, liver and lymph nodes. RT-PCR analysis of cell lines and freshly isolated cells demonstrated that CLM5 mRNA was preferentially expressed in monocytes s, dendritic cells and RAW264.7 macrophage cell line. The selective expression of CLM5 in antigen-presenting cells (APC) indicated that CLM5 may be involved in the innate immune response against pathogens.To identify the function of CLM5, we investigated whether CLM5 could affect RLH and TLR-triggered type I IFN and proinflammatory cytokine production in macrophages. We found that CLM5 can promote TLRs ligand-induced IFN-βand IL-6 production in macrophages. After mouse peritoneal macrophages were transfected with CLM5 siRNA for 48h, the CLM5-silenced macrophages were treated with LPS(0.1μg/ml), polyI:C (10μg/ml), CpG ODN (0.3μM/L) for 6 hrs. Concentrations of IFN-βand IL-6 in the supernatants were measured by ELISA. The concentrations of IFN-βand IL-6 were found to be decreased after kncokdown of CLM5 in macrophages. These results indicate that CLM5 promotes TLR-triggered type I IFN and proinflammatory cytokine production in macrophages.As the same, The concentrations of IFN-βwas decreased after knockdown of CLM5 when infect with VSV for 24 hour,and TCID50 also increased.This indicate that CLM5 also participate in RLH-triggered signaling.To explore the underlying mechanism of CLM5 in the promotion of type I IFN and proinflammatory cytokine production, we cotransfected HEK293 with TRIF (or MDA5, RIG-I), pCMV-CLM5 plasmid and IFN-βluciferase reporter plasmid, and then the luciferase activity was measured after 24hrs. Overexpression of CLM5 enhanced TRIF, MDA5, RIG-I-induced IFN-βactivation, indicating that CLM5 positively participates in TLRs and RLH signaling. Co-immunoprecipitation experiments improved that CLM5 can associate with FcRγ,indicate the possible role of FcRγin CLM5 regulate RLH and TLR signaling.In conclusion, we cloned CLM5 which belongs to CLM family. CLM5 was selectively expressed in macrophages and dendritic cells. Knockdown of CLM5 in macrophages inhibited the production of IFN-βand IL6 induced by the stimulation with TLRs ligands,and the production of IFN-βwas also decreased after VSV infection. Furthermore, CLM5 enhanced TRIF, MDA5, RIG-I-induced IFNβactivation. Taken together, these results suggest that CLM5 may be a positive regulator of TLRs and RLH-triggered type I IFN and proinflammatory cytokine production in macrophages.