Expression of SALM5 protein in mouse tissues. Fig. that SALM5 suppressed lipopolysaccharide-induced inflammatory responses in the CNS and that a SALM-specific monoclonal antibody promoted inflammation in the CNS, and thereby aggravated clinical symptoms of mouse experimental autoimmune encephalomyelitis. In addition, we identified herpes virus entry mediator as a functional receptor that mediates SALM5s Rabbit Polyclonal to PKA-R2beta suppressive function. Our findings reveal a molecular link between the neuronal system and the immune system, Piboserod and provide potential therapeutic targets for the control of CNS diseases. gene family (Fig. 1B). SALMs, also known as LRFNs (leucine-rich and fibronectin III domainCcontaining proteins), are a group of newly characterized adhesion molecules predominantly expressed in the CNS. The five members of the SALM family are type I transmembrane proteins, with a typical extracellular structure composed Piboserod of leucine-rich repeats (LRRs), an Ig-like domain name, and a fibronectin type III (FN) domain name. Members of the SALM family are known for their involvement in neurite outgrowth and synapse formation (as a gene specifically expressed in the CNS.(A) Strategy used to identify molecules with Ig-like domains Piboserod that are enriched in immune-privileged organs. (B) Expression profile of the SALM family in different human organs or cell types. (C) Staining of lymphocytes (as indicated from normal mice) for SALM5 fusion protein binding by flow cytometry. (D) SALM5 mRNA expression in different mouse tissues determined by reverse transcription polymerase chain reaction (RT-PCR). (E) SALM5 mAb (clone 7A10) staining of human embryonic kidney 293T (HEK293T) cells transfected with mouse SALM5 (mSALM5) full-length (right panel) or control (left panel) plasmid. (F) Expression of SALM5 in normal tissues. Paraffin-embedded na?ve mouse tissues (as indicated) were stained using a biotin-labeled SALM5 mAb. Ultimately, we selected SALM5 for further study because recombinant SALM5-Ig fusion protein Piboserod showed clear staining with several types of immune cells, including CD4+ T cells, CD8+ T cells, and B cells (Fig. 1C). This result implied the presence of a putative counter-receptor for SALM5 on these cells, and the SALM5-mediated conversation might be involved in regulating immune responses within immune-privileged tissues. As shown in Fig. 1D, SALM5 mRNA was only detected in the brain, but not in other organs, including the heart, spleen, lung, liver, and skeletal muscle. We then generated a SALM5 mAb (clone 7A10) by immunization of a hamster, and exhibited that this mAb is highly specific to both mouse and human SALM5 (Fig. 1E). Immunohistochemical analysis of mouse tissues with 7A10 exhibited that SALM5 protein is constitutively expressed in the brain and spinal cord, but not in the spleen (Fig. 1F); the staining pattern was similar to two commercially available SALM5 antibodies (fig. S2). In addition, Western blot analysis of mouse tissues further exhibited that SALM5 is usually restrictively expressed in the brain (fig. S3). Our results thus indicate that SALM5 is usually constitutively and selectively expressed in the CNS. SALM5 inhibits microglia/macrophage-mediated neuroinflammation To determine whether SALM5 is indeed involved in CNS inflammation, we administered lipopolysaccharide (LPS) systemically, which induces CNS inflammation by activating microglial cells ( 0.05 (unpaired Students test). HVEM is the counter-receptor for SALM5 To identify the counter-receptor for SALM5, we screened a receptor-ligand proteome with a human SALM5-Ig fusion protein (= 7). Clinical scores of EAE were measured daily. Representative results from three impartial experiments are shown. * 0.05 (unpaired Students test). (B) Pathology of spinal cord sections from mice on day 19 after EAE induction. Inflammatory infiltrates in spinal cords were revealed using H&E staining. The infiltrates were further visualized by staining with mAb against CD3 for T cells or with mAb against MAC3 for macrophages. (C and D) Quantification of infiltrating mononuclear cells in the CNS. The mouse brains and spinal cords were prepared and extracted on day 19 after EAE induction. Total numbers of mononuclear cells (C), as well as the respective numbers of CD4+ T cells, CD8+ T cells, B cells, macrophages, and microglia (D) in the CNS were counted using flow cytometry. Data are representative of three impartial experiments with five mice in each group. * 0.05 (unpaired Students test). (E) RT-PCR detection of the proinflammatory cytokine mRNA levels in the spinal cords of na?ve mice or mice treated with SALM5 mAb or control antibody after EAE induction. (F) Immunohistochemical staining of activated microglia.