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Background Triggering receptor expressed on myeloid cells 2 (TREM2) signaling is considered to regulate anti-inflammatory responses in macrophages, dendritic cell maturation, osteoclast development, induction of obesity, and Alzheimer’s disease pathogenesis. However, little is known regarding the effect of TREM2 on natural killer (NK) cells. Results Here, we demonstrated for the first time that CD3−CD122+NK1.1+ precursor NK (pNK) cells expressed TREM2 and their population increased in TREM2-overexpressing transgenic (TREM2-TG) mice compared with that in female C57BL/6 J wild type (WT) mice. Both NK cell-activating receptors and NK cell-associated genes were expressed at higher levels in various tissues of TREM2-TG mice than in WT mice. In addition, bone marrow-derived hematopoietic stem cells (HSCs) of TREM2-TG mice (TG-HSCs) successfully differentiated into NK cells in vitro, with a higher yield from TG-HSCs than from WT-HSCs. In contrast, TREM2 signaling inhibition by TREM2-Ig or a phosphatidylinositol 3-kinase (PI3K) inhibitor affected the expression of the NK cell receptor repertoire and decreased the expression levels of NK cell-associated genes, resulting in significant impairment of NK cell differentiation. Moreover, in melanoma-bearing WT mice, injection of bone marrow cells from TREM2-TG mice exerted greater antitumor effects than that with cells from WT control mice. Conclusions Collectively, our data clearly showed that TREM2 promoted NK cell development and tumor regression, suggesting TREM2 as a new candidate for cancer immunotherapy.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell surface receptor expressed on macrophages, microglial cells, and pre-osteoclasts, and that participates in diverse cellular function, including inflammation, bone homeostasis, neurological development, and coagulation. In spite of the indispensable role of the TREM2 protein in the maintenance of immune homeostasis and osteoclast differentiation, the exact ligand for TREM2 has not yet been identified. Here, we report a putative TREM2 ligand which is secreted from MC38 cells and identified as a cyclophilin A (CypA). A specific interaction between CypA and TREM2 was shown at both protein and cellular levels. Exogenous CypA specifically interacted and co-localized with TREM2 in RAW264.7 cells, and the physical interactions were shown to regulate TREM2 signaling transduction. The Pro144 residue in the extracellular domain of TREM2 was found to be the specific binding site of CypA. When considered together, this provides evidence that CypA interacts specifically with TREM2 as a potent ligand.

Axl receptor tyrosine kinase is involved in the tumorigenesis and metastasis of many cancers. Axl expression was markedly higher in human papilloma virus type 16E6 (HPV16E6)-overexpressing HeLa (HE6F) cells and lower in HPV16E6-suppressing CaSki (CE6R) cells than in the controls. SiRNA-mediated knockdown of E6 expression led to increased phosphatase and tensin homolog (PTEN) phosphorylation at Ser380 and attenuated AKT phosphorylation. Expression of membrane-associated guanylate kinase inverted-2 (MAGI-2), an E6-induced degradation target, was induced in E6-siRNA-transfected cells. Moreover, myeloid zinc finger protein 1 (MZF1) binds directly to the Axl promoter in HE6F cells. Axl expression was regulated by HPV16E6-mediated PTEN/AKT signalling pathway, and Axl promoter activity was regulated through MZF1 activation in cervical cancer, which promoted malignancy. Axl silencing suppressed the metastasis of Caski cells and enhanced the susceptibility to NK cell-mediated killing of HE6F cells. In addition, the expression of Axl and MZF1 was highly correlated with clinical stage of cervical cancer and HPV16/18 infection. Taken together, Axl expression was induced by HPV16E6 in cervical cancer cells, suggesting that blockade of Axl signalling might be an effective way to reduce the progression of cervical cancer.

Natural killer (NK) cells have been well known to play a critical role in innate immunity, but they are also capable of regulating adaptive immunity through the induction of T cell-mediated memory response and B cell-mediated autoimmune response. NK cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow (BM), and a series of surface molecules are expressed on NK cells in a differentiation stage-specific manner. Axl receptor tyrosine kinase is originally identified as homeostatic regulators for antigen-presenting cells, and its ligand, growth-arrest-specific gene 6 (Gas6), has been reported to promote cell survival, proliferation, and migration, but their regulatory role in the development and effector function of NK cells is not yet fully understood. In this study, to investigate whether Axl is required for the regulation of NK cell development, the expression of mature NK (mNK) cell-specific receptors and NK cell-associated genes was analyzed in the differentiated HSCs-derived NK cells in vitro and the NK cells harvested from Axl-/- mice. We found that agonistic anti-Axl antibody or recombinant Gas6 specifically upregulated the expression of mNK cell-specific receptors, such as LY49A, Ly49G2, Ly49C/F/I, NKG2A/C/E (1.5- to 3.5-fold increase), and NK cell-associated genes, such as IL-2Rβ (2.3- or 2.4-fold increase), Perforin (4.1- or 2.1-fold increase), IL-15Rα (2.14- or 2.04-fold increase), and IFN-γ (3.3- or 2.8-fold increase) compared to each isotype control, whereas it was abrogated by treatment of Axl-Ig. Anti-Axl antibody or rGas6 also induced a 2.5- or 1.9-fold increase in the proliferation of developing NK cells compared to each control, respectively. mNK cell populations expressing mNK cell-specific receptors were reduced about twofold in NK cells differentiated from HSCs of Axl-/- mice compared with those of wild-type mice. Furthermore, the triggering of Axl signaling by agonistic anti-Axl antibody promoted the cytolytic activity (1.5- to 1.9-fold increase) against target tumor cells. In B16F10 melanoma-bearing mice, the number of metastatic colonies was decreased by 83 % by the administration of mNK cells treated with anti-Axl antibody compared to control Ig. These data suggest that Axl plays an essential role in the regulation of NK cell development as well as NK effector function.

BiP and other endoplasmic reticulum (ER)-resident proteins are thought to be metabolically stable and to function primarily in the ER lumen. We sought to assess how the abundance of these proteins dynamically fluctuates in response to various stresses and how their subpopulations are relocated to non-ER compartments such as the cytosol. We showed that the molecular chaperone BiP (also known as GRP78) was short-lived under basal conditions and ER stress. The turnover of BiP was in part driven by its amino-terminal arginylation (Nt-arginylation) by the arginyltransferase ATE1, which generated an autophagic N-degron of the N-end rule pathway. ER stress elicited the formation of R-BiP, an effect that was increased when the proteasome was also inhibited. Nt-arginylation correlated with the cytosolic relocalization of BiP under the types of stress tested. The cytosolic relocalization of BiP did not require the functionality of the unfolded protein response or the Sec61- or Derlin1-containing translocon. A key inhibitor of the turnover and Nt-arginylation of BiP was HERP (homocysteine-responsive ER protein), a 43-kDa ER membrane-integrated protein that is an essential component of ER-associated protein degradation. Pharmacological inhibition of the ER-Golgi secretory pathway also suppressed R-BiP formation. Finally, we showed that cytosolic R-BiP induced by ER stress and proteasomal inhibition was routed to autophagic vacuoles and possibly additional metabolic fates. These results suggest that Nt-arginylation is a posttranslational modification that modulates the function, localization, and metabolic fate of ER-resident proteins.

TREM2 plays a critical role in the alleviation of Alzheimer’s disease by promoting Aβ phagocytosis by microglia, but the detailed molecular mechanism underlying TREM2-induced direct phagocytic activity of Aβ remains to be revealed. We found that learning and memory functions were improved in aged TREM2 TG mice, with the opposite effects in KO mice. The amount of phagocytosed Aβ was significantly reduced in the primary microglia of KO mice. CD36 expression in primary microglia was greater in TG than in WT mice but was substantially decreased in KO mice. The expression of C/EBPα, an upstream transcriptional activator of CD36, was also elevated in primary microglia of TG mice but decreased in KO mice. The transcription of CD36 was markedly increased by TREM2 overexpression, and this effect was suppressed by a mutation of the C/EBPα binding site on the CD36 promoter. The TREM2-induced expression of CD36 and C/EBPα was inhibited by treatment with PI3K/AKT signaling blockers, and phosphorylation of AKT was elevated in TREM2-overexpressing BV2 cells. The present study provides evidence that TREM2 is required for preventing loss of memory and learning in Alzheimer’s disease by regulating C/EBPα-dependent CD36 expression and the consequent Aβ phagocytosis.

Vascular calcification is a hallmark of cardiovascular disease. Interleukin-24 (IL-24) has been known to suppress tumor progression in a variety of human cancers. However, the role of IL-24 in the pathophysiology of diseases other than cancer is unclear. We investigated the role of IL-24 in vascular calcification. IL-24 was applied to a β-glycerophosphate (β-GP)-induced rat vascular smooth muscle cell (VSMC) calcification model. In this study, IL-24 significantly inhibited β-GP-induced VSMC calcification, as determined by von Kossa staining and calcium content. The inhibitory effect of IL-24 on VSMC calcification was due to the suppression of β-GP-induced apoptosis and expression of calcification and osteoblastic markers. In addition, IL-24 abrogated β-GP-induced activation of the Wnt/β-catenin pathway, which plays a key role in the pathogenesis of vascular calcification. The specificity of IL-24 for the inhibition of VSMC calcification was confirmed by using a neutralizing antibody to IL-24. Our results suggest that IL-24 inhibits β-GP-induced VSMC calcification by inhibiting apoptosis, the expression of calcification and osteoblastic markers, and the Wnt/ β-catenin pathway. Our study may provide a novel mechanism of action of IL-24 in cardiovascular disease and indicates that IL-24 is a potential therapeutic agent in VSMC calcification.

Bacterial β-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield β-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial β-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial β-(1,3)-glucan at daily doses of 2.5 or 5mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial β-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1β, IL-6 and IL-17A/F, were markedly decreased in the colon of β-(1,3)-glucan-pretreated mice. β-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, β-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial β-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production.

Introduction Interleukin-18 (IL-18), a member of the IL-1 cytokine super-family, has been known to interferon-g-inducing factor and plays a critical role in angiogenesis, bone metabolism, metabolic syndrome, inflammatory and immune response. However, the role of IL-18 in the melanin synthesis of B16F10 melanoma cells has not been investigated. Methods B16F10 cells were treated with IL-18 and/or for 5 days, and were then dissolved in 1N NaOH in 10% DMSO at 80 °C for 1 h. The relative melanin contents were determined by measuring the absorbance at 405 nm using an ELISA reader. Expression of IL-18, IL-18R and melanogenesis-related genes was examined by real time PCR. Results Here, we found that IL-18 significantly induced the melanin synthesis in B16F10 cells, and anti-IL-18 abrogated IL-18-induced melanin synthesis. We also observed that anti-IL-18 decreased gene and protein expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and dopachrome tautomerase (Dct) stimulated by IL-18. In addition, anti-IL-18 reduced IL-18-induced phosphorylation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK). Conclusion Taken together, these results suggest that IL-18 may be involved in regulation of the melanin synthesis in B16F10 cells via the ERK pathway.

Introduction The mulberry belongs to the Morus genus of the Moraceae family. There are 24 species of Morus, with at least 100 known varieties. Methods RAW 264.7 cells were pretreated with mulberry or mulberry derivatives for 2 h, and then stimulated with lipopolysaccharide (LPS). Gene expression of proinflammatory cytokines were determined by RT-PCR. The levels of NO were determined by measuring the amount of nitrate using Griess reagents, and absorption was measured at 570 nm. Intracellular ROS levels were evaluated using a DCFH-DA fluorescent probe by flow cytometry. Results We found that mulberry derivatives inhibited the gene expression of proinflammatory cytokines including IL-6, IL-1b, TNF-aand COX-2 in Raw264.7 cells stimulated with lipopolysaccharide (LPS). In addition, LPS-induced intracellular ROS and NO levels were decreased in Raw264.7 cells treated with mulberry derivatives compared with those in mulberry. Conclusion These data suggest that mulberry derivatives showed anti-inflammatory activity by reducing intracellular ROS and NO production via suppression of proinflammatory cytokine expression, and mulberry derivatives may be useful for the prevention and therapy of inflammation-related diseases.

Introduction Axl receptor tyrosine kinase (Axl) has been known to play a crucial role in cell survival, proliferation, and migration, but little is known about its regulatory role in the migration of gd T cells. Here we investigated the role of Axl in the migration of intraepithelial gd T cells. Methods Primary intraepithelial lymphocytes (IELs) and gdT cells were isolated from 6 to 8 weeks old WT and Axl-deficient mice. Gene expression of IL-7Ra, CCR5, CCR9 and integrin aEb7 was examined by realtime-PCR. IEL phenotype was analyzed by flow cytometry. For adoptive transfer, primary IELs isolated from donors were injected into orbital vein of recipient mice after 6 weeks. Results The gd T cell population and absolute cell number was increased in IELs of Axl-deficient mice compared to them of wild type mice but not in other tissues. In addition, up-regulation of IL-7Ra, CCR5, CCR9 and integrin aEbexpression was observed in both IELs and gd T cells purified from IELs of Axl-deficient mice. The adoptive transfer of IELs of Axl-deficientmice to RAG-deficientmice induced the increased migration of gdT cells. Conclusion Our findings suggest that Axl is required for the migration of intestinal gd T cells by the transcriptional regulation of IL-7Ra, CCR5, CCR9 and integrin aEb7.

Introduction Triggering receptor expressed on myeloid cells-2 (TREM-2) is a cell surface receptor that participates in diverse cellular functions including inflammation, bone homeostasis and neurological development. We examined the effects of TREM-2 on experimentally induced inflammatory bowel disease (IBD) in mice. Methods Wild type (WT) and TREM2 transgenic (TREM2 TG) mice were administrated with dextran sulfate sodium (DSS) to induce IBD. The gene expression of proinflammatory cytokines were examined using RT-PCR and histopathological analysis was performed by H&E staining. The population of regulatory T cells was analyzed by flow cytometry. Results We found that body weight and colon length were remarkably decreased by treatment of DSS in TREM2-TG mice compared with WT littermates. Gene expression of proinflammatory cytokines including TNF-a, IL-1b, IL-17F, and IL-6 was increased in colonic tissue of DSS-treated TREM2-TG mice. In addition, recruitment of immune cells into colon tissue was also increased by DSS treatment in TREM2-TG mice, but population of regulatory T cells was decreased. Conclusion Our data suggest that TREM2 may be involved in the pathogenesis of IBD by upregulating proinflammatory cytokines due to the reduction of regulatory T cells.

Introduction The abnormal growth of vascular smooth muscle cells induced by reactive oxygen species is considered as a major pathogenic process in vascular diseases. Interleukin-10 family of cytokines is known to inhibit cancer cell growth through induction of cell cycle arrest and apoptosis. However, the role of interleukin-10 family of cytokines in ROS-induced VSMC growth has not been investigated yet. Methods Mouse vascular aorta smooth muscle cells were treated with H 2 O 2 . The cell cycle and intracellular ROS production was examined using flow cytometry analysis, and mRNA expression of VEGF and PDGF was investigated by RT-PCR and Real time PCR. Protein expression of PI3K/Akt and Erk was determined by western blot analysis Results We found that interleukin-10 family of cytokines inhibited the growth of mouse vascular aorta smooth muscle cells treated with H 2 O 2 by inducing the arrest of cell cycle at G0/G1 phase through the regulation of p21 and cyclin D1. Furthermore, interleukin-10 family of cytokines suppressed the mRNA expression of VEGF and PDGF, which subsequently reduced an elevated level of migration in response to H 2 O 2 . Interestingly, interleukin-10 family of cytokines attenuated H 2 O 2 -induced ROS production due to the upregulated mRNA expression of TRX-1 and PRX-2. Signaling through protein tyrosine kinases and mitogen-activated protein kinases is required for ROS-mediated activation of VSMCs. Consistently, we also showed that H 2 O 2 -induced PI3K/Akt and Erk signaling pathways were blocked by interleukin-10 family of cytokines. Conclusion Our data suggest a novel mechanism that interleukin-10 family of cytokines suppresses growth of normal VSMCs stimulated by H 2 O 2 through inhibiting ROS production via PI3K/Akt and Erk signaling pathways.