Insulin or insulin-like growth factor 1 (IGF-1) promotes the activation of phosphoinositide 3 kinase (PI3K)/Akt signaling in immune cells including dendritic cells (DCs), the most potent professional antigen-presenting cells for naive T cells. Klotho, an anti-aging protein, participates in the regulation of the PI3K/Akt signaling, thus the Ca²⁺-dependent migration is reduced in klotho-deficient DCs. The present study explored the effects of insulin/IGF-1 on DC function through klotho expression. To this end, the mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were treated with insulin or IGF-1 and followed by stimulating with lipopolysaccharides (LPS). Tumor necrosis factor (TNF)-α formation was examined by enzyme-linked immunosorbent assay (ELISA). Phagocytosis was analyzed by FITC-dextran uptake assay. The expression of klotho was determined by quantitative PCR, immunoprecipitation and western blotting. As a result, treatment of the cells with insulin/IGF-1 resulted in reducing the klotho expression as well as LPS-stimulated TNF-α release and increasing the FITC-dextran uptake but unaltering reactive oxygen species (ROS) production in BMDCs. The effects were abolished by using pharmacological inhibition of PI3K/Akt with LY294002 and paralleled by transfecting DCs with klotho siRNA. In conclusion, the regulation of klotho sensitive DC function by IGF-1 or insulin is mediated through PI3K/Akt signaling pathway in BMDCs.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells for naive T cells to link innate and acquired immunity. Klotho, an anti-aging protein, participates in the regulation of Ca2+ dependent migration in DCs. Vitamin E (VitE) is an essential antioxidant to protect cells from damage and elicits its inhibitory effects on NF-κB-mediated inflammatory response. However, the roles of VitE on mouse DC functions and the contribution of klotho to those effects both are unknown. The present study explored the effects of VitE on klotho expression, maturation, ROS production and migration in DCs. The mouse bone marrow cells were isolated and cultured with GM-CSF to attain bone marrow-derived DCs (BMDCs). Cells were stimulated with LPS (100 ng/ml) in the presence or absence of VitE (500 µM). RT-PCR and immunoprecipitation methods were employed to determine klotho expression, ELISA to determine cytokine release, flow cytometry to analyze number of CD86+CD11c+ cells, the intracellular expression of cytokines and reactive oxygen species (ROS) production and a transwell migration assay to trace migration. Klotho transcript level and this hormone secretion in DC supernatant were enhanced by VitE treatment and further increased in the presence of NF-κB inhibitor Bay 11-7082 (10 µM). Moreover, VitE treatment inhibited IL-12p70 protein expression of, ROS accumulation in and CCL21-dependent migration of LPS-triggered mature DCs, these effects were reversed following klotho silencing. The up-regulation of klotho by VitE could contribute to the inhibitory effects of VitE on NF-κB-mediated DC functional maturation. The events might contribute to immunotherapeutic effect of VitE on the pathophysiology of klotho-related disease.

Dendritic cells (DCs) are professional antigen presenting cells involved in the induction of T cell-mediated adaptive immunity. Plasmacytoid DCs (pDCs) originate from lymphoid precursors and produce type I interferons (IFNs) in response to pathogens. A20 is considered as a negative regulator of toll-like receptor (TLR) signaling pathways, in which Toxoplasma gondii- derived profilin (TgPRF) is a TLR11/12 ligand recognised by DCs to stimulate their maturation/activation. Little is known about contributions of A20 to changes in biological properties of pDCs. The present study, therefore, explored whether pDC functions are influenced by A20. To this end, bone marrow cells were isolated and cultured with Flt3L to attain CD8DCs, CD11bDCs and pDCs and followed by challenge with TgPRP in the presence or absence of A20 siRNA. Expression of maturation markers were analysed by flow cytometry, and secretion of inflammatory cytokines by ELISA, cell migration by a transwell migration assay and expression of signalling molecules by western blotting. As a result, treatment with A20 siRNA enhanced activations of IκB-α and STAT-1, leading to increases in expressions of maturation markers and cytokine productions as well as migration of TgPRP-treated pDCs, while mature CD11bDCs produced at higher levels of TNF-α and IL-6 only. In addition, functions of CD8DCs remained unaltered following A20 silencing. The effects of A20 on pDC maturation and activation were completely abolished by IKK inhibitor and partially blunted by fludarabine. In conclusion, the inhibitory effects of A20 on pDC functions are expected to affect the immune response in T. gondii infection.

Chronic myelogenous leukemia (CML) is characterised by the translocation of regions of the BCR and ABL genes, leading to the fusion gene BCR-ABL forming the Philadelphia (Ph) chromosome. Vinblastine (Vinb) and Vincristine (Vinc) are Vinca alkaloids and frequently used in combination chemotherapy in leukemias and lymphomas. Deubiquitinating enzyme (DUB) genes such as A20, Otubain 1 and CYLD are known as inhibitors of functional activation of immune cells mediated through the NF-κB/STAT pathway. Little is known about the regulatory role of Vinb/Vinc on the function of CML cells and the contribution of the DUBs to those effects. In the end, the gene expression profile was determined by quantitative RT-PCR, physiological properties of CML cells by flow cytometry and cytokine production by ELISA. As a result, inactivated expression of the DUBs A20, CYLD, Otubain 1 and Cezanne and enhanced activation of CD11b+ and CD4T cells were observed in CML patients. Importantly, Vinc enhanced the expression of A20 and CYLD and inhibited the proliferation and survival of CML (K562) cells. The effects were abolished in the presence of A20 siRNA, while cell proliferation only depended on the presence of CYLD. In conclusion, the up-regulation of A20 by Vinc could involve inhibitory effects on the proliferation and survival of K562 cells. The events might contribute to the anticancer effect of Vinc on A20-sensitive CML cells.

Klotho (KL) encodes a single-pass transmembrane protein and is predominantly expressed in the kidney, parathyroid glands, and choroid plexus. Genetic studies on the KL gene have revealed that DNA hypermethylation is one of the major risk factors for aging, diseases, and cancer. Besides, KL exerts anti-inflammatory and anti-tumor effects by regulating signaling pathways and the expression of target genes. KL participates in modulation of the insulin/insulin-like growth factor-1 (IGF-1) signaling, which induces the growth hormone (GH) secretion. Accordingly, KL mutant mice display multiple aging-like phenotypes, which are ameliorated by overexpression of KL. Therefore, KL is an important contributor to lifespan. KL is further identified as a regulator of calcium (Ca2+) channel-dependent cell physiological processes. KL has been also shown to induce cancer cell apoptosis, thus, it is considered as a potential tumor suppressor. Our recent studies have indicated that KL modulates an influx of Ca2+ from the extracellular space, leading to a change in CCL21-dependent migration in dendritic cells (DCs). Interestingly, the regulation of the expression of KL was mediated through a phosphoinositide 3-kinase (PI3K) pathway in DCs. Moreover, downregulating of KL expression by using siRNA knockdown technique, we observed that the expression of Ca2+ channels including Orai3, but not Orai1, Orai2, TRPV5 and TRPV6 was significantly reduced in KL-silenced as compared to control BMDCs. Clearly, additional research is required to define the role of KL in the regulation of organismic and cellular functions through the PI3K signaling and the expression of the Ca2+ channels.

Psoriasis is a common chronic, immune-mediated inflammatory disease of the skin. PSORS1C3 is a non-protein coding gene, of which the RNA transcript is found in psoriatic patients. CARD14 is mainly expressed in epidermal keratinocytes. TLR4 is a transmembrane protein to recognize microbial antigens. Our study aimed to assess the relationship among PSORS1C3, CARD14 and TLR4 polymorphisms, inflammatory expression and psoriasis susceptibility. To the end, 71 patients with psoriasis and 46 healthy individuals with the well-characterized clinical profiles were enrolled. Gene polymorphisms were determined by Sanger DNA sequencing and secretion of cytokines by ELISA. As a result, genetic analysis of PSORS1C3 gene identified nine SNPs and three haplotype blocks. Sequencing of the CARD14 gene determined eight SNPs and one haplotype block. Sequencing of TLR4 gene identified nine SNPs, in which a SNP rs1018673641 was found to exert deleterious effect. The linkage disequilibrium analysis showed that seven variants in PSORS1C3 gene and three SNPs in CARD14 gene were in tightly linked. More importantly, a significant association between IL-6 level and rs1018673641 AT genotype in TLR4 gene was detected in psoriatic patients. In conclusion, the PSORS1C3, CARD14 and TLR4 polymorphisms and haplotypes may be correlated with risk of suffering psoriasis and the IL-6-mediated chronic inflammation in psoriasis could be partially regulated by the TLR4 functional variant.