The effect of everolimus treatment on the expression of inflammatory and anti-inflammatory factors in monocyte-derived dendritic cells

Abstract

Dendritic cells are specialized antigen-presenting cells that play an essential role in bridging the gap between innate and acquired immune responses. In addition to the exceptional capacity of antigen presentation, these cells are able to initiate and control immunogenic and tolerogenic immune responses. Immunogenic DCs interact with T lymphocytes and play a role in defense against cancer cells, and tolerogenic DCs participate in suppressing immune responses by producing and expressing inhibitory factors. Cancer has affected all societies today. Considering the side effects of common cancer treatments, including surgery, chemotherapy, and radiation therapy, such as damage to healthy body cells, the use of immunotherapy in cancer treatment has received attention in recent years. On the other hand, the defect in self-antigen tolerance leads to immune reactions against self-antigens, which leads to autoimmune diseases. As a result, specific immune suppression methods can be used in the treatment of autoimmune diseases. mTOR is one of the effective signaling pathways in DCs, which plays a vital role in regulating immune responses by participating in the recognition and processing of immunological stimuli. The functions of various immune cells including neutrophils, NK cells, mast cells, macrophages, DCs, B and T cells are regulated by mTOR. Disruption of the mTOR system has been observed in several types of human cancers. Everolimus is a derivative of rapamycin that inhibits mTOR and has an immunosuppressive and antiproliferative role. This study investigated the effect of treatment with Affinitor drug (Everolimus) on the expression of inflammatory and anti-inflammatory factors in monocyte-derived dendritic cells.

Methods: First, peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll solution. Then, monocytes were separated from PBMC and using IL-4 and GM-CSF cytokines, monocyte-derived DCs were produced. Expression of surface markers of DCs such as CD11c, HLA-DR and CD86 was performed by flow cytometry. DCs were then incubated with lipopolysaccharide to mature and activate. In order to obtain the optimum dose of everolimus drug, doses of 10 and 50 nM were investigated, then the amount of apoptosis and necrosis induced by different doses was measured with Annexin and PI and read by flow cytometry. Then mDCs were treated with cabergoline drug and the relative expression of IL-10, IL-12, TNF-α, TGF-β, NF-KB and IDO genes was obtained by Real Time PCR method. Results: The expression levels of CD11c and HLA-DR markers, which are related to maturation and antigen presentation, were lower and higher, respectively, in DCs treated with everolimus compared to the control group. On the other hand, the expression levels of inflammatory factors IL-12, IL-1β, TNF-α, and NF-κB in the treated DCs increased significantly compared to the control group, and conversely, the expression levels of anti-inflammatory factors IL-10 and IDO decreased.