A secondary messenger generated by bacteria, is reported to bind STING
A secondary messenger generated by bacteria, is reported to bind STING directly [6]. In the course of recognition of intracellular DNA, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) functions as a cytosolic DNA sensor activating reaction of GTP and ATP to kind cGAMP, an endogenous secondary messenger that binds STING and stimulates the synthesis of type I IFN [7sirtuininhibitor]. Inappropriate recognition of self-DNA leads to generation of autoantibodies and overproduction of cytokines including CXCL10, IFN-, and TNF-. Phagocytosed apoptotic and necrotic DNA which can be incompletely digested as a consequence of deficiency of lysosome function dysregulate innate immune responses by way of a TLR-independent pathway and mediate interferonopathy and autoimmune diseases, including systemic lupus erythematosus (SLE) and chronic polyarthritis (reviewed in [10]). In an earlier study, DNase II knock-out mice with markedly enhanced levels of IFN-sirtuininhibitorand other cytokines died or exhibited signs of arthritis [11]. STING is proposed to be involved in over-production of inflammatory cytokines in response to selfDNA due to the fact cytokine levels and polyarthritis lesions are remarkably decreased in Dnase II and STING double knock-out mice [12]. Mutations in 3′ repair exonuclease1 (TREX1), previously referred to as Dnase III, also appear to trigger autoimmune diseases through interaction with STING. TREX1 degrades intracellular double-stranded DNA and negatively regulates STING-dependent innate immune responses [13]. Functional deficiency of TREX1 has been shown to cause accumulation of DNA and consistent activation of immune responses. Aicardi-Gouti es syndrome (AGS) is amongst the IFN-associated autoimmune ailments caused by mutation on the TREX1 gene [14, 15]. Autoimmune ailments attributable to TREX1 mutations can be rescued by functional deficiency of IRF3 or type I IFN receptor (IFNR) [3]. Consequently, targeting STING to suppress the type I IFN response against self-DNA seems to present an efficient approach to treat autoimmune disease. In vitro SDF-1 alpha/CXCL12, Human (68a.a) screening of medicinal plant extracts led to the identification of a 70 ethanol extract of Cephalotaxus koreana that specifically inhibits STING-induced, but not TBK1- or IRF3-induced IFN- promoter activation. The effects of two key ester alkaloids isolated from the genus Cephalotaxus on STING-induced type I IFN signaling pathway had been further investigated.Materials and techniques Cell IdeS, Streptococcus pyogenes (His) culture, plasmids, reagents and plant materialsHuman embryonic kidney 293T (HEK293T) cells and human monocytic leukemia cell line THP-1 cells have been obtained from Korean Cell Line Bank (Seoul, Korea). HEK293T cells have been cultured in Dulbecco’s Modified Eagle Medium(DMEM) (Biowest, Nuaille, France) supplemented with 10 fetal bovine serum and 1 penicillin/streptomycin. THP-1 cells had been cultured in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA) supplemented with ten fetal bovine serum, 1 penicillin/streptomycin and 0.05mM 2-mercaptoethanol. Human STING (hSTING), TBK1 and IRF3 were cloned into a pEF-based location vector from the pENTRhSTING, pENTR-hTBK1, and pENTR-hIRF3 plasmids, respectively, working with LR clonaseTM enzyme mix (Invitrogen, Carlsbad, CA). 2’3′-cGAMP was acquired from InvivoGen (San Diego, CA). Homoharringtonine was purchased from Sigma-Aldrich (St. Louis, MO) and harringtonine from Santa Cruz Biotechnology (Dallas, TX). Cephalotaxine was obtained from Glentham Life Sciences (Corsham, UK). OmicsFectTM in vitro tran.