Bserved variations had been not resulting from distinctive cell viability (Figure S3). These data demonstrate that NLRC3 attenuates cytokine response to intracellular DNA with no affecting cell viability.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptImmunity. Author manuscript; readily available in PMC 2015 March 20.Zhang et al.PageNLRC3 deficiency causes enhanced IFN- and IL-6 production in response to c-di-GMP and c-di-GMPNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptC-di-GMP, a compact di-nucleotide monophosphate, is often a second messenger of bacteria which include Listeria monocytogenes and Burkholderia thaildensis, and activates the IFN-I response via interaction with STING (Burdette et al., 2011; Jin et al., 2011; Sauer et al., 2011). Nlrc3-/- MEFs made far more IFN- and IL-6 proteins in response to transfected c-di-GMP (Figure 2A ). In addition, Nlrc3-/- MEFs produced elevated IFN-I and IL-6 in response to infection with c-di-GMP making L. monocytogenes (Figure 2C ). Increased IFN was also observed in Nlrc3-/- cells infected with an additional c-di-GMP generating bacteria, B. thaildensis (Figure 2F). As a result Nlrc3-deficiency leads to elevated innate immune response to cytoplasmic DNA, c-di-GMP, and bacteria that produce c-di-GMP.2-Methylquinoline-4,6-diamine Chemscene NLRC3 inhibits the STING-dependent pathway Cytoplasmic DNA and c-di-GMP induce IFN-I via the STING molecule, which led us to examine each functional and molecular interactions amongst NLRC3 and STING (Burdette et al., 2011; Huang et al., 2012; Ouyang et al., 2012; Shang et al., 2012; Shu et al., 2012). To investigate if NLRC3 affects the STING pathway, we examined the influence of NLRC3 around the activation of IFN- promoter-luciferase by STING. This reporter assay was internally controlled by the co-transfection of a Renilla luciferase construct. NLRC3 inhibited IFN- promoter activation by STING by 9.72 fold. STING operates by interaction and activation of its downstream kinase, TBK1 (Tanaka and Chen, 2012). NLRC3 drastically lowered IFN- promoter activation by TBK1. On the other hand NLRC3 had no direct impact on the downstream interferon regulatory transcription factor 3 (IRF3), indicating that NLRC3 most likely functions at the upstream STING-TBK level (Figure 3A).41102-25-4 site As a specificity control, one more NLR, NLRP11, did not minimize IFN- promoter activation by TBK1 (Figure 3B).PMID:23659187 NLRC3 also inhibited a second promoter driven by the canonical interferon-stimulated responsive element (ISRE), that is recognized to become activated by STING and TBK1 (Ishikawa and Barber, 2008; Zhong et al., 2008) (Figure 3C). However NLRC3 had no impact around the activation from the ISRE promoter by mitochondrial antiviral signaling protein (MAVS) (also known as interferon-beta promoter stimulator 1 (IPS-1), virus-induced signaling adapter (VISA) and CARD adaptor inducing IFN- (CARDIF)), which is important for RNA sensing, nor did it have an effect on promoter activation by the downstream IRF3 (Figure 3C). Furthermore, NLRC3 inhibited NF-B promoter activated by STING, and decreased MAVS activation slightly but didn’t affect retinoic acid-inducible gene 1 (RIG-I)(Figure 3D). We also observed that NLRC3 inhibited c-di-GMP and poly(dA:dT)-induced ISRE activation (Figure 3E). These experiments indicate that the predominant impact of NLRC3 is around the STING pathway. As an additional specificity control for NLR proteins, overexpression of NLRC5, which has been reported to inhibit different innate immune pathways when tested in an overexpression technique (Cu.