These murine models facilitate a dissection of the role of STING in specific tissues and provide tools for evaluating STING inhibitors for the treatment of SAVI patients. = 40 mice per group. murine models facilitate a dissection of the role of STING in specific tissues and provide tools for evaluating STING inhibitors for the treatment of SAVI patients. = 40 mice per group. (= 5 mice per genotype were used. Representative images are shown at 20 resolution. (= 8 mice per genotype at 4C6 wk of age. Black bar represents littermate control for N153S (NS) HET mice and gray bar is the littermate E7820 control for V154M (VM) HET mice. * 0.05; ** 0.01; **** 0.0001; ns, not significant. Because N154S and V155M are the most common SAVI mutations identified in patients, we used CRISPR/Cas9 genome editing to introduce the corresponding N153S or V154M alleles into mice. Sanger sequencing confirmed the expected base substitutions in heterozygous mice (and and = 6 mice per group. ( 0.05; ** 0.01; ns, not significant. To evaluate the IFN signature more thoroughly, we analyzed total bone marrow (BM) from the mutant lines. We detected an increase in ISGs and other immune genes in both strains and found that the BM E7820 of V154M mutant E7820 showed higher expression of some genes compared with the N153S mutant (and and E7820 and and and and and and = 8 mice per group. ( 0.05; ** 0.01; *** 0.001; **** 0.0001; ns, not significant. SAVI Mutations Cause Abnormal Lymphocyte Development. To determine whether the reduced number of mature T and B cells in the SAVI spleens reflected defects in lymphoid cell development, we evaluated the thymus and the BM. The total number of thymocytes, including the early CD4?CD8? double-negative thymocytes, were reduced (Fig. 4and and and and and = 8 mice for each WT and mutant genotype. * 0.05; ** 0.01; *** 0.001; E7820 **** 0.0001; ns, not significant. IRF3, IFNaR, and MLKL Deficiency Fail to Rescue V154M SAVI Phenotype. Because STING activates type I interferons through IRF3, we hypothesized that blocking IRF3 to limit the production of Rabbit Polyclonal to Cytochrome P450 39A1 type I interferons could rescue the lethality of SAVI mice. Therefore, we crossed the more potent SAVI V154M mutant strain to mice deficient in IRF3 or the type I IFN/ receptor. (IFNaR). Consistent with other reports (16, 17), we found that the death observed in SAVI HET mice was not rescued by deletion of either of IRF3 or IFNaR (Fig. 5 and that still showed increased expression in SAVI mutant mice that lacked IFNaR (= 15 mice. (= 10 mice. (= 5 mice 8C12 wk aged per group. * 0.05, ** 0.01, *** 0.001, **** 0.0001. Besides type I interferons, several studies have linked cytosolic DNA-sensing pathways to necroptosis downstream of STING activation. IFN production upon STING activation was reported to engage RIPK3 and MLKL, leading to oligomerization of MLKL and MLKL-dependent necroptotic cell death (22C24). Because we observed significant loss of lymphoid cells in these SAVI strains, we considered the possibility that necroptosis led to cell death in SAVI mice. V154M HET mice were crossed to MLKL KO mice to generate V154M HET MLKL KO mice and littermate controls. MLKL-deficiency did not change the immune abnormalities of the V154M HET mice (Fig. 5and and and and = 8C10 mice per group compiled from two impartial experiments. ** 0.01; *** 0.001. When we evaluated the activation status of the CD45.1 and CD45.2 TCR-+ cells, we found that the donor-derived T cells from V154M mutant were activated, as indicated by increased expression of CD44 (Fig. 6= 5 mice per group analyzed 8C10 wk after BM reconstitution. Discussion A variety of mutations in genes associated with the metabolism or sensing of endogenous nucleic acids lead to an assortment of clinical syndromes that are collectively referred to as type I interferonopathies, because these patients share an elevated IFN gene signature (3, 26). To what.