Figure 7

STING agonist induction of IFN-β. Bone marrow-derived macrophages (BMDMs) derived from the femurs of WT or Tmem173^gt mice were polarized in vitro into M1 or M2 subtypes. (a,b) Using iNOS/NOS2 mRNA induction as a marker for M1 cells, and Arg-1 mRNA induction as a marker for the M2 subtype, we observed no differences in the expression of either marker when these cells from the WT and Tmem173^gt were compared. (c) Increased IFN-β expression or secretion in response to STING agonists (or cytoplasmic DNA exposure) is a commonly used marker to show that the STING pathway has been activated. In this regard, when M1 or M2 polarized cells from the WT or Tmem17^Gt were incubated for 6-hr with DMXAA, it was observed that IFN-β mRNA expression was only induced in the WT macrophages. (d) Similarly, when the 3 subtypes of macrophages were exposed to the non-canonical ligand 2′3′-cGAMP or the bacterial second messenger c-di-AMP, IFN-β expression was increased solely in the WT macrophage polarized subsets. Significance was assessed by one-way ANOVA followed by Tukey’s multiple comparisons post-hoc test between all groups. Values represent the means ± SEM of the fold changes in the respective mRNAs; *P < 0.05, **P < 0.01, or ***P < 0.001 vs. the corresponding Tmem17^Gt group (eg. WT M1, DMXAA treated vs. Tmem17^Gt M1, DMXAA treated). For macrophage polarization: M0, untreated; M1, 50 ng/ml of LPS and IFNγ; M2, 40 ng/ml IL-4 for 48 hrs. STING agonists: DMXAA 20 μg/ml; c-di-AMP 20 μg/ml plus lipofectamine; 2′3′-cGAMP 20 μg/ml plus lipofectamine for 6 hrs. Data was normalized to β-actin mRNA and experimental transcripts expressed as the relative fold change in each mRNA selected.