Supplementary Figure 7: Apoptotic cells and SLE microparticles drive AhR activation and IL-10 production in human macrophages. | Nature Immunology

Supplementary Figure 7: Apoptotic cells and SLE microparticles drive AhR activation and IL-10 production in human macrophages.

From: Apoptotic cell–induced AhR activity is required for immunological tolerance and suppression of systemic lupus erythematosus in mice and humans

Supplementary Figure 7

(a) Irradiated, apoptotic jurkat cells were cultured with PBDM (Ap-PBDM) for 8h. Culture supernatants were collected and IL-10 and IL-6 were measured by ELISA and mRNA was collected for assessment of CYP1A1 (normalized against BACTIN) by sqPCR. (b) Staurosporine treated, apoptotic jurkat cells were treated with DNAse and cultured with PBDM for 8h (Ap-PBDM). Culture supernatants were collected indicated proteins were measured by ELISA and mRNA was collected for assessment of CYP1A1 sqPCR. (c) Serum concentrations of kynurenine (Kyn) and indole-3-propionic acid (IPA) were measured by HPLC from samples described in Supplementary Tables 1,2,3. Bars are mean +/- standard deviation. *=P≤ 0.05, ns= not significant as determined by the Wilcoxon rank-sum test. (d) Microparticles purified from plasma of patient samples described in Supplementary Table 4,5 were quantified by flow cytometry. (e) Representative electron micrographs illustrating morphology and size distribution of microparticles from plasma of SLE patients and controls. Bar is 500nm and magnification is 50,000x. (f) Microparticles were assessed by flow cytometry for surface expression of the markers indicated. MFI ratio represents the relative mean fluorescence intensity (MFI) for the indicated CD marker versus the MFI for isotype controls. (g) Microparticls origin was analyzed by expression of endothelial (CD31), haematopoietic (CD45), neutrophil (CD66b), B cell (CD19), and T cell (CD3) markers. % positive indicates the % of microparticles that were positive for the markers compared to isotype controls. (h) Microparticles isolated from the plasma of SLE patients were added to cultures of healthy donor PBDM (20% volume/volume) +/- AhR antagonist CH223191. Eight hours later, culture supernatants were collected to measure cytokine production by ELISA. For a, b, h n=5 biologically independent samples per group. Bars are mean values +/- standard deviation and *=P≤0.05, **=P≤0.01 as determined by two sided Student’s t-test. All Experiments were repeated three times with similar results.

Back to article page