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Figure 1. Susceptibility of A1R-KO mice to PIL.C57BL/6 WT and A1R-KO mice were injected with pristane and monitored for alopecia, chronic wounds, or death for 36 weeks. (A) Rate of disease appearance * p <0.05 (n=8-14). Mantel-Cox test sighs of disease graphs. After 36 weeks, mice were sacrificed and analyzed for (B) anti-dsDNA levels in serum of surviving mice by ELISA, and (C) spleen size of surviving mice was measured. *p <0.05, ** p <0.01 (n=3-8). Values are mean ± SE.
Figure 2. Lymphopenia following pristane injection. Following pristane injection to C57BL/6, WT mice, and A1R-KO mice (n=3-6). Blood counts were performed at the indicated time points. (A) WBC, (B) lymphocyte, (C) neutrophils, and (D) monocytes. *p <0.05, ** p <0.01, compared to control (WT at T=0). Values are mean ± SE.
Figure 3. A1R and A2AR mRNA levels in PIL. Pristane was injected into C57BL/6 WT and A1R-KO mice. To examine the dynamic expression of the two high-affinity adenosine receptors, A1R and A2AR, the spleen was removed at indicated time points (6h, 24h, 48h, and 10 days). A1R and A2AR mRNA levels in adherent splenocytes were analyzed by real-time PCR and normalized by housekeeping RPL-12 levels. Results are median + interquartile range, (n=3-6) * p <0.05, between expression levels of each receptor to expression at time 0. least two independent experiments performed
Figure 4. Adenosine receptors and susceptibility to induced autoimmune type-1 diabetes. Mice were injected with low dose streptozotocin (STZ, 50mg/kg) for five consecutive days. Mice were considered diabetic when glucose remained above 200mg/dl. The experiment was ended when, in one of the groups, all animals were sick. (A) C57BL/6 WT and A1R-KO mice, (B) Balb/C WT and A2AR-KO mice. Mantel-Cox test sighs of disease graphs. (n=5-7).
Figure 5. cfDNA in autoimmune diseases. Peritoneal lavage was collected to examine the levels of cfDNA in (A) PIL model at several time points after pristane injection, and in (B) T1D model at day 10 after first STZ injection. Analyzed for cfDNA levels by a direct rapid fluorometric assay with the fluorochrome SYBR Gold (lower panel). Results are median + interquartile range, (n=3-6) *p <0.05, ** p <0.01, between expression levels of each receptor to expression at time 0. ^^p <0.01, ^^^ p <0.001 compared to WT at the same time point (n=5-7). At least two independent experiments performed
Figure 6. Adenosine receptors regulate NET production. (A) HL-60 cells differentiated by retinoic acid (RA) to neutrophil-like cells (2x105 cells/well) in triplicates were pre-exposed to A1 adenosine receptor agonist (CCPA, 1nM) or A2A adenosine receptor agonist (GCS, 30nM), and then cells were stimulated with PMA (200nM) and H202 (0.03%) in the presence of DNA fluorescent dye (5 µM SYTOX Green) for 3h. Relative NETs production was measured by fluorescence in 96-well plates and normalized to NETs production by cells treated with only H2O2+PMA. (B) Neutrophils (2x105 cells/well) in triplicates were isolated from bone marrow of WT and A1R-KO mice (C57BL/6 background) and WT and A2AR-KO mice (BALB/c). Relative NETs production was measured by fluorescence and normalized to NETs production by WT neutrophils treated with H2O2+PMA.*p < 0.05; **p < 0.01. Values are mean ± SE. At least two independent experiments performed