Results

Autoimmunity

Pristane induced lupus (PIL) model

Kinetics of disease development
We have previously shown that elevated adenosine in severe inflammation causes A1R depletion associated leukopenia (13). To study the role of A1R in autoimmunity, we compared the appearance of pristane-induced lupus-like disease between a group of A1R-KO mice and a group of WT mice. In contrast to WT mice that remained without any sign of disease, KO mice started to exhibit the classic pathological signs of lupus: alopecia, chronic skin wounds, and death starting at 10 weeks from induction. At 36 weeks following pristane injection, 43% of A1R-KO mice suffered from lupus-like disease; five died, three suffered from alopecia, and one suffered from chronic wounds, while WT mice had no physical signs of disease (Figure 1A, p <0.05), although they did develop Anti-dsDNA, which is a hallmark of lupus. Anti-dsDNA levels in vehicle-treated WT mice were 12670.15±4712.31 ng/ml compared to 39775.08±19352.7 ng/ml in pristane-treated WT mice (Figure 1B, p <0.05). Spleens were removed and measured at the experience endpoint and were significantly larger following pristane injection in WT mice 0.136±0.016 vs. 0.29±0.099 gr. (Figure 1C, p <0.01). Differences in Anti-dsDNA and in spleen size did not reach significance between WT and A1R-KO mice following pristane injection, as the mice with the most severe illness died before the experience endpoint and therefore are not included in these results.
As expected, the basal WBC counts were lower in A1R-KO mice compared to WT mice (Figure 2A, p <0.05). In blood counts of WT mice, following pristane injection, we observed a deep reduction in WBC number, and the main leukocyte population to be affected was lymphocyte (Figure 2B). At 6h, lymphocyte counts were reduced by 85% from 7.44±3.48 to 1.05±0.73 cells x103/μl, (Figure 2B,p <0.05). All other cell populations were not significantly affected by pristane injection (Figure 2C, 2D). Partial recovery in lymphocyte counts was observed in the WT group 48h after injection.
We followed the A1R and A2AR mRNA levels normalized to RPL-12), at several time points, for 10 days (Figure 3). In WT mice, parallel to the decline of WBC, A1R mRNA levels dropped 6h after pristane injection (Figure 3A,p <0.05), and returned to basal level 24h after treatment. In these mice, A2AR was induced following the injection, reaching significance at 10 days (240h, Figure 3B,p <0.05). In contrast, A1R-deficient mice failed to upregulate the immunosuppressive receptor A2AR and stayed low for 10 days (Figure 3B,p <0.05).

Type-1 diabetes model

In accordance with the lupus model, the absence of both A1 and A2A receptors was found to accelerate the induction of type-1 diabetes (T1D). At 15 days, after the first STZ injection, all A1R-KO mice were diabetic, while the WT group at this time point remained free of disease (Figure 4A). Similarly, A2AR-KO mice (BALB/c background) were also more susceptible than WT mice to early development of T1D. At day 20, all A2AR-KO mice had elevated blood glucose levels, while only 40% of WT mice propagated T1D (Figure 4B).
Cell-free DNA (cfDNA) underpins the progression of autoimmune diseases (18). In the PIL model, in both groups, we observed the elevation of cfDNA levels in peritoneal lavage with a peak at 24h, (significant in the WT group, p <0.01; Figure 4A). A decline in cfDNA levels was observed at 48h. Ten days (240h) after injection, cfDNA levels stabilized, cfDNA levels in A1R-KO were significantly higher compared to WT mice (531±120 vs. 267±60 ng/ml, respectively,p <0.01; Figure 5A). Similarly, in the T1D model at day ten, cfDNA levels in A1R-KO mice were elevated compared to basal WT cfDNA levels (360.04±107.64 vs. 99.62±62.43 ng/ml, respectively, p <0.01; Figure 5B).

Regulation of NETosis by Adenosine Receptors

DNA released from neutrophils that undergo NETosis is a major source of cfDNA. We explored the regulation of NETosis by adenosine receptors using differentiated HL-60 cells stimulated for NETosis by PMA+H2O2. Stimulation of neutrophil-like HL-60 cells with CCPA (1nM), a specific A1R agonist prior to induction of NETosis, increased NETs production by 18% compared to untreated control cells. In contrast, pre-treatment with the A2AR agonist CGS21680 (30nM) diminished NETs production by 30% (*p <0.05, **p <0.001; Figure 6A). In accordance with these results, neutrophils isolated from A1R-KO mice produced 35% less NETs compared to WT mice (p <0.05, Figure 6B), and neutrophils from A2AR-KO mice without the suppressive effect of A2AR produced 70% more NETs (p <0.01, Figure 6B).