Discussion

In previous studies, we found that elevated adenosine either from uncontrolled systemic inflammation or by pharmacological treatment, downregulates and desensitize the adenosine A1R and upregulates the immunosuppressive A2AR (27). So what is the benefit of this acute immunosuppressive mechanism for recovery from a severe inflammatory damage? We hypothesized that, under destructive pathological conditions, normal immune response is probably unwanted, and such induction of lymphopenia and immunosuppression is a beneficial physiological mechanism to reduce the prevalence of autoimmunity.
Our data from mice with PIL-like disease and T1D show that without the presence of A1R these autoimmune diseases exacerbate. Similar to our study, Tsutsui et al. showed in experimental allergic encephalomyelitis that compared to WT mice, A1R-KO mice developed a severe progressive-relapsing form of the disease (30). We suggest that early response of A1R is the trigger of a protective mechanism, and without this receptor, mice are prone to severe autoimmunity.
Following pristane injection, in WT animals, we observed an acute reduction of A1R and leukocyte counts. Pristane in the peritoneum is known to cause inflammation and damage (reviewed in (35)) and rapid desensitization of A1R observed immediately after pristane injection is probably due to elevated levels of adenosine.
We believe that the fast depletion of A1R in the presence of elevated adenosine removes its anti-apoptotic protection and by reduction of Gi activity enables an early lymphotoxic effect by elevation of cAMP (13). The effect of A1R depletion is transient, and after 48h, lymphocyte counts begin to recover. The long-term suppression of immunity probably mediated by the elevated A2AR (36), which is induced by A1R and peaks at 48h and remains high in WT animals even 10 days after pristane injection. These findings are consistence with the findings that following adjuvant-induced arthritis, adenosine concentration in plasma stays high for weeks (37). The critical role of early A1R stimulation in upregulation of A2AR was shown by our group previously (38). Accordingly, in the current study in A1R-KO mice, pristane injection fails to upregulate A2AR, and mRNA level of A2AR is lower in A1R-KO also at T=0, which probably enables stronger lymphocyte reactivity in A1R-depleted animals.
Additional support for the role of low A2AR in the development of autoimmunity is our observation that A2AR-KO mice were predisposed to TID development. Similar to our findings, Deaglio et al. showed that A2AR-KO mice were more susceptible to STZ-induced diabetes with the presence of hyper-proliferative T cells (45), and Zhang et al. showed that A2AR activation suppressed inflammation in the progression of lupus nephritis (46).
In the present study, we suggest that adenosine regulates the release of DNA by NETosis and that the same A1R/A2AR dependent immunosuppressive mechanism reduces cfDNA levels. The presence of double-stranded DNA (dsDNA) was shown to be more than a distinct marker for illness severity; dsDNA is also a STING activator (47) and recent studies show a clear association between elevated cfDNA levels and autoimmunity (48).
We used differentiated HL-60 cells to study regulation of NETosis by agonists of adenosine receptors. Similar to the effect on other neutrophil functions, Such as adherence to endothelium, chemotaxis, activation and trafficking (8-12), the A1R agonist enhanced NETs production (118%). A2AR is a negative regulator of NETosis- stimulation of cells with a specific A2AR agonist decreased NETs production to 70% of untreated cells. In accordance, neutrophils isolated from A1R–KO mice produced fewer NETs (65%) compared to those isolated from WT mice, and vice versa: neutrophils isolated from A2AR–KO were stronger producers of NETs (170%). In support of our data, Liu et al. showed that activation of A2AR inhibits neutrophil cell death, and suggest that this finding is part of the anti-inflammatory role of A2AR in modulating neutrophil survival during SIRS (49). In addition a recent study by Ali et al. has shown that A2A adenosine receptor agonist attenuates NETosis (50).
In the T1D model, cfDNA levels in A1R-KO mice were elevated compared to basal levels of WT mice. In both models, the higher levels of cfDNA were in accordance with the proportion of animals’ sickness. In the PIL model, we followed the levels of cfDNA in detail during the first 10 days after pristane injection. At day 10, A2AR levels were low in A1R-KO mice, compared to WT mice while cfDNA levels were significantly higher than the WT mice, which might contributed to the severe development of the disease in this group.
To conclude, adenosine initiates diverse cellular responses directed to prevent excessive inflammation in order to restore immune homeostasis. Our data from PIL and T1D propose that A1 and A2A receptors have a protective role in autoimmunity development. The acute elimination of lymphocytes and reduction of DNA release from NETosis depends on A1R desensitization and long-term suppression maintained by A1R-dependent elevation of A2AR. We believe that severe traumatic events trigger adenosine mediated protective mechanism in order to reduce reaction against self-antigens.