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.