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).