Adenosine/A(2A)R/PKA signaling regulates HO-1-mediated anti-inflammatory responses during Leishmania donovani infection.

Adenosine receptor A(2A)R plays a pivotal role in dampening pro-inflammatory cytokine levels in Leishmania donovani-infected macrophages, thus promoting infection. However, the specific regulatory pathway remains unidentified. In this study, we showed that blocking A(2A)R signaling reduces the expression of heme oxygenase-1 (HO-1), an enzyme earlier implicated in reducing pro-inflammatory cytokine levels. A(2A)R, being a G-protein-coupled receptor (GPCR), increased intracellular cAMP, thereby activating protein kinase A (PKA) activity. Inhibition of the A(2A)R/PKA pathway impacted two major transcription factors of HO-1, cAMP response element-binding protein (CREB) and nuclear factor erythroid 2-related factor 2 (NRF2). PKA directly activated CREB through phosphorylation, and the ChIP assay further validated the involvement of PKA in p-CREB-mediated HO-1 transcription. On the other hand, PKA-mediated glycogen synthase kinase-3 beta (GSK-3β) phosphorylation at the Ser-9 position rendered it inactive and removed its inhibitory effect on NRF2, thus allowing its nuclear translocation during infection. Macrophages transfected with constitutively active nonphosphorylated GSK-3β showed reduced nuclear localization of NRF2 and decreased parasite survival. Administering the A(2A)R inhibitor in infected mice decreased HO-1 levels, liver and spleen parasite burden, and increased pro-inflammatory cytokine levels. Our findings revealed Leishmania exploits adenosine-A(2A)R signaling to activate PKA-mediated CREB- and NRF2-dependent HO-1 upregulation, reducing pro-inflammatory cytokine levels and favoring pathogenesis. IMPORTANCE: Visceral leishmaniasis, caused by the protozoan parasite Leishmania donovani, is a major health concern affecting over a million people worldwide. An increase in host ATP production and its efflux benefits the survival of Leishmania parasites and prolongs the infection. Effluxed ATP is converted to adenosine, which activates adenosine-A(2A)R signaling to provide an immunosuppressive milieu, necessary for infection propagation. This study identified cAMP/PKA as the essential components of A(2A)R signaling, which further differentially activate two transcription factors to induce the antioxidant enzyme HO-1, responsible for creating the anti-inflammatory environment. Our findings highlight A(2A)R as a promising drug target against visceral leishmaniasis and other inflammation-related diseases, offering us the opportunity to alleviate inflammatory responses, thereby broadening the impact on disease management and therapy.