Invariant natural killer T (iNKT) cells are activated at sites of local tissue injury, or globally during vaso-occlusive episodes of sickle cell disease (SCD). Tissue damage stimulates production of CD1d-restricted lipid antigens that activate iNKT cells to produce Th1- and Th2-type cytokines. Here, we show that circulating iNKT cells in SCD patients express elevated levels of the ectonucleoside triphosphate diphosphosphohydrolase, CD39, as well the adenosine A2A receptor (A2AR). We also investigated the effects of stimulating cultured human iNKT cells on the expression of genes involved in the regulation of purinergic signaling. iNKT cell stimulation caused induction of ADORA2A, P2RX7, CD38, CD39, ENPP1, CD73, PANX1, and ENT1. Transcription of ADA, which degrades adenosine, was reduced. Induction of CD39 mRNA was associated with increased ecto-ATPase activity on iNKT cells that was blocked by POM1. Exposure of iNKT cells to A2AR agonists during stimulation reduced production of IFN-γ and enhanced production of IL-13 and CD39. Based on these findings, we define purinergic Th2-type cytokine bias as an antiinflammatory purinergic response to iNKT cell stimulation resulting from changes in the transcription of several genes involved in purine release, extracellular metabolism, and signaling.

Allosteric enhancers of the adenosine A1 receptor amplify signaling by orthosteric agonists. Allosteric enhancers are appealing drug candidates because their activity requires that the orthosteric site be occupied by an agonist, thereby conferring specificity to stressed or injured tissues that produce adenosine. To explore the mechanism of allosteric enhancer activity, we examined their action on several A1 receptor constructs, including (1) species variants, (2) species chimeras, (3) alanine scanning mutants, and (4) site-specific mutants. These findings were combined with homology modeling of the A1 receptor and in silico screening of an allosteric enhancer library. The binding modes of known docked allosteric enhancers correlated with the known structure-activity relationship, suggesting that these allosteric enhancers bind to a pocket formed by the second extracellular loop, flanked by residues S150 and M162. We propose a model in which this vestibule controls the entry and efflux of agonists from the orthosteric site and agonist binding elicits a conformational change that enables allosteric enhancer binding. This model provides a mechanism for the observations that allosteric enhancers slow the dissociation of orthosteric agonists but not antagonists.

We characterized a novel population of tolerogenic myeloid dendritic cells (tmDCs) defined as CD11c+ CD11b+ CD14+ CD4+ and immunoglobulin-like transcript receptor (ILT)-4+ that are significantly more abundant in the circulation of infants and young children than in adults. TmDCs secrete the immunosuppressive lymphokine interleukin (IL)-10 when stimulated with the heavy constant region of immunoglobulins (Fc) and express high levels of the adenosine A2A receptor (A2A R), which, when activated by adenosine, inhibits the release of pro-inflammatory cytokines from most immune cells. Here we show that stimulation of the A2A R on tmDCs by regadenoson or N-ethylcarboxamidoadenosine (NECA) rapidly increases cyclic AMP accumulation and enhances IL-10 production under Fc stimulatory conditions. In co-culture experiments, tmDCs inhibit the differentiation of naïve T cells to a pro-inflammatory phenotype. In conclusion, although DCs are classically viewed as antigen presenting cells that activate T cells, we show an independent role of tmDCs in pediatric immune regulation that may be important for suppressing T cell responses to neoantigens in infants and young children.

Abstract Background Severe Clostridium difficile toxin-induced enteritis is characterized by exuberant intestinal tissue inflammation, epithelial disruption and diarrhea. Adenosine, through its action on the adenosine A2A receptor, prevents neutrophillic adhesion and oxidative burst and inhibits inflammatory cytokine production. Alanyl-glutamine enhances intestinal mucosal repair and decreases apoptosis of enterocytes. This study investigates the protection from enteritis by combination therapy with ATL 370, an adenosine A2A receptor agonist, and alanyl-glutamine in a rabbit and murine intestinal loop models of C. difficile toxin A-induced epithelial injury. Methods Toxin A with or without alanyl-glutamine was administered intraluminally to rabbit ileal or murine cecal loops. Animals were also given either PBS or ATL 370 parenterally. Ileal tissues were examined for secretion, histopathology, apoptosis, Cxcl1/KC and IL-10. Results ATL 370 decreased ileal secretion and histopathologic changes in loops treated with Toxin A. These effects were reversed by the A2A receptor antagonist, SCH 58261, in a dose-dependent manner. The combination of ATL 370 and alanyl-glutamine significantly further decreased ileal secretion, mucosal injury and apoptosis more than loops treated with either drug alone. ATL 370 and alanyl-glutamine also decreased intestinal tissue KC and IL-10. Conclusions Combination therapy with an adenosine A2A receptor agonist and alanyl-glutamine is effective in reversing C. difficile toxin A-induced epithelial injury, inflammation, secretion and apoptosis in animals and has therapeutic potential for the management of C. difficile infection.