NF-κB Activation Mediates Induction Of Anti-Inflammatory Adenosine A2A Receptors In iNKT Cells Of Sickle Cell Patients During Vaso-Occlusive Episodes and Upon Activation Of Cultured Human iNKT Cells

Abstract

iNKT cells are activated in mice and people with vaso-occlusive sickle cell disease (SCD) and play a pivotal role in initiating an inflammatory cascade. In mice, iNKT cell activation requires NF-κB signaling [Stanic, AK et al. J Immunol. 172:4667, 2004]. In mice with SCD, lung inflammation is associated with induction in iNKT cells of IFN-γ and anti-inflammatory adenosine A2A receptor (A2AR) mRNAs [Wallace, KL et al. Blood 116:5010, 2010]. The A2AR is one of four G protein coupled adenosine receptors (A1, A2A, A2B and A3) and it is the most abundant adenosine receptor expressed in T cells. In patients with SCD, painful vaso-occlusive crisis (pVOC) is associated with NF-κB activation and increased expression of A2AR immunoreactivity detected with the monoclonal Ab, 7F6-G5-A2, in circulating iNKT cells. Infusion of a moderate dose (6 nM) of the A2AR agonist, regadenoson during pVOC reduced iNKT cell activation over time [Field, J. et al. Blood 121:3329, 2013]. Since A2AR activation inhibits iNKT cell activation, we reasoned that iNKT cell activation during pVOC might occur preferentially in iNKT cells with low A2AR expression. However, in the current study we show that activated human Vα24+ CD4+ iNKT cells concordantly (i.e. on the same cells) expressed Ser-536 phosphorylated/activated NF-κB (p-NFκB), cytokines and A2ARs. These findings suggest that NF-κB signaling increases A2AR expression as a counter-regulatory mechanism to render activated iNKT cells highly sensitive to inhibition by A2AR activation. Spare receptor theory predicts that enhanced receptor expression will increase the functional potency of agonists. To test this hypothesis we infused into stable SCD patients at steady state a low dose of regadenoson (2 nM) well below its receptor binding KD that was measured by competition for radioligand binding to be 12 nM. Low-dose regadenoson infusion reduced the percentage of p-NF-κB-high/activated iNKT cells by an average of 69% over 12 hours (P = 0.005). These findings suggest that A2AR induction is a counter-regulatory mechanism that limits iNKT cell activation by increasing the sensitivity of activated iNKT cells to inhibition by adenosine. In order to further test the hypothesis that NF-κB activation stimulates A2AR transcription, we examined cultured human iNKT cells. Blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of cytokine and A2AR mRNA and protein upon iNKT activation by plate-bound anti-CD3 antibodies. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells serves as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. We conclude that A2AR agonists have therapeutic potential for treating inflammatory diseases associated with iNKT cell activation, including SCD. Disclosures:Off Label Use: Ragadenoson is used to inhibit activation of iNKT cells.