NF-κB Is Activated in CD4+ iNKT Cells by Sickle Cell Disease and Mediates Rapid Induction of Adenosine A2A Receptors

Abstract

Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11–7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.

A key role of the mitochondrial citrate carrier (SLC25A1) in TNFα- and IFNγ-triggered inflammation

“…Several approaches were employed to test this possibility. First, we used a well-known inhibitor of the IκB kinase, whose activity is required for NFkB function, specifically IKK inhibitor VII [31]. We found that the addition of inhibitor VII to U937/PMA cells co-treated with TNFα led to a reduction of both the SLC25A1 mRNA and protein levels compared to treatment with TNFα alone (Fig.…”

Adenosine A2A Receptors Intrinsically Regulate CD8+ T Cells in the Tumor Microenvironment

“…The A 2A R is upregulated in multiple immune cell types upon activation (5, 32, 33). Blockade or deletion of A 2A Rs in NK cells (2) or myeloid cells (Cekic et al unpublished data) significantly inhibits tumor growth and metastasis and is associated with transactivation of cytotoxic lymphocytes.…”

Simultaneous assessment of NF-κB/p65 phosphorylation and nuclear localization using imaging flow cytometry

“…The functional significance of the different phosphorylation sites with regards to NF-kB transcriptional activity differ. The use of phospho-specific antibodies to detect expression levels of the various phosphorylated p65 motifs by flow cytometry has been used as a parameter of response to assess the activity of the NF-κB pathway in relation to disease states or drug intervention (Hernandez Mde et al, 2011; Lin et al, 2013; Simard et al, 2014; Vafadari et al, 2013). Alternatively, since activation of NF-κB is associated with relocation from the cytoplasm to nucleus, the nuclear localization of NF-κB has also been used as a parameter for activation.…”