IL-32 isoforms differentially impact coronary artery endothelium functions and potential to recruit inflammatory cells


BACKGROUND: HIV-induced inflammation leads to the premature development of cardiovascular diseases (CVD). We have previously shown that interleukin-32 (IL-32), a proinflammatory cytokine that is expressed in multiple isoforms (a, β, γ, D, ε, θ, ζ, η, and small/sm) with different inflammatory potential, is chronically upregulated in HIV-1 infection, even under ART and is associated with CVD. However, the mechanistic role of these different IL-32 isoforms in CVD is yet to be identified. In this study, we aimed to investigate the potential impact of the different IL-32 isoforms on coronary artery endothelial cells (CAEC), the dysfunction of which is a major driver for atherosclerotic plaque formation.
METHODS: Recombinant IL-32 isoforms (a, β and γ; the only commercially available isoforms) were used at 500ng/ml to stimulate primary CAEC (pCAEC/Creative Bioarray). pCAEC dysfunction in response to IL-32 stimulation was measured by studying the upregulation of VCAM-1 and ICAM-1 using flow cytometry, expression of the chemokines CCL2, CXCL1 and CXCL8 using both qRT-PCR (for gene expression) and ELISA for protein expression in supernatant as well as chemoattraction abilities by transwell assays.
RESULTS: IL-32 isoforms showed differential effects on pCAEC. While IL-32β and to a lesser extent IL-32γ, upregulated the expression of ICAM-1 and VCAM-1 (p=0.0347 and p=0.0019, respectively) in pCEAC, the impact of IL-32a was not significant. Furthermore, both IL-32β and IL-32γ significantly upregulated the expression of the chemoattractants CCL2, CXCL1 and CXCL8 at the protein level (CCL2: p=0.002 and p=0.002, CXCL1: p=0.002 and p=0.002, and CXCL8: p=0.002 and p=0.0039, respectively). In contrast, IL-32a significantly downregulated CCL2 (p=0.0068) and CXCL1 (p=0.0020). In line with these results, classical monocytes isolated with negative selection (StemCell) from Peripheral blood mononuclear cells (PBMCs) showed significantly higher migration towards pCEAC cells stimulated with IL-32β and IL-32γ, but not IL-32a in transwell assays (p=0.0004, p=0.0008 and p=0.356, respectively).
CONCLUSIONS: Our results suggest that IL-32β and IL-32γ isoforms induce coronary artery endothelial cell dysfunction and enhance their potential to recruit inflammatory cells such as monocytes. These IL-32 isoforms are upregulated in HIV infection and likely contributing to endothelial cell inflammation and CVD and may represent a therapeutic target.

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