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

Title
IL-32 isoforms differentially impact coronary artery endothelium functions and potential to recruit inflammatory cells
Presenter
Rémi Bunet
Authors
R. Bunet * (1,2), H. Ramani (1,2), M. Durand (3,2), C. Chartrand-Lefebvre (4,2), J.-P. Routy (5), T. Rejean (6), B. Trottier (7), P. Ancuta (1,2), A. Landay (8), M. El Far (2), C. Tremblay (1,2)
Institutions
(1) Université de Montréal, Département de Microbiologie, Infectiologie et Immunologie, Montréal, Canada, (2) Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montréal, Canada, (3) Université de Montréal, Département de Médecine, Montréal, Canada, (4) Université de Montréal, Département de Radiologie, Radio-Oncologie et Médecine Nucléaire, Montréal, Canada, (5) Research Institute of McGill University Health Centre, Montréal, Canada, (6) Clinique Médicale l''Actuel, Montréal, Canada, (7) Centre de Médecine Urbaine du Quartier Latin, Montréal, Canada, (8) Rush University Medical Center, Department of Internal Medicine, Chicago, United States

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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