Session Type: Abstract Submissions (ACR)
Reverse signalling seems to play a role in the mode of action of anti-TNF-α agents but this pathway and its relevance are poorly understood. CD36 is a scavenger receptor implicated in the phagocytosis of senescent cells at the end of the inflammatory process. We previously demonstrated that TNF-α inhibitors increase CD36 expression in human monocytes and our results suggest a reverse signalling pathway. Casein kinase 1 phosphorylation of serine localized in the intra-cytoplasmic tail of membrane TNF-α (tmTNF) has been reported to play a role in reverse signalling. Our aim was to clarify the pathway engaged in reverse signalling induced by certolizumab-pegol and its relevance on TNF receptor (TNFR) and TLR4 signalling.
Human monocytes were isolated from peripheral blood mononuclear cells of healthy donors by plastic adherence or negative selection. The consequence of dephosphorylation of tmTNF in CD36 induction was assessed using a specific casein-kinase 1 inhibitor D4476. We searched for a putative phosphatase using cyclosporine A, a specific inhibitor of calcineurin phosphatase. An inhibitor of the endopeptidase SPPL2b, ZLL-2 ketone, was used to asses intra-membrane proteolysis of tm-TNF. CD36 and expression was analyzed by RT-qPCR and flow cytometry. IL-1 expression was induced by LPS and analysed by RT-PCR and western blot. Experiments were done at least 3 times in duplicate. Data were analyzed by Wilcoxon test matched-pairs signed-rank test and Student’s paired t-test.
Certolizumab-pegol and D4476 used separately respectively increased the expression of CD36. The association of both compounds potentiated this increase (p=0.02). This suggested the role of casein kinase I in the down-regulation tmTNF. The calcineurin inhibitor cyclosporine strongly inhibited the induction of CD36 observed with certolizumab-pegol (p=0.02), However, the inhibitor of intra-membrane clivage of tmTNF, ZLL-2, did not modify the induction of CD36 induced by certolizumab-pegol (p=0.2). Moreover a transfection of TNF-α linked with GFP in its intracytoplasmic tail failed to show any nuclear translocation in presence of certolizumab-pegol. These data demonstrated the absence of intramembrane proteolysis in reverse signalling induced by anti-TNF-α. IL-1 expression induced by LPS was significantly decreased by certolizumab-pegol, and this effect was inhibited by the calcineurin inhibitor cyclosporine A. Furthermore, TNFR2 did not play a role in the reverse signalling induced by tmTNF.
Certolizumab-pegol induced reverse signalling in human monocytes through tm-TNF. This resulted in increased CD36 expression, down-modulation of IL-1 production induced byTLR4, but had no effect on TNFR2 signalling. Dephosphorylation of tmTNF as well as calcineurin activation were involved in certolizumab-pegol-induced reverse signalling. Taken together, these results suggest a role of reverse signalling in the regulation of inflammation by anti-TNF-α.
J. F. Boyer,
A. L. Constantin,
A. G. Cantagrel,
J. L. Davignon,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/certolizumab-pegol-induces-reverse-signaling-through-membrane-tnf-via-calcineurin-activation-in-human-monocytes-relevance-in-inflammation/