Background/Purpose:

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.

Methods:

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.

Results:

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.

Conclusion:

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.

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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/