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Resolvin D1 promotes the targeting and clearance of necroptotic cells

Abstract

Inflammation-resolution is a protective response that is mediated by specialized pro-resolving mediators (SPMs). The clearance of dead cells or efferocytosis is a critical cellular program of inflammation-resolution. Impaired efferocytosis can lead to tissue damage in prevalent human diseases, like atherosclerosis. Therefore understanding mechanisms associated with swift clearance of dead cells is of utmost clinical importance. Recently, the accumulation of necroptotic cells (NCs) was observed in human plaques and we postulated that this is due to defective clearance programs. Here we present evidence that NCs are inefficiently taken up by macrophages because they have increased surface expression of a well-known “don’t eat me” signal called CD47. High levels of CD47 on NCs stimulated RhoA-pMLC signaling in macrophages that promoted “nibbling”, rather than whole-cell engulfment of NCs. Anti-CD47 blocking antibodies limited RhoA-p-MLC signaling and promoted whole-cell NC engulfment. Treatment with anti-CD47 blocking antibodies to Ldlr−/− mice with established atherosclerosis decreased necrotic cores, limited the accumulation of plaque NCs and increased lesional SPMs, including Resolvin D1 (RvD1) compared with IgG controls. Mechanistically, RvD1 promoted whole-cell engulfment of NCs by decreasing RhoA signaling and activating CDC42. RvD1 specifically targeted NCs for engulfment by facilitating the release of the well-known “eat me signal” called calreticulin from macrophages in a CDC42 dependent manner. Lastly, RvD1 enhanced the clearance of NCs in advanced murine plaques. Together, these results suggest new molecules and signaling associated with the clearance of NCs, provide a new paradigm for the regulation of inflammation-resolution, and offer a potential treatment strategy for diseases where NCs underpin the pathology.

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Acknowledgements

We thank the AMC imaging core facility for the use of the Zeiss LSM880 confocal microscope. IMARIS software by Bitplane Inc was purchased using funding from NSF #1725984.

Funding

This work was supported by NIH grants HL119587 (G.F.), HL141127 (G.F.), HL119047 (K.J.R), HL106173 (M.S.), GM095467 (M.S.; Core B), and BRG-CA207725 (M.B.), NIH R35 HL144475 (N.J.L.), HL110951 (D.D.T), HL113208 (D.D.T), and HL130304 (D.D.T). BES was supported by a National Research Service Award from the NIH (HL136044), and KJR by the Canadian Institutes for Health Research.

Author information

GF and KJR designed research. GF, BDG, MM, and MB conducted and analyzed in vitro imaging experiments. GF, JH, NR, SS, ZH, YK, and NJL conducted and analyzed atherosclerosis experiments. BES, COR, and MS performed LC-MS/MS analysis. GF, BDG, and MM wrote the paper and all authors including DDT reviewed/edited the manuscript.

Correspondence to Gabrielle Fredman.

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Edited by S. Nagata

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