Soluble CD93 is an Apoptotic Cell Opsonin Recognized by the α_xβ₂Integrin

Abstract

Abbreviations: ADAM -a disintegrin and metalloproteinase. BSA -bovine serum albumin. CHO -Chinese hamster ovary cells. CTLD -C-type lectin-like domain. FBS -fetal bovine serum. MMPmatrix metalloproteinase. PBMC -peripheral blood mononuclear cells. PFA -paraformaldehyde. sCD93 -soluble CD93. ABSTRACTEfferocytosis -the phagocytic removal of apoptotic cells -is essential for the maintenance of homeostasis and prevention of the inflammatory and autoimmune diseases which can follow the lysis of uncleared apoptotic cells. CD93 is a transmembrane glycoprotein previously implicated in efferocytosis and angiogenesis, and upon mutation, results in the onset of efferocytosis-associated diseases such as atherosclerosis and rheumatoid arthritis. CD93 is produced as a cell surface protein which is shed as soluble CD93, but it is unknown how CD93 mediates efferocytosis or whether its efferocytic activity is mediated by the soluble or membrane-bound form. Herein, we demonstrate that the membrane bound form of CD93 has no phagocytic, efferocytic, or tethering activity, whereas soluble CD93 potently opsonizes apoptotic cells but not a broad range of Gram-Negative, Gram-Positive or fungal microorganisms. Using mass spectrometry, we identified the αxβ2 integrin as the receptor required for soluble CD93-mediated efferocytosis, and via deletion mutagenesis determined that soluble CD93 binds to apoptotic cells via its C-Type Lectin-Like domain, and to αxβ2 by its EGF-like repeats. This bridging of apoptotic cells to the αxβ2 integrin markedly enhanced efferocytosis by macrophages, and could be abrogated by knockdown of αxβ2 integrin. Combined, these data elucidate the mechanism by which CD93 regulates efferocytosis and identify a previously unreported opsonin-receptor system utilized by the immune system for the efferocytic clearance of apoptotic cells.Clearly, there is data supporting three distinct and mutually exclusive models of CD93 efferocytic function. In this study, we directly asses these three models of CD93 function, using in vitro models of binding, efferocytosis and opsonization. These experiments disfavor the efferocytic receptor and tethering receptor models of CD93 function, and instead demonstrate a strong opsonic effect of sCD93 against apoptotic cells but not against a panel of microorganisms. Using mass-spectrometry we then identified αxβ2 integrin as the efferocytic receptor which recognizes sCD93 opsonized targets, with apoptotic cells "bridged" by the CD93 CTLD domain to αxβ2 integrin on phagocytes via the CD93 EGFlike domain. This represents a previously undescribed opsonin-receptor system which aids in the efferocytosis of apoptotic cell by phagocytes such as macrophages. MATERIALS AND METHODS MaterialsDH5a Escherichia coli was a gift from Dr. John McCormick (University of Western Ontario), Rhodotorula minuta and Candida glabrate were gifts from Dr. Andre Lachance (University of Western Ontario). Escherichia coli K29 + and K29 -, and Burkholderia cenocepacia were gifts from Dr. Susan Koval (University of Wester...

First-in-Human Study of Mivebresib (ABBV-075), an Oral Pan-Inhibitor of Bromodomain and Extra Terminal Proteins, in Patients with Relapsed/Refractory Solid Tumors

“…DCXR encodes for a protein that plays an important role in glucose metabolism (36,37). CD93 is known as a myeloid marker involved in cell adhesion and clearance of apoptotic cells (38). The mechanism of mivebresib-induced modulation of these biomarkers remains to be elucidated.…”

GATA2 Expression by Intima-Infiltrating Macrophages Drives Early Atheroma Formation

“…Our gene expression analysis identified a reduction in the expression of genes involved in the efferocytosis of apoptotic cells and the phagocytosis of microbial pathogens (Figures S4). RT-PCR confirmed that the αx integrin, a receptor for both complement-opsonized pathogens and CD93-opsonized ACs 48 , was downregulated in intima-infiltrating macrophages ( Figure 4A). Unexpectedly, integrin-linked kinase, which was downregulated in the patients analyzed in our microarray analysis ( Figure S4, Table S1) was not downregulated in the patient cohort used for our RT-PCR assays ( Figure 4B).…”

The Injured Sciatic Nerve Atlas (iSNAT), Insights into the Cellular and Molecular Basis of Neural Tissue Degeneration and Regeneration

“…CFD is a serine protease required for the formation of C3 convertase. Soluble C1qR1/CD93 functions as a bridging molecule that aids apoptotic cell binding to professional phagocytes for removal by efferocytosis (Blackburn et al, 2019). Taken together, we provide validation for many injury-regulated gene products identified by scRNA-seq and show that serum proteins that function in opsonization feature prominently in the PNS following nerve crush injury.…”

“…CFD is a serine protease required for the formation of C3 convertase. Soluble C1qR1/CD93 functions as a bridging molecule that aids apoptotic cell binding to professional phagocytes for removal by efferocytosis (Blackburn et al, 2019).…”