Thrombospondin-1, CD47, and SIRPα display cell-specific molecular signatures in human islets and pancreata

Abstract

Background: Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα) and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Methods: Fresh islets and pancreatic tissue from non-diabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression determined. Results: CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, while SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by beta cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Conclusions: Human pancreatic exocrine and endocrine tissue expressed CD47 whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.

Regulation of STAT1 Signaling in Human Pancreatic β-Cells by the Lysine Deacetylase HDAC6: A New Therapeutic Opportunity in Type 1 Diabetes?

“…In previous work, we provided firm evidence that SIRPα is increased in expression when human β-cells are exposed to IL-13 (22). However, it has been argued that SIRPα is not an abundant constituent of the human β-cell proteome under control conditions since a recent study failed to detect it by Western blotting or immunohistochemistry in human islet cells (32).…”

“…Gene expression array analysis indicated a large-scale proportional upregulation of SIRPα in response to IL-13 in β-cells but the apparent extent of this increase is magnified because of the low basal expression of the gene. Given that gene expression profiles often provide only a poorly accurate estimate of protein levels (and noting that a recent study (32)), we considered it imperative to verify its expression in human pancreatic islets at the protein level. This goal was achieved using two independent antisera which allowed the ready detection of SIRPα in islet endocrine cells of the human pancreas using immunohistochemical approaches.…”

“…To address this discrepancy, we re-examined SIRPα expression in human pancreas sections from control subjects using two different antisera, including that employed in the recent negative report (32). Immunohistochemical staining revealed a strongly positive signal in islets immunostained with either of two different anti-SIRPα reagents (obtained from Abcam and Origene respectively (Figure 1A)).…”

Insulitis in human type 1 diabetes: lessons from an enigmatic lesion

“…In addition, one of the β-cell responses to cytokine (notably interferon-α) exposure is induction of PDL-1, 72 an immune checkpoint protein which can bind to its ligand, PD-1, displayed on CD8+ T-cells to initiate T-cell death. Furthermore, β-cells also express a second immune checkpoint protein, CD47, 73 , 74 which can both deflect attacks by incoming macrophages and also actively promote their own (or neighbouring β-cell) viability via interaction with signal regulatory protein-α (SIRPα). There has been debate about whether β-cells display SIRPα under normal conditions 73 , 74 but, despite the apparently limited mRNA expression, functional and histological evidence suggest that SIRPα is present at the protein level (and is induced by cytoprotective agents such as interleukin-13 75 ) such that it may also be involved in repelling autoimmunity.…”

Tolerating CD47

“…The role of CD47 in the disglycaemia was presumed but not shown. As fresh human islets from individuals without diabetes lacked SIRPα 53 (Figure 2B), the relevance of the finding is unclear. Further miring things, human‐derived beta‐like EndoC‐βH1 cells, generated by multiple viral transfections, were employed to assess SIRPα 54 .…”