Abstract
Aims/hypothesisInflammasome activation and subsequent IL-1β production is a driver of islet pathology in type 2 diabetes. Oligomers, but not mature amyloid fibrils, of human islet amyloid polypeptide (IAPP), which is co-secreted with insulin, trigger NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome activation. C4b-binding protein (C4BP), present in serum, binds to IAPP and affects transition of IAPP monomers and oligomers to amyloid fibrils. We therefore hypothesised that C4BP inhibits IAPP-mediated inflammasome activation and IL-1β production.MethodsMacrophages were exposed to IAPP in the presence or absence of plasma-purified human C4BP, and inflammasome activation was assessed by IL-1β secretion as detected by ELISA and reporter cell lines. IAPP fibrillation was assessed by thioflavin T assay. Uptake of IAPP–C4BP complexes and their effects on phagolysosomal stability were assessed by flow cytometry and confocal microscopy. The effect of C4BP regulation of IAPP-mediated inflammasome activation on beta cell function was assessed using a clonal rat beta cell line. Immunohistochemistry was used to examine the association of IAPP amyloid deposits and macrophage infiltration in isolated human and mouse pancreatic islets, and expression of C4BP from isolated human pancreatic islets was assessed by quantitative PCR, immunohistochemistry and western blot.ResultsC4BP significantly inhibited IAPP-mediated IL-1β secretion from primed macrophages at physiological concentrations in a dose-dependent manner. C4BP bound to and was internalised together with IAPP. C4BP did not affect IAPP uptake into phagolysosomal compartments, although it did inhibit its formation into amyloid fibrils. The loss of macrophage phagolysosomal integrity induced by IAPP incubation was inhibited by co-incubation with C4BP. Supernatant fractions from macrophages activated with IAPP inhibited both insulin secretion and viability of clonal beta cells in an IL-1β-dependent manner but the presence of C4BP during macrophage IAPP incubation rescued beta cell function and viability. In human and mouse islets, the presence of amyloid deposits correlated with higher numbers of infiltrating macrophages. Isolated human islets expressed and secreted C4BP, which increased with addition of IL-1β.Conclusions/interpretationIAPP deposition is associated with inflammatory cell infiltrates in pancreatic islets. C4BP blocks IAPP-induced inflammasome activation by preventing the loss of macrophage phagolysosomal integrity required for NLRP3 activation. The consequence of this is the preservation of beta cell function and viability. C4BP is secreted directly from human pancreatic islets and this increases in response to inflammatory cytokines. We therefore propose that C4BP acts as an extracellular chaperone protein that limits the proinflammatory effects of IAPP.
Highlights
Type 2 diabetes has an important inflammatory component [1] that drives insulin resistance in peripheral tissues and contributes to pancreatic islet dysfunction
Islet amyloid polypeptide (IAPP)–C4b-binding protein (C4BP) uptake assays monocyte-derived macrophages (MDMs) or THP1 cells were incubated with labelled IAPP or C4BP, harvested and uptake assessed by flow cytometry
C4BP inhibits IAPP-mediated IL-1β production Addition of IAPP to primed THP1 cells resulted in the processing of 35 kDa pro-IL-1β and secretion of 17 kDa mature IL-1β, detected by western blot (Fig. 1a)
Summary
Proteins Plasma C4BP and recombinant monomeric C4BP α-chain were purified as described [14, 15]. Electron microscopy During ThT assays, samples (1–10 μl) were removed and placed on formvar-coated Cu grids. IAPP–C4BP uptake assays MDMs or THP1 cells were incubated with labelled IAPP or C4BP, harvested and uptake assessed by flow cytometry. Viability and insulin secretion INS-1 cells were incubated with conditioned supernatant fractions from activated or unactivated THP1 cells or with medium alone and cell death was measured by flow cytometry. Islet staining Pancreas sections from human donors and human IAPP (hIAPP) transgenic mice were stained for macrophages using CD68/SR-D1 antibody (Novus Biologicals, Littleton, CO, USA), or using guinea pig anti-insulin (DAKO) mouse anti-C4BP, sheep anti-pancreatic polypeptide (Bio-Rad) or Congo Red. See ESM Methods for further details. All data show means ± SD of three independent repeats, unless otherwise noted
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