Introduction
Type 2 diabetes has an important inflammatory component [
1] that drives insulin resistance in peripheral tissues and contributes to pancreatic islet dysfunction. The cytokine IL-1β plays an important role, and IL-1β-targeted therapy has shown promise in animal models [
2] and clinical trials [
3,
4]. IL-1β is produced by the inflammasome, where NOD-like receptor activation leads to formation of intracellular aggregates of apoptosis-associated speck-like protein containing a CARD domain (ASC) and pro-caspase 1, leading to caspase 1 activation and cleavage of pro-IL-1β to the mature active cytokine. Islet amyloid polypeptide (IAPP), which is co-secreted with insulin [
5], forms oligomers that induce inflammasome activation [
6]. In type 2 diabetes, IAPP and insulin secretion is increased to compensate for decreased insulin sensitivity in peripheral tissues, leading to increased local IAPP concentrations and formation of islet amyloid deposits. Over 90% of individuals with type 2 diabetes have detectable deposits at autopsy, and amyloid deposition correlates with beta cell loss [
7]. Only low-molecular-weight IAPP species, not mature amyloid fibrils, cause inflammasome activation [
6] or disrupt lipid bilayers [
8,
9]. Destabilisation of the phagolysosomal membrane is the proposed mechanism by which IAPP and other particulates activate the inflammasome [
6,
10].
Previously, we found that the serum protein C4b binding protein (C4BP) binds to human IAPP and co-localises to amyloid deposits in the pancreatic islets of individuals with type 2 diabetes [
11]. Therefore, we decided to test the hypothesis that C4BP affects IAPP-induced inflammasome activation. Inflammasome activation by endogenous protein aggregates or amyloids is involved in many important diseases [
12,
13] and identification of novel modes of inflammasome inhibition is therefore an important research goal.
Discussion
We have found that human C4BP is secreted from isolated human pancreatic islets and inhibits IAPP-mediated inflammasome activation and secretion of the diabetogenic cytokine, IL-1β. C4BP is a 500 kDa multimer, with seven identical α-chains and one β-chain, forming an ‘octopus’-like structure. C4BP is unusual among complement proteins in that there is no reported human deficiency [
23], suggesting an important conserved role outside traditional complement regulation. By binding to apoptotic cells and regulating complement activation, C4BP is involved in the non-inflammatory clearance of cellular debris [
24]. Here, we describe a novel mechanism whereby C4BP also maintains ‘silent’ clearance of endogenous material. Previously, we identified IAPP binding sites on C4BP α-chain domains 2 and 8 [
11]. C4BP allowed increased IAPP uptake by macrophages but did not alter localisation of IAPP to phagolysosomes. However, C4BP did prevent phagolysosomal membrane destabilisation, a recognised mechanism of particulate-mediated inflammasome activation [
10,
19]. Current models indicate that active nucleation of amyloid is required for IAPP-mediated membrane disruption, while mature fibrils do not cause loss of barrier function [
8,
9,
25], consistent with our findings that mature fibrils caused limited toxicity and no inflammasome activation. This explains how the observed C4BP-mediated inhibition of fibrillation limits the proinflammatory potential of IAPP. However, we cannot rule out that C4BP also ‘coats’ IAPP aggregates and limits their membrane interactions, as C4BP also limited inflammasome activation by MSU and SiO
2 crystals. Using IAPP from a different supplier under differing buffer conditions, we previously described that C4BP was able to enhance fibril formation, but we now show convincing evidence that C4BP inhibits IAPP fibrillation when tested in cell culture conditions. Preliminary results using recombinantly expressed IAPP were consistent with this action of C4BP. Further studies are required to elucidate in detail the effects of C4BP on the kinetics of IAPP fibril formation under various experimental conditions.
The role of IL-1β in type 2 diabetes was demonstrated by clinical trials using the recombinant IL-1 receptor antagonist anakinra: it improved glycaemic control in individuals with type 2 diabetes [
4] and improved inflammation and beta cell function for months after therapy cessation [
3]. In animal models, targeting IL-1β decreased islet inflammation and improved glycaemic control [
2,
26]. Islet macrophage infiltration and expression of inflammasome components and pro-IL-1β are increased in type 2 diabetes [
1]. Beta cells express extremely high levels of the IL-1 receptor [
27] and we and others show that IL-1β leads to beta cell dysfunction [
28‐
32]. The expression of C4BP within the human islet, the site of IAPP and IL-1β production, is therefore of interest as a local mechanism of inflammasome regulation. C4BP α-chain expression is highest in liver, the main source of circulating serum C4BP, but was also high in isolated islets. In comparison, CD59, a ubiquitously expressed cell surface complement inhibitor, was comparably expressed in all tested tissues/cells, although highest in pancreatic islets, where it is required for insulin secretion [
33]. IL-1β increases C4BP secretion from isolated islets, and serum C4BP levels are also increased in diabetic individuals [
34,
35]. Locally produced C4BP could therefore act in islet homeostasis, protecting beta cells against the deleterious effects of IAPP. Accordingly, C4BP co-localises with IAPP amyloid in vivo in pancreatic islets from humans with type 2 diabetes [
11]. Further work with
C4bp-knockout mice will be needed to fully determine how C4BP affects IAPP-mediated inflammation and amyloid deposition in pancreatic islets in situ.
C4BP also interacts with other amyloidogenic proteins such as Aβ peptide [
36] and neocortical plaques in individuals with Alzheimer’s disease [
37]. We have also described binding of C4BP to the truncated form of thioredoxin, Trx80 [
38], which forms aggregate deposits in human brains [
39] and have shown that C4BP binds to Aβ peptide [
40], prion β-oligomers and fibrils [
41]. In Alzheimer’s disease, C4BP binds to amyloid deposits and local C4BP expression is also upregulated [
40]. The implications for protective effects of C4BP in amyloid-mediated diseases are clear, especially as inflammasome activation is proposed as a common disease mechanism in many amyloid- or protein aggregate-induced diseases [
12,
13]. We propose that C4BP could function as an extracellular chaperone protein for neutralisation of amyloidogenic proteins in human diseases.
Acknowledgements
The authors thank E. Nilsson, Lund University, for preparing cryosections, the Human Tissue Lab/Nordic Islet Network for providing human tissue samples and S. Linse, Lund University, for advice.
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