Differential Activation of Innate Immune Pathways by Distinct Islet Amyloid Polypeptide (IAPP) Aggregates

Abstract

Aggregation of islet amyloid polypeptide (IAPP) contributes to beta cell dysfunction in type 2 diabetes and islet transplantation. Like other amyloidogenic peptides, human IAPP induces macrophage IL-1␤ secretion by stimulating both the synthesis and processing of proIL-1␤, a pro-inflammatory cytokine that (when chronically elevated) impairs beta cell insulin secretion. We sought to determine the specific mechanism of IAPP-induced proIL-1␤ synthesis. Soluble IAPP species produced early during IAPP aggregation provided a Toll-like-receptor-2-(TLR2-) dependent stimulus for NF-B activation in HEK 293 cells and bone marrow-derived macrophages (BMDMs). Nonamyloidogenic rodent IAPP and thioflavin-T-positive fibrillar amyloid produced by human IAPP aggregation failed to activate TLR2. Blockade of TLR6 but not TLR1 prevented hIAPP-induced TLR2 activation, consistent with stimulation of a TLR2/6 heterodimer. TLR2 and its downstream adaptor protein MyD88 were required for IAPP-induced cytokine production by BMDMs, a process that is partially dependent on autoinduction by IL-1. BMDMs treated with soluble but not fibrillar IAPP provided a TLR2-dependent priming stimulus for ATP-induced IL-1␤ secretion, whereas late IAPP aggregates induced NLRP3-dependent IL-1␤ secretion by LPS-primed macrophages. Moreover, inhibition of TLR2 and depletion of islet macrophages prevented up-regulation of Il1b and Tnf expression in human IAPP-expressing transgenic mouse islets. These data suggest participation by both soluble and fibrillar aggregates in IAPPinduced islet inflammation. IAPP-induced activation of TLR2 and secretion of IL-1 may be important therapeutic targets to prevent amyloid-associated beta cell dysfunction.

IAPP and type 1 diabetes: implications for immunity, metabolism and islet transplants

“…After 6 h of incubation, IAPP immunoreactivity can be found loosely packed in phagosome-like organelles, and from 24 to 72 h, IAPP-reactive fibrils accumulate in dense lysosome-like organelles. This timeline is consistent with that of IL1B release in hIAPP-treated cells: secretion of IL1B in unprimed or LPS-primed macrophages appears after approximately 6 h of incubation and continues thereafter (Masters et al 2010, Sheedy et al 2013, Westwell-Roper et al 2016. The requirement for longer incubation for maximal IL1B secretion indicates that the process of NLRP3 activation by hIAPP aggregates is different from that of ligands like ATP, which activate the inflammasome more rapidly.…”

“…Deletion of either Nlrp3 or Casp1 abrogates hIAPP-induced IL1B secretion in cells, regardless of whether they are first primed with LPS or hIAPP aggregates (Masters et al 2010, Sheedy et al 2013, Westwell-Roper et al 2016. Furthermore, pharmacological inhibition of NLRP3 with glyburide inhibited hIAPP-induced IL1B secretion (Masters et al 2010, Westwell-Roper et al 2011.…”

“…Though hIAPP can act as a priming stimulus, it is notably less potent than LPS; LPS priming results in a 10-fold greater induction of TNF relative to hIAPP over 24 h (Westwell-Roper et al 2011). Moreover, LPS-primed cells subsequently treated with hIAPP secrete approximately 10-fold greater levels of IL1B than cells treated with hIAPP only (Westwell-Roper et al 2016); this may explain why others have not observed robust IL1B secretion from cells stimulated with hIAPP alone compared to LPS-primed cells treated with hIAPP (Masters et al 2010, Sheedy et al 2013. The relative contributions of hIAPP-induced NFKB1 and NLRP3 activation in islet inflammation and ensuing beta cell dysfunction in vivo remain to be tested, though IL1 clearly plays a critical role, as IL1 antagonism ameliorates inflammation, and beta cell dysfunction and death, in hIAPP transgenic mice and isolated human islets (Westwell-Roper et al 2011, Hui et al 2017, Jin et al 2017.…”

“…Early, pre-fibrillar hIAPP species are the most pro-inflammatory (Masters et al 2010, Westwell-Roper et al 2011 and most toxic to INS1 cells (Abedini et al 2016). It should be noted that fibrillar hIAPP species can, however, activate the inflammasome in LPS-primed macrophages, though to a lesser extent than freshly dissolved hIAPP (Westwell-Roper et al 2016). Furthermore, through size fractionation, Masters and colleagues identified that smaller pre-fibrillar hIAPP aggregate species of <100 nm induced the highest level of IL1B secretion from BMDCs after overnight incubation (Masters et al 2010).…”

“…We have reported that activation of NFKB1 and priming by hIAPP is dependent on toll-like receptor (TLR) 2 or a TLR 2/6 heterodimer and myeloid differentiation primary response 88 (MYD88)-dependent signalling hIAPP induction of NFKB1 activity and pro-inflammatory cytokines is abrogated in Tlr2 −/− and Myd88 −/− BMDMs, and in a human TLR2-expressing reporter cell line treated with TLR2-or TLR6-neutralizing antibodies (WestwellRoper et al 2016). Furthermore, Tlr2 knockout islets express less Il1b in response to hIAPP application, and immunoneutralization of TLR2 reduces Il1b expression in islets from hIAPP transgenic rodents (Westwell-Roper et al 2016). Though hIAPP can act as a priming stimulus, it is notably less potent than LPS; LPS priming results in a 10-fold greater induction of TNF relative to hIAPP over 24 h (Westwell-Roper et al 2011).…”