Autoimmune inner ear disease patient–associated 28-kDa proinflammatory IL-1β fragment results from caspase-7–mediated cleavage in vitro
Shresh Pathak, Andrea Vambutas
Shresh Pathak, Andrea Vambutas
Published February 13, 2020
Citation Information: JCI Insight. 2020;5(3):e130845. https://doi.org/10.1172/jci.insight.130845.
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Research Article Otology

Autoimmune inner ear disease patient–associated 28-kDa proinflammatory IL-1β fragment results from caspase-7–mediated cleavage in vitro

Abstract

Interleukin-1β (IL-1β) is a key proinflammatory cytokine involved in the progression of many autoinflammatory and autoimmune diseases, including autoimmune inner ear disease (AIED). IL-1β inhibition has been shown to result in clinical hearing improvement in a small cohort of corticosteroid-resistant patients with AIED. Canonical processing of pro–IL-1β by caspase-1 generates an active 17-kDa fragment, capable of instigating a proinflammatory microenvironment. However, in response to LPS, PBMCs from patients with AIED uniquely express a 28-kDa IL-1β fragment, as compared with PBMCs from control subjects. We synthesized and compared the biologic activity of the 28-kDa fragment to the 17-kDa IL-1β product and the pro–IL-1 31-kDa protein. The 28-kDa IL-1β fragment induces IL-6, TNF-α, and CCL3 in PBMCs. Uniquely, only caspase-7 treatment showed a dose- and time-dependent increase in 28-kDa band generation. Mass spectrometry confirmed the putative caspase-7 cleavage site of pro–IL-1β, which was used to generate the 28-kDa fragment used for PBMC stimulation studies. Collectively, these results provide insight into the function of a poorly understood, processed 28-kDa form of IL-1β in patients with AIED that is uniquely generated by caspase-7 and is capable of activating further downstream proinflammatory cytokines. Further investigation may provide novel pharmacologic targets for the treatment of this rare disease.

Authors

Shresh Pathak, Andrea Vambutas

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Figure 1

PBMCs from patients with AIED uniquely express a 28-kDa fragment in response to LPS.

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PBMCs from patients with AIED uniquely express a 28-kDa fragment in resp...
(A) Western blot analysis of PBMCs of several representative patients with AIED (n = 5 out of 30 patients with AIED with 28-kDa band observed) and control subjects (n = 4 out of 24 control subjects studied). PBMCs were treated with 1 μg/mL LPS, and processing of IL-1β was determined by Western blot using anti–IL-1β antibody (it identifies a proform doublet at 33/31 kDa and bands at 28 kDa and 17 kDa, R&D Systems). These representative samples demonstrate generation of a 28-kDa IL-1β band in LPS-stimulated PBMCs from patients with AIED. Generation of the 28-kDa IL-1β is not the result of variability in the amount of detectable caspase-7 (34 kDa) or pro–caspase-1 (a doublet of 50/48 kDa). The majority of samples were analyzed twice; however, in several instances the AIED patient samples were exhausted (>66% run in duplicate). (B) Relative quantification of the 28-kDa band, as normalized to actin in both groups, by densitometry analysis software ImageJ (NIH) revealed statistically significant differences analyzed by Mann-Whitney U test (P = 0.0004) between patients with AIED (n = 30) and control subjects (n = 24).