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

Shresh Pathak,Andrea Vambutas

doi:10.1172/jci.insight.130845

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.

Highlights

Autoimmune inner ear disease (AIED) is a rare, yet-to-be-classified orphan disease [1] characterized by periods of acute hearing decline triggered by unknown stimuli [2]
We have identified that the 28-kDa fragment of IL-1β is expressed in PBMCs from patients with AIED in response to LPS, and this fragment is capable of inducing other downstream proinflammatory cytokines
When cultured PBMCs were stimulated with LPS and protein fragments separated by molecular weight using Western blot analysis, we observed that the 28-kDa fragment generated during LPS-induced IL-1β processing is unique to patients with AIED (n = 30) when compared with normal healthy subjects (n = 24), because normal healthy subjects did not express the band or had minimal expression of this band (Figure 1A)

Summary

Introduction

Autoimmune inner ear disease (AIED) is a rare, yet-to-be-classified orphan disease [1] characterized by periods of acute hearing decline triggered by unknown stimuli [2]. Treatment consists of corticosteroids, which are initially effective in 70% of patients; the responsiveness dramatically declines over time (14% remain responsive at 3 years) [3]. For those patients who do not respond to corticosteroids, alternative immunosuppressive agents have not been efficacious [4, 5]. We initially studied human patients with AIED undergoing cochlear implantation by evaluating their PBMCs stimulated with autologous cochlear perilymph to gain insight as to the potential inflammatory pathways in this disease.

Methods

Results

Conclusion