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Toll-like receptor agonists, poly(I:C) and flagellin, lead to IL-36γ induction with divergent release kinetics and differentially alter autophagy in primary human keratinocytes Volume 33, issue 2, 2022-06-01

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Authors
1 Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA
2 The Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, New York, USA
3 Department of Pediatrics, Steven and Alexandra Cohen Children’s Medical Center of New York, Barbara and Donald Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA
4 Department of Otolaryngology, Long Island Jewish Medical Center, Barbara and Donald Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA
5 Department of Molecular Medicine, Barbara and Donald Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY
Correspondence: B. Steinberg
* These authors contributed equally to this work as co-first authors. Current address: The Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, New York, USA

IL-36γ, a pro-inflammatory member of the IL-1 cytokine superfamily, can be induced and secreted by normal human foreskin keratinocytes (HFKs) in response to pathogenic stimuli, however, the mechanisms underlying the secretion are unknown. In this study, we demonstrate that stimulation with the TLR3 agonist, poly (I:C), led to a delayed secretion of IL-36γ compared to stimulation with the TLR5 agonist, flagellin, despite equal levels of the cytokine (p = 0.006). IL-36γ was shown to be released from HFKs in its inactive, uncleaved form, based on western blotting. Moreover, recombinant IL-36γ in its activated, cleaved form induced endogenous IL-36γ 10-fold (p = 0.004) and CXCL8 five-fold (p = 0.003) over baseline levels compared to unactivated full-length recombinant IL-36γ. The ratio of LC3b-II/LC3b-I was significantly higher in poly(I:C)-treated cells compared to flagellin-treated and unstimulated controls without a change in SQSTM1/p62 after 24 hours of stimulation (p = 0.043). Under fluorescence microscopy, poly(I:C) led to a two-fold increase at eight hours and four-fold increase at 24 hours in accumulated autophagosomes post-stimulation (p = 0.032). In contrast, autophagosomes were unchanged relative to baseline in response to flagellin. Bafilomycin A1 treatment enhanced poly(I:C)-mediated IL-36γ secretion (p = 0.044) while rapamycin led to a noticeable, but non-significant, increase in flagellin-mediated IL-36γ secretion, indicating that interrupting autophagic flux can alter IL-3γ grelease from HFKs. Finally, we show that, compared to clinically normal laryngeal tissue, there were significantly higher levels of LC3b-II in HPV-infected respiratory papilloma tissue, indicating a higher number of autophagosomes; a signature of disrupted autophagic flux.