Induction and secretion of IL-36γ in HFKs in response to TLR agonists. HFKs were stimulated with poly(I:C) (1,500 ng/mL) or flagellin (1;000 ng/mL) over the course of three days or left unstimulated as control, after which cell lysates were subject to western blotting.
A) Representative western blot showing full induction of IL-36γ by 24 hours and maintenance of intracellular levels up to 72 hours in response to both TLR agonists. B) Quantification of IL-36γ induction showing a nearly four-fold increase in IL-36γ in response to either agonist by 24 hours (bars represent mean ± SD based on three experiments). C) Time-dependent secretion of IL-36γ in HFKs stimulated with 1,500 ng/mL poly(I:C) measured by ELISA showing no elevation in IL-36γ by 48 hours post-stimulation. D) Time-dependent secretion of IL-36γ in HFKs stimulated with 1,000 ng/mL flagellin measured by ELISA showing a nearly three-fold increase in IL-36γ by 48 hours post-stimulation (bars represent mean ± SD based on three experiments). Measurement of IL-36γ in response to poly(I:C) stimulation at 24 hours was not performed as previous studies have shown baseline levels at 48 hours. ** p<0.01; two-tailed Student’s T-test. n.s.; not significant.
IL-36γ in HFKs is associated with intracellular membranes and is secreted as a full-length, unprocessed form.
A) HFKs were stimulated for 48 hours with poly(I:C) (1,500 ng/mL) or flagellin (1,000 ng/mL) followed by homogenization and ultra-centrifugation to separate membranes from the cytosol. The representative western blot shows effective separation based on LAMP1 and LC3b-II signal in the membrane fraction. The majority of IL-36γ resides in the cytosol while a fraction pellets with internal membranes, suggesting an association with intracellular vesicles prior to secretion (data are representative of three experiments). B) HFKs were stimulated for 96 hours with poly(I:C) (1,500 ng/mL) or flagellin (1,000 ng/mL), conditioned media was collected, and total protein precipitated. Precipitated protein was subjected to western blotting in parallel with recombinant full-length (amino acids: 1-169) and cleaved (amino acids: 17-169) IL-36γ standards. IL-36γ released in response to poly(I:C) and flagellin was of a similar size to the full-length standard. C-D) HFKs were stimulated with recombinant full-length (rFL) or cleaved (rCL) IL36g (as used in [A] and [B], at 10 ng/mL), or poly(I:C)-stimulated or flagellin-stimulated HFK-conditioned media for 18 hours, and IL-36γ (C) and CXCL8 (D) transcript levels were measured by qPCR, relative to unstimulated controls and normalized to GAPDH. *** <0.00; Student’s T-test.
Differential effect of poly(I:C) and flagellin on LC3b-I to LC3b-II conversion and p62/SQSTM1. HFKs were stimulated with 1,500 ng/mL poly(I:C) or 1,000 ng/mL flagellin and lysed after 8 or 24 hours. Total protein was subjected to western blotting and probed for LC3b and p62 to assess early effects on autophagy.
A) Representative western blot showing stimulus-specific changes in the LC3b-II/-I ratio as an early response to poly(I:C) or flagellin stimulation. B) Quantified changes in LC3b-II/-I ratios of unstimulated or poly(I:C) or flagellin-stimulated HFKs, relative to time zero, indicating a poly(I:C)-specific increase in autophagosome membrane formation at 24 hours. C) Representative western blot showing p62/SQSTM1 levels after 8- and 24hour stimulations with poly(I:C) or flagellin along with unstimulated controls. D) Quantified p62/SQSTM1 levels, normalized to β-actin, in unstimulated or poly(I:C) or flagellin-stimulated HFKs relative to time zero, showing no evidence of p62/SQSTM1 decrease after 24 hours of stimulation. * p<0.05; two-tailed Student’s T-test. n.s.; not significant.
Poly(I:C) but not flagellin treatment leads to autophagosome accumulation. HFKs were stimulated with poly(I:C) (1,500 ng/mL) or flagellin (1,000 ng/mL), or CQ (50 μM) as a positive control, or were left unstimulated for the indicated time points. Cells were stained with an autophagy monitoring kit and fluorescent signals were visualized.
A) Representative micrographs showing steady state, poly(I:C)-, flagellin- or CQ-induced autophagosome presence at 0, 8 and 24 hours. B) Quantification of fluorescent autophagosomes post-treatment, expressed as CTCF relative to time zero. Scale bars represent 40 μm. Data represent three independent experiments after passaging HFKs three times. GDR; green detection reagent; CQ; chloroquine. * p< 0.05; two-tailed Student’s T-test.
IL-36γ secretion in HFKs is modulated by compounds that affect autophagy. HFKs were stimulated with poly(I:C) or flagellin as described previously for 24 hours followed by a media change containing bafilomycin A1 (50 nM), rapamycin (1 μM) or sucrose (100 mM). IL-36γ accumulated for 48 hours, after which cells were lysed, and conditioned media was collected and subjected to IL-36γ-specific ELISA. Total protein was subjected to western blotting to confirm effects on autophagic protein expression and adequate IL-36γ induction.
A) Representative western blot showing IL-36γ induction after TLR stimulation, an elevated LC3b-II/-I ratio, and accumulation of p62/SQSTM1 (indicative of autophagy blockage by bafilomycin A1). B) IL-36γ accumulation, measured by ELISA, showing that blocking autophagy increases poly(I:C)-mediated, but not flagellin-mediated IL-36γ secretion. C) Representative western blot showing IL-36γ induction after TLR stimulation as well as LC3b-I, -II, and p62/SQSTM1 levels 48 hours after rapamycin introduction. D) IL-36γ accumulation, measured by ELISA, showing a trend in the increase of IL-36γ secretion in the presence of flagellin , but not poly(I:C), in response to rapamycin, compared to vehicle. E) Representative western blot showing IL-36γ induction after TLR stimulation, and elevated LC3b-II, -I and p62/SQSTM1 levels, 48 hours after sucrose-mediated mTOR independent autophagy induction. F) IL-36γ accumulation, measured by ELISA, showing no evidence of altered IL-36γ secretion in response to sucrose for either agonist. Bars represent mean ± SD from three experiments. * p < 0.05; two-tailed Student’s T-test.
The level of autophagosome membrane-associated LC3b-II is altered in laryngeal papillomas compared to clinically normal laryngeal tissue.
A) Representative western blot of extracts from clinically normal (CN) and papilloma (P) laryngeal tissue, showing IL-36γ over-expression and low levels of the autophagosomal marker, LC3b-II, in papilloma laryngeal tissue (β-actin was used as a loading control). Data are representative of three different tissue pairs analysed. B) LC3b-II/β-actin ratios are significantly reduced in RRP biopsies compared to clinically normal tissues as a surrogate, relative measure of autophagy. Bars represent mean ± SD based on three papilloma and three clinically normal tissues. * p<0.05; two-tailed Student’s T-test.
Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA
The Institute of Molecular Medicine, The Feinstein Institutes for Medical Research, Manhasset, New York, USA
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
Department of Otolaryngology, Long Island Jewish Medical Center, Barbara and Donald Zucker School of Medicine at Hofstra/Northwell, Hempstead, NY, USA
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.