Figure 2.

Human keratinocytes undergo NLRP1-dependent ASC speck formation, lytic cell death and mature IL-1β secretion upon DPP8/9 inhibition or depletion. A, immortalized human keratinocytes (N/TERT-1) or primary foreskin keratinocytes were treated with 3 μm talabostat or 1 μg/ml LPS for 24 h. Culture media were harvested, clarified by centrifugation, and analyzed by IL-1β ELISA. Statistical significance was calculated using one-way ANOVA with Bonferroni's multiple comparison test between all treatment groups. Bar graph represents biological triplicates. B, immortalized human keratinocytes were treated with talabostat for 16 h. Cell culture media were collected and analyzed by Luminex assay. Heat map represents all cytokines or chemokines that showed >2-fold increase in the 3 μm talabostat treated group versus control-treated cells. Cytokines/chemokines previously shown to be up-regulated in MSPC patient-derived keratinocytes are shown in red (17). C, immortalized human keratinocytes (N/TERT-1) were treated 30, 3, and 0.3 μm of talabostat or DMSO for 24 h. In parallel, cells were transfected with an empty vector of NLRP1 a.a. 1213–1474, and media were harvested 24 h post-transfection. The cell culture media were concentrated 10 times using Amicon columns with a molecular mass cutoff of 3 kDa before SDS-PAGE. D, immortalized human keratinocytes were treated with 3 μm talabostat at the indicated times. In parallel, the cells were transfected with NLRP1 a.a. 1213–1474 and harvested 24 h later. Cell pellets were lysed in radioimmune precipitation assay buffer and insoluble materials were cross-linked with 1 mm DSS in PBS to visualize ASC polymers by SDS-PAGE. E, immortalized human keratinocytes (N/TERT-1) stably expressing ASC-GFP were treated with 2 μm talabostat or DMSO in the presence of propidium iodide. The cells were imaged using a wide-field fluorescent microscope equipped with a temperature- and CO₂-controlled chamber at 20× magnification. Images were acquired every 10 min for 20 h. Left panel, cells before and ∼20 h after talabostat addition. Right panel, a close-up, time-course view of a cell undergoing pyroptotic cell death following talabostat treatment. Yellow dotted line delineates cell periphery. Green arrow, ASC speck; white arrow, membrane rupture; red arrow, PI influx. F, immortalized human keratinocytes (N/TERT-1) were treated with siRNAs against NLRP3, NLRP1, and ASC/PYCARD for 48 h before 3 μm talabostat treatment for 24 h. The cell pellets were lysed in radioimmune precipitation assay buffer, and the insoluble materials were cross-linked with 1 mm DSS in PBS. Cell culture media were concentrated 10 times with Amicon columns with a molecular mass cutoff of 3 kDa before SDS-PAGE. The asterisk indicates a nonspecific band with the NLRP1 antibody. G, immortalized keratinocytes stably expressing Cas9 or Cas9 with two independent guide RNAs against NLRP1 and ASC were treated with 3 μm talabostat for 24 h. IL-1β in the culture media was analyzed by ELISA. Statistical significance was calculated using one-way ANOVA with Bonferroni's multiple comparison test between all treatment groups. Bar graph represents four independent experiments. H, control or NLRP1 KO keratinocytes were treated with siRNAs against NLRP3, DPP8, DPP9, and DPP8 and DPP9 together for 72 h. IL-1β in the culture media was analyzed by ELISA. Statistical significance was calculated using one-way ANOVA with Bonferroni's multiple comparison test between all treatment groups. Bar graph represents two biologically independent experiments.