Induction of ASC pyroptosis requires gasdermin D or caspase-1/11-dependent mediators and IFNβ from pyroptotic macrophages

Abstract

Mesenchymal stem cells (MSCs) have been used in cell-based therapies for a variety of disorders. Some factors such as inflammatory mediators in the diseased area might damage the survival of MSCs and affect their efficacy. Pyroptosis is a form of programmed necrosis as a response for immune cells to cytosolic pathogenic stimuli. Whether MSCs develop pyroptosis under pathological stimulation, its underlying mechanism and biological significance are still unclear. Here, we found that LPS, flagellin, dsDNA, nigericin (NIG), or LPS combined with nigericin (LPS/NIG) could not induce pyroptosis in adipose-tissue-derived mesenchymal stem cells (ASCs). However, when applied the culture media collected from LPS/NIG-induced pyroptotic bone marrow-derived macrophages (BMDMs) to incubate ASCs, ASCs developed pyroptosis. Inhibition of caspases or deletion of Caspase-1/11 in ASCs did not affect the pyroptotic macrophage media-triggered ASC pyroptosis while ablation of Caspase-1/11 abolished BMDM pyroptosis induced by LPS/NIG. Media collected from LPS/NIG stimulated Gsdmd-/- or Caspase-1/11-/- BMDMs could not induce pyroptosis of ASCs. In addition, RNA-seq analysis showed that interferon (IFN)-stimulated genes were upregulated in pyroptotic ASCs. Adding IFNβ could boost LPS/NIG stimulated BMDM media-induced ASC pyroptosis. Surprisingly, the pyroptotic ASCs had a lower bactericidal ability to P. Aeruginosa. Taken together, induction of ASC pyroptosis requires gasdermin D or caspase-1/11-dependent mediators and IFNβ from pyroptotic macrophages.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. LPS, flagellin, dsDNA or nigericin cannot induce ASC pyroptosis.

ASCs were primed with 0.5 μg/ml LPS for 4 h, prior to challenging with 1 μg/ml LPS, 0.25 μg/ml flagellin, 2 μg/ml dsDNA, and 10 μM nigericin respectively, with or without 0.025% v/v FuGENE HD. Pyroptosis was detected by immunoblot analysis (a) of GSDMD-NT and mature IL-1β in ASCs, ELISA detection of mature IL-1β (b) and LDH release (c) in cell supernatant. n = 3. Tubulin was used as an internal control for immunoblot analysis. Data are representative of at least three independent experiments. Statistical significance between the control group and treated groups was determined using one-way analysis of variance. Data are shown as mean ± SD. ns: p > 0.05, *p < 0.05. LPS lipopolysaccharide, FLA flagellin, NIG nigericin, T transfect, ASCs adipose tissue-derived mesenchymal stem cells, GSDMD gasdermin D, GSDMD-NT N-terminal gasdermin D, ELISA enzyme-linked immunosorbent assay, LDH lactate dehydrogenase.

Fig. 2. Supernatant collected from LPS/NIG-treated BMDMs induces pyroptosis of ASCs.

The table showed the different supernatants collected from BMDMs or 3T3 cells. a LPS-primed ASCs were treated with supernatants of 3T3 cells and BMDMs for 2 h. After treating with related supernatants, the cells were collected and lysed for immunoblot analysis or replaced the supernatant with fresh media for another 2 h. At the end of experiment, the medium was harvested for detecting LDH, mature IL-1β, and caspase-1 p20 release of ASCs. Immunoblot analysis of caspase-11, proCaspase-1, GSDMD, and IL-1β in cell lysates of ASCs. b–d immunoblot analysis of caspase-11, proCaspase-1, and GSDMD, IL-1β in cell lysates of ASCs after treating with indicated supernatant. Data are representative of at least three independent experiments. LPS-primed ASCs were treated with 1 μg/ml LPS plus 10 μM nigericin or the indicated supernatants of 3T3 cells and BMDMs for 2 h. After the above treatments, collected the cells for immunoblot analysis or replaced the supernatant with fresh media for another 2 h. e Representative microscopic photographs of ASCs treated with SC3, SCB, SLN3, and SLNB respectively. As stated in a, photographs were taken by at 2 h after treatment. Objective magnification: ×40, bar 25 μm. Red arrows indicated vacuoles; black arrows indicated cell swelling. f–h As stated in a, the medium was harvested for detecting LDH (f), mature IL-1β (g), and caspase-1 p20 (h) release levels of ASCs. Data are representative of at least three independent experiments. n = 3. Statistical significance was determined using one-way analysis of variance. Data are shown as mean ± SD. ns: p > 0.05, ****p < 0.0001. LPS lipopolysaccharide, NIG nigericin, ASCs adipose tissue-derived mesenchymal stem cells, BMDMs Bone-marrow-derived macrophages, SN supernatant, GSDMD gasdermin D, GSDMD-NT N-terminal gasdermin D, LDH lactate dehydrogenase.

Fig. 3. The cleavage of GSDMD and proIL-1β in ASCs do not rely on the caspases of ASCs.

a ASCs were pretreated with VX765 or zVAD of indicated concentrations for 0.5 h before being primed with 0.5 μg/ml LPS for 4 h. Then ASCs were stimulated by SLNB mixed with VX765 or zVAD of indicated concentrations for 2 h. After the above treatments, replace the supernatant with fresh media for another 2 h-incubation or collect cells for immunoblot analyses (b, e) of indicated proteins. Data are representative of at least three independent experiments. c, f LDH release level of ASCs in the medium of a. n = 3. d, g ELISA detection of mature IL-1β in the media of a. n = 3. h–j LPS-primed WT and caspase-1/11−/− ASCs were treated with SCB and SLNB for 2 h. Then replace the supernatant with fresh media for another 2-h-incubation or collect cells for immunoblot analyses (out of 3 performed) h of indicated proteins. LDH (i) and mature IL-1β (j) were detected in the medium. n = 3. Statistical significance was determined using one-way analysis of variance. Data are shown as mean ± SD. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. zVAD z-VAD-FMK, LPS lipopolysaccharide, ASCs adipose tissue-derived mesenchymal stem cells, BMDMs Bone-marrow-derived macrophages, SN supernatant, GSDMD gasdermin D, GSDMD-NT N-terminal gasdermin D, SLNB supernatant collected from LPS/NIG-treated BMDMs, NT no treatment of inhibitor, DMSO dimethyl sulphoxide, LDH lactate dehydrogenase, ELISA enzyme-linked immunosorbent assay.

Fig. 4. The supernatants from LPS/NIG-treated caspase-1/11−/− or Gsdmd−/− macrophages cannot cause pyroptosis of ASCs.

a, b WT BMDMs, caspase-1/11−/− BMDMs, WT iBMDMs, and Gsdmd−/− iBMDMs were stimulated with 1 μg/ml LPS for 5 h prior to challenging with 10 μM nigericin for 1 h. The supernatants were collected for detecting caspase-1 p20 by immunoblotting analysis (a, b). Data are representative of at least three independent experiments. c–h Treat LPS-primed ASCs with the above supernatants for 2 h. After the treatments of related supernatants, collect ASCs for immunoblot analysis (out of at least three performed) of GSDMD and IL1β (c, f) or replace the supernatant with fresh media for another 2 h. Harvest the medium for evaluating LDH (d, g) and mature IL-1β release levels (e, h) of ASCs. n = 3. Statistical significance between the control group and treated groups was determined using one-way analysis of variance. Data are shown as mean ± SD. ns: p > 0.05, ***p < 0.001, ****p < 0.0001. LPS lipopolysaccharide, NIG nigericin, ASCs adipose tissue-derived mesenchymal stem cells, BMDMs Bone marrow-derived macrophages, iBMDMs immortalized bone marrow-derived macrophages, WT wildtype, GSDMD gasdermin D, GSDMD-NT N-terminal gasdermin D, SN supernatant, SCB supernatant collected from BMDMs, SLNB supernatant collected from LPS/NIG-treated BMDMs, LDH lactate dehydrogenase.

Fig. 5. SLNB treatment modulates ASC’s transcriptome and triggers ASCs’ responses to interferon.

LPS-primed ASCs were treated with SC3, SLN3, SCB, and SLNB respectively for 2 h. a RNA-seq heat map for ASCs exposed to SC3, SLN3, SCB, and SLNB respectively (n = 3). The heat map showed 114 significantly up-regulated genes (q < 0.001) in SLNB-treated ASCs compared to SCB-treated and SLN3-treated ASCs. Red (12) to blue (0) were ranked by values of log2(value of gene expression+1). b The significant KEGG pathways (q < 0.05) of the genes in a. c Gene Ontology analysis of the genes in a showing the biological process for SLNB treatment (q < 0.05). ASCs adipose tissue-derived mesenchymal stem cells, SC3 supernatant collected from 3T3 cells, SLN3 supernatant collected from LPS/NIG-treated 3T3 cells, SCB supernatant collected from BMDMs, SLNB supernatant collected from LPS/NIG-treated BMDMs, KEGG Kyoto Encyclopedia of Genes and Genomes, Num number.

Fig. 6. IFNβ expression increases in LPS/NIG-stimulated macrophages and IFNβ promotes pyroptosis of ASCs.

a Heat map from RNA-seq analysis for ASCs exposed to SC3, SLN3, SCB, and SLNB respectively (n = 3). The heat map showed the expression level of 15 IFN-stimulated genes significantly up-regulated in SLNB-treated ASCs compared to SLN3-treated ASCs. Red (highest) to green (lowest) represent the values of gene expression level. b–e 3T3 cells, BMDMs, WT iBMDM, and Gsdmd−/− iBMDM were incubating in LPS (1 μg/ml) for 5 h before being challenging with nigericin (10 μM) for 1 h. Then Cells were collected and evaluated for expression of IFNβ and IFNγ at mRNA levels by RT-PCR. Values were showed as the relative ratio of mRNA of IFNβ or IFNγ to mRNA of GAPDH. n = 3. f–h ASCs were pretreated with IFNβ (0.4 ng/ml) and LPS (0.5 μg/ml) for 4 h before being stimulated with LPS/NIG, SCB, or SLNB for 2 h. And then collected ASCs for immunoblot analysis (out of at least three performed) of GSDMD and IL-1β (e) or replaced with fresh medium for another 2 h. Harvest the medium for detecting LDH (f) and mature IL-1β (g) released by ASCs. n = 3. The statistical significance of the differences between various treatments was determined by a two-tailed t test for two groups or one-way analysis for three or more groups. Data are shown as mean ± SD. ns: p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. IFN interferon, LPS lipopolysaccharide, NIG nigericin, ASCs adipose tissue-derived mesenchymal stem cells, BMDMs bone marrow-derived macrophages, iBMDMs immortalized bone marrow-derived macrophages, WT wildtype, GSDMD gasdermin D, GSDMD-NT N-terminal gasdermin D, SCB supernatant collected from BMDMs, SLNB supernatant collected from LPS/NIG-treated BMDMs, RT-PCR real-time reverse transcriptase-polymerase chain reaction, LDH lactate dehydrogenase.

Fig. 7. Pyroptosis reduces the antibacterial ability of ASCs.

LPS-primed ASCs were incubated with SC3, SLN3, SCB, and SLNB respectively for 2 h, followed by infected with GFP-PAO1 at MOI of 1 for an additional 2 h. n = 3. a CFU counts were performed to determine the number of viable P. aeruginosa in the supernatants. b The intracellular GFP-fluorescence of ASCs was measured by a FACScan flow cytometer. The median fluorescence intensity represents the intracellular GFP-PAO1. n = 3. Statistical significance was determined using one-way analysis of variance. Data are shown as mean ± SD. ns: p > 0.05, *p < 0.05, **p < 0.01. LPS lipopolysaccharide, ASCs adipose tissue-derived mesenchymal stem cells, SC3 control supernatant collected from 3T3 cells, SLN3 supernatant collected from LPS/NIG-treated 3T3 cells, SCB control supernatant collected from BMDMs, SLNB supernatant collected from LPS/NIG-treated BMDMs, CFU colony-forming units, MOI multiplicities of infection.