Abstract
Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases1,2,3,4. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin-1β (IL-1β). The dominant effect of IL-1β in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system in which macrophages are induced to undergo pyroptosis without IL-1β or IL-1α release (denoted Pyro−1), we identify unexpected beneficial effects of the Pyro−1 secretome. First, we noted that the Pyro−1 supernatants upregulated gene signatures linked to migration, cellular proliferation and wound healing. Consistent with this gene signature, Pyro−1 supernatants boosted migration of primary fibroblasts and macrophages, and promoted faster wound closure in vitro and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro−1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound-healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that its synthesis is induced de novo during pyroptosis, downstream of caspase-1 activation and cyclooxygenase-2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.
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Data availability
All raw and processed sequencing data generated in this study have been deposited in the NCBI GEO (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE262847. The publicly available dataset used in this study is available at GEO under the accession number GSE119509. The gating strategy for flow cytometry and raw, uncropped images of western blots are provided in the Supplementary Information. Source data are provided with this paper.
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Acknowledgements
We thank members of the laboratory of K.S.R. for inputs and suggestions; A. Wullaert for Il18-KO mice; P. Vandenabeele for Rosa26-TdT Tg;Sox2-cre mice; and the VIB Protein Core, VIB Flow Cytometry Core and VIB Bioimaging Core for contributions. K.S.R. is supported by the FWO (Odysseus grant G0F5716N, EOS DECODE 30837538), the Special Research Fund UGent (iBOF BOF20/IBF/037), the European Research Council research and innovation programme (grant agreement number 835243) and BJC Investigator Funds from the Washington University School of Medicine. M.L. is supported by FWO research grants (FWO-EOS-GOI5722N (CD-INFLADIS), G014221N and G017121N), the Special Research Fund UGent (BOF23/GOA/001) and the European Research Council grants 683144 (PyroPop) and 101101075 (PyroScreen). E.H. is supported by the LEO Foundation EMEA Award 2022. A.S. is supported by the Priority 2030 Federal Academic Leadership Program of the Russian Federation. Additional support was received through the FWO Postdoctoral Fellowship (1227220N to P.M.; 1225421N to C.J.A.; 12Y2122N to C.M.), a German Research Foundation Postdoctoral fellowship (MA7770/1-1 to C.M.), an EMBO postdoctoral fellowship (ALTF 409-2020 to R.T.) and a Marie Skłodowska-Curie Actions individual fellowship (800446 to S.M.). U.J. is supported by a Boehringer Ingelheim transition grant and a Lawrence C. Pakula, MD IBD Innovation Fund Award. pLEX_307 was a gift from D. Root (Addgene plasmid number 41392). psPAX2 was a gift from D. Trono (Addgene plasmid number 12260). pCMV-VSV-G was a gift from B. Weinberg (Addgene plasmid number 8454). Graphics in Figs. 1a,c,d, 2b–e, 3d,e,g and 4a,b,d,k,l and Extended Data Figs. 1c, 3b and 6a were created using BioRender.com as specified in the captions.
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Contributions
P.M. and K.S.R. designed the experiments and wrote the manuscript with input from all co-authors. P.M. carried out most of the experiments. S.M. carried out the skin wound healing experiments, assisted in several experiments and provided critical inputs for the overall manuscript. E.H. provided advice on skin wound healing experiments and the manuscript. L.B., C.M., R.T. and L.V.W. assisted with several experiments, and aided in the experimental design. Y.I., B.N.K. and B.H. assisted in several experiments. C.J.A. provided critical experimental suggestions. A.G. carried out microscopy. N.V.O. provided technical help. U.J. conceptualized and carried out colonic wound healing experiments. J.P. carried out the bioinformatic analysis of the RNA-seq data. J.B.C. analysed the GSE119509 dataset and carried out Pyro−1 signature mapping. A.S. and V.S. carried out unbiased searches of public databases. M.L. provided mice, and critical scientific inputs on experiment design and the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Proliferation/migration gene signatures induced by Pyro−1 secretome.
a) (left) Flow cytometry plots depicting uptake of 7-AAD by BMDMs from C57BL/6(Control), Nlrp1bTg (Pyro−1), Nlrp1bTg Casp1/11KO (Apop) with or without exposure to LeTox (120 min). (right) Annexin V staining of C57BL/6 Nlrp1bTg Casp1/11KO BMDMs, exposed to LeTox for 180 min. b) Pyro−1 cells without TLR triggers do not release IL-1β/α. Bar graph denoting the release of IL-1α and IL-1β from Control, Pyro−1, Apop cells in the absence or presence of LPS pretreatment ahead of the LeTox trigger. Data represents mean ± s.e.m. of n = 3 independent biological replicates analyzed via ordinary one-way ANOVA, using the Holm-Sidák’s multiple comparison test. ****P < 0.0001, ns- not significant using. c) No differential gene expression signature was induced by live cell supernatants. Supernatants were collected from live BMDMs (no LeTox treatment) of C57BL/6 Control mice, Nlrp1bTg mice or Nlrp1bTg C1/11KO mice and incubated with Mo target macs for 4 h. This was followed by gene expression analysis of Mo target macs via RNA sequencing. No differences in gene expression were observed between the 3 treatments. d) Pyro−1 supernatants do not induce inflammatory cytokine secretion from Mo target macrophages. Supernatants from Control or Pyro−1 cells (with or without LPS) were incubated with Mo target macs for 4 hr. Control or Pyro−1 supernatants do not induce the release of inflammatory cytokines in the absence of LPS based triggers. mean ± s.e.m. of n = 3 independent biological replicates. e) Key GO categories induced by Pyro−1 supernatants in Mo macs. Pie charts highlighting representative dominant GO terms collectively, and the number of associated genes within these terms. B.V., blood vessel. f) Frequency of differentially expressed genes induced by Pyro−1 supernatants in the top GO terms. Bar graph showing the frequency at which differentially expressed genes are represented within each of the top GO categories identified by gene set enrichment analysis. g) Differential gene expression data. Table representing log2fold change and adjusted p-value for genes differentially expressed by Pyro−1 treated Mo target macs. The genes listed in the table represent genes occurring with the highest frequency within the top GO categories (Extended Data Fig. 1f). h) Gene ontogeny (GO) categories representing genes downregulated by Pyro−1 supernatants. Bar graph representing the downregulated genes in Mo target macs exposed to Pyro−1 supernatants, organized as GO categories. i) Confirmation of proteinase K activity. Western blot denoting the degradation of the protein component of Pyro−1 supernatants after incubation with Proteinase-K for 1 h. Data represents 1 biological replicate. j) Cx3cl1 induction by Pyro−1 supernatants is sensitive to Proteinase K treatment. (left) Control, Pyro−1, Pyro−1-18, or Apop supernatants either treated with Proteinase K or untreated and incubated with Mo target macs. Gene expression was analyzed after 4 hr via qPCR. Data represent mean ± s.e.m. of n = 3 independent biological replicates, analyzed using ordinary one-way ANOVA, using the Holm-Šídák’s multiple comparision test. ****P < 0.0001, ***P < 0.001, ns- not significant. The graphics of cells and tubes in c were created with BioRender.com.
Extended Data Fig. 2 Identification of soluble metabolic mediators in Pyro−1 supernatants.
a) Schematic of the unbiased search of public gene expression datasets matching Pyro−1 signature. A compendium of 24970 mouse gene expression datasets was gathered from gene expression omnibus dataset. For each of the dataset, GESECA gene set analysis algorithm from FGSEA R-package was run, using previously obtained Pyro−1 signature as the input gene set. Finally, the datasets were ranked by GESECA p-value. b) Enhanced in vitro wound healing induced by Apop supernatants. (left) Wounds were generated on a monolayer of primary mouse ear fibroblasts. Control and Apop supernatants were added, and the closure of the wounds were monitored using live microscopy for 24 h. (right) Quantification of wound closure with Apop supernatants compared to Control supernatants at 24 h post wound generation. Data represent n = 4 independent biological replicates analyzed via unpaired 2 tailed t-test with Welch’s correction. *P = 0.0489. c) Lower IL-1β levels in primary human monocyte supernatant. Human blood derived monocytes were isolated and either left untreated or triggered using NeedleTox (NdTox). Post pyroptotic death, the supernatant was collected and IL-1β was measured via ELISA. Mouse BMDMs were treated with LPS for 4 h, followed by LeTox trigger. Supernatants were accessed for IL-1β using ELISA. Data represents mean ± sem of n = 4 independent biological replicates for human samples and n = 3 independent biological replicates for mouse samples, analyzed using one-way ANOVA using Šídák’s multiple comparisons test. ****P < 0.0001.
Extended Data Fig. 3 De novo synthesis of PGE2 during pyroptosis can promote wound healing in vivo.
a) Properties of NSIADs used in the study. Table depicting specificity of Aspirin, Ibuprofen, NS-389, Indomethacin towards COX1 or COX2 and whether the activity of the drug is reversible or irreversible. b) (left) Schematic representing the treatment regimen used. Nlrp1bTg mouse BMDMs were incubated overnight with either aspirin, ibuprofen, NS-398 or indomethacin. Control, Nlrp1bTg, and Nlrp1bTg cells treated with inhibitors were then triggered with LeTox in the presence of the drugs. Supernatants were collected 120 mins post trigger and PGE2 was measured using ELISA. (right) PGE2 levels in the supernatants of the mentioned conditions. n = 4(Pyro−1; aspirin), n = 3 (control), n = 2 (NS-398, ibuprofen), n = 1(indomethacin) independent biological replicates, analyzed with one-way ANOVA using Sidak multiple comparison test. ****P < 0.0001. c) Kinetics of inducible caspase mediated pyroptosis in THP1 human monocytes. THP-1-iC1 Cells were either left untreated or treated with B/B dimerizer. Cells quickly responded via loss of plasma membrane integrity as indicated by positivity for 7-AAD. Plasma membrane permeability and phosphatidylserine exposure occurred simultaneously Depicted is the merge of fluorescence recorded in the channels for Hoechst 33342, Annexin-VI-FITC and 7-AAD (FL) or the corresponding differential interference contrast (DIC) image. The scale bar represents 20 µm. Data is representative of images obtained from 2 independent biological replicates. d) Pyro−1 supernatant treated wounds exhibit increased Claudin-4 staining. Data represents quantification of Claudin-4 staining intensity in NS-398 treated intestinal punch biopsy wounds, either treated with Control or Pyro−1 supernatants. The data is represented as fold change with respect to Claudin-4 intensity observed in untreated wounds. n = 7(Pyro−1), n = 6 (Control) independent colonic mouse wounds analyzed using unpaired, two-tailed t-test. ****P < 0.0001. Violin plots show the entire range of values, and the center line denotes the median. The graphics of mice, pipettes and cells in b were created with BioRender.com.
Extended Data Fig. 4 Pyro−1 supernatants induce changes in wound bed that contribute to wound healing.
a) Cell migration is not induced by Pyro−1 supernatants in the epithelial tongue. Mice were treated with Pyro−1 or control supernatants after full-thickness wounding with an 8 mm punch biopsy. Skin sections at day 4 post-wounding were stained with Itga5 (green) and Anti-Cytokeratin K14 (red). Quantification (right) and representative immunofluorescent images (left). Data represent n = 5 mice wound sections for Control and n = 4 mice wound sections for Pyro−1, analyzed using unpaired two-tailed t-test. Violin plots show the entire range of values and the center line denotes the median, with the two side lines representing quartiles. Scale bar: 150 μm. b) Cell proliferation is not induced by Pyro−1 supernatants in the epithelial tongue. Mice were treated with Pyro−1 or control supernatants after full-thickness wounding with an 8 mm punch biopsy. Skin sections at day 4 post-wounding were stained with Ki67 (green) and Anti-Cytokeratin K14 (red), and nuclei were stained with DAPI (blue). Quantification (right) and representative immunofluorescent images (left). Violins plots show minimum to maximum values with all independent replicates, centre denotes median, analyzed using unpaired two-tailed t-test. Data represent n = 4 mouse wound sections from Control and Pyro−1 supernatant treated groups. Violin plots show the entire range of values and the center line denotes the median, with the two side lines representing quartiles. Scale bar: 150 μm. c) Pyro−1 sups induce changes in granulation tissue. Mice were treated with Pyro−1 or Control supernatants after full-thickness wounding with an 8 mm punch biopsy. Skin sections at day 4 post-wounding were subjected to Hematoxylin and eosin-staining. (left) wound sections (right) quantification of granulation tissue thickness, analyzed using unpaired two-tailed t-test. ***P < 0.001. Data represent n = 4 mouse wound sections from Control and Pyro−1 supernatant treated groups. Violin plots show the entire range of values and the center line denotes the median, with the two side lines representing quartiles. d) Pyro−1 sups induce proliferation and differentiation in the granulation tissue. Mice were treated with Pyro−1 or Control supernatants after full-thickness wounding with an 8 mm punch biopsy. Skin sections at day 4 post-wounding were stained with Ki67 (green) and DAPI (blue) or with CD31 (green), α-SMA (magenta) and DAPI (blue). Representative immunofluorescent images (left) and quantification representing ratios of pixels from the individual cell populations to the entire tissue pixel count (right). Floating bars show the range of values, open circles show independent replicates (n = 5 mice) and the line denotes the mean Data analysed by unpaired two-tailed t-test. (*P = 0.0210 for Ki-67; *P = 0.0146 for α-SMA *P = 0.0178 for CD31). e) Apop supernatants induce wound closure. Dermal punch biopsy wounds were treated with either Control or Apop supernatants from day 0 to 2 post wounding and wound closure was measured. Representative images for Control, Pyro−1, Apop treated mouse wounds at day 1 and day 2 post wounding.
Extended Data Fig. 5 Pyro−1 cells secrete metabolites over the course of death.
a) Metabolites enriched in Pyro−1 supernatants. Bar graphs for fold enrichment of selected signaling metabolites in Pyro−1 or Apop supernatants compared to Control supernatants as deciphered by untargeted metabolomics. Spermidine, taurine, g3P and malate were enriched in both Pyro−1 and Apop supernatants (but significantly more in Pyro−1 with respect to Apop supernatants). Spermine, PEP, 5’MTA and creatinine were uniquely enriched in Pyro−1 supernatants. Data represent the of 4 independent biological replicates analyzed via unpaired two-tailed t-test. Spermidine **P = 0.0015, Taurine *P = 0.0142, Malate **P = 0.0088, G3P **P = 0.0025, Spermine **P = 0.006, 5’MTA *P = 0.424, Creatinine ****P < 0.0001, PEP ****P < 0.0001. The calculated using unpaired t-test. PEP: phosphoenolpyruvate, MTA 5’-Methylthioadenosine. Box plots represent range of min to max values with a line at mean. b) Absolute metabolite concentration in Pyro−1 supernatants. Control, Nlrp1bTg, Nlrp1bTg Casp1/11KO mice derived BMDMs were triggered with LeTox, and supernatants were collected- Control, Pyro−1, Apop respectively Representative graph depicting absolute quantities of a few metabolites in the supernatants of Control and Pyro−1 supernatants, as measured by mass spectrometry. Bar graph represents mean ± sem of n = 4 independent biological replicates. c) Kinetics of metabolite release from Pyro−1 cells. Supernatants from Pyro−1 cells were collected either at 60, 90 min or 120 min after LeTox trigger and untargeted metabolomics was performed. (Left) Bar graph representing log2fold change in the quantities of metabolites released during Pyro−1 compared to Control supernatants. Molecular weights of the compounds represented in the bar graph are mentioned. (Right) Representative graphs of selected metabolites demonstrating the gradual increase in metabolite concentration in the supernatants of Pyro−1 cells from 60 to 120 min post LeTox trigger. Data represents 4 biological replicates analyzed via ordinary one-way ANOVA. Bar graphs represent min to max value with line at mean for n = 4 independent biological replicates. Spermidine **P = 0.0023, *P = 0.0136; Spermine **P = 0.0082, *P = 0.0112; Taurine *P = 0.0120; Putrescine *P = 0.118; MTA ***P = 0.0002, ***P = 0.0003; PEP ****P < 0.0001.
Extended Data Fig. 6 Pyro−1 supernatants induce alterations in cellular infiltration at the wound site.
a) Schematic for the gating strategy to identify macrophage subpopulations at the wound. b) Pyro−1 sups increase the number of non-immune cells at the wound site. The number of CD45− cells/cm2 of wound area at day 3 after wounding and supernatant treatment. Data represent 10 biological replicates, analyzed using unpaired two tailed t-test. Violin plots show the entire range of values, and the center line denotes the median. ****P < 0.0001. c) Macrophage polarization is dependent on GsdmD. Mo target macrophages were treated with supernatants from Control, Pyro−1, Pyro−1-18 or Nlrp1bTg GsdmDKO HoxB8 derived macrophages (GsdmDKO) for 24 h. The expression of CD301 was examined using flow cytometry. Live cells were gated on CD11b+ expression and probed for either the % of cells expressing CD301(left) or the MFI of CD301 expression (right). Data represent mean ± sem of n = 4 independent biological replicates, analyzed using RM one-way ANOVA test, represented as fold change over Control supernatants. For percentage for CD301 expressing cells *P = 0.032(Control vs Pyro−1), *P = 0.015(Control vs Pyro−1-18). For MFI ***P = 0.0008, *P = 0.0105. d) Pyro−1 supernatants do not induce Ly6C expression. (left) MFI of Ly6C in CD45+/CD64+ cells at day 3 after wounding and supernatant treatment. Data represent 10 biological replicates, analyzed using unpaired t-test. P = 0.5450. (right) MFI of Ly6C in CD45+/F4-80+ cells at day 3 after wounding and supernatant treatment. Data represent 10 biological replicates, analyzed using unpaired t-test. P = 0.0526. e) CD301 expression is enhanced in Lyc6Cintermediate but not in Ly6Chi populations. (left) CD301 MFI measured in CD45+/CD64+/Ly6Cintermediate (CD45+/CD64+/Ly6Cint) at the wound site. (right) CD301 MFI measured in CD45+/CD64+/Ly6Chi populations at the wound site. Data represent 10 biological replicates, analyzed using unpaired t-test, ns = 0.0572, *P = 0.0372. f) IL-1β levels at wound site.IL-1β levels were measured on days 0, 2 and 8 post wounding in skin sections. Data represents 3 biological replicates, analyzed using one-way ANOVA. Values on the comparison bars denote P values. g) Mouse dermal skin wounds were injected with either Pyro−1 supernatants, IL-1β Blocking antibody or the Isotype Control. Rate of wound closure was measured. Data represents 7 biological replicates, analyzed via 2-way ANOVA. The graphics of mice and cells in a were created with BioRender.com.
Supplementary information
Supplementary Figures
Supplementary Figs. 1–4, which include the gating strategies and the uncropped blot.
Supplementary Table 1
This table has the following worksheets. WS1: Description of WS2–WS13. WS2: List of genes enriched in M0 target macrophages treated with Pyro−1 supernatant compared to control supernatants. WS3: List of GO categories identified to be enriched in M0 target macrophages treated with Pyro−1 supernatants versus control supernatants. The columns represent P value, Padj and log10[Padj]. significance: Padj < 0.01. WS4: List of GO categories identified to be downmodulated in M0 target macrophages treated with Pyro−1 supernatant versus control, as in WS3. WS5: GO categories that were significantly enriched in M0 target macrophages treated with Pyro−1 supernatants versus control supernatants, setting the Padj threshold to be <0.01 or log10[Padj] < 2. WS6: GO categories associated with cellular migration identified in WS3, with their Padj. The differentially expressed genes that are represented in that particular GO category are also listed next to each category. WS7: GO categories associated with cellular proliferation identified in WS3, with their Padj values. The differentially expressed genes that are represented in that particular GO category are also listed. WS8: GO categories associated with inflammation and immune response identified in WS3, with their Padj. The differentially expressed genes that are represented in that particular GO category are also listed. WS9: GO categories associated with signalling response identified in WS3, with their Padj. WS10: Number of GO categories each of the genes were present in (columns a and b). The GO categories for each gene are listed in column c. WS11: Input Pyro−1 signature used for unbiased search of public databases. WS12: Datasets identified by the unbiased search of public databases. WS13: Top-100 databases listed in WS12.
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Mehrotra, P., Maschalidi, S., Boeckaerts, L. et al. Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair. Nature 631, 207–215 (2024). https://doi.org/10.1038/s41586-024-07585-9
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DOI: https://doi.org/10.1038/s41586-024-07585-9
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