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Group 3 innate lymphoid cell pyroptosis represents a host defence mechanism against Salmonella infection

Abstract

Group 3 innate lymphoid cells (ILC3s) produce interleukin (IL)-22 and coordinate with other cells in the gut to mount productive host immunity against bacterial infection. However, the role of ILC3s in Salmonella enterica serovar Typhimurium (S. Typhimurium) infection, which causes foodborne enteritis in humans, remains elusive. Here we show that S. Typhimurium exploits ILC3-produced IL-22 to promote its infection in mice. Specifically, S. Typhimurium secretes flagellin through activation of the TLR5-MyD88-IL-23 signalling pathway in antigen presenting cells (APCs) to selectively enhance IL-22 production by ILC3s, but not T cells. Deletion of ILC3s but not T cells in mice leads to better control of S. Typhimurium infection. We also show that S. Typhimurium can directly invade ILC3s and cause caspase-1-mediated ILC3 pyroptosis independently of flagellin. Genetic ablation of Casp1 in mice leads to increased ILC3 survival and IL-22 production, and enhanced S. Typhimurium infection. Collectively, our data suggest a key host defence mechanism against S. Typhimurium infection via induction of ILC3 death to limit intracellular bacteria and reduce IL-22 production.

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Fig. 1: Distinct ILC responses during early S. Typhimurium infection.
Fig. 2: IL-22+ ILC3s are specifically induced in early S. Typhimurium infection.
Fig. 3: S. Typhimurium flagellin protein promotes IL-22+ ILC3s.
Fig. 4: Presence of ILC3s increases susceptibility to S. Typhimurium infection in mice.
Fig. 5: Pyroptosis in gut ILC3s is induced by S. Typhimurium independent of flagellin.
Fig. 6: Caspase-1 controls ILC3 cell death in a cell-intrinsic manner during S. Typhimurium infection.

Data availability

All data supporting the findings of this study are available within the Article and its supplementary materials. The Mus musculus reference genome (GRCm38/mm10 assembly) https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/ was used for RNA-seq read alignment. The accession GEO number of the RNA-seq data in this paper is GSE201292. Source data are provided with this paper.

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Acknowledgements

We thank the entire Zhou lab for help and suggestions; L. N. Howell at Animal Care Services of the University of Florida for the hydrodynamic injection, and Y. Sun from Molecular Pathology Core, Department of Pathology of the University of Florida for tissue section and immunofluorescence staining; M. Cella at Washington University for kindly providing the MNK-3 cell line, and the Genomics Facility (University of Chicago) for sequencing services and assistance; F. Vacca at the University of Edinburgh for sharing the protocol for proteinase K treatment of the bacterial culture supernatant. L.Z. is an Investigator in the Pathogenesis of Infectious Disease supported by the Burroughs Wellcome Fund. The work was supported by the National Institutes of Health grants AI132391 and DK105562 (to L.Z.), AI60557 (to R.C.) and AI126172 (to S.W.).

Author information

Authors and Affiliations

Authors

Contributions

L.X. and L.Z. designed the study. L.X. performed experiments. S.W. and K.N.O. assisted with experiments. J.W.D. performed RNA-seq data analysis. C.J. and R.C. provided reagents and suggestions. L.Z. supervised the study. L.X. and L.Z. wrote the manuscript with input from the other authors.

Corresponding author

Correspondence to Liang Zhou.

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Competing interests

R.C. is a founder and part owner of Curtiss Healthcare, Inc., which is involved in developing vaccines against infectious diseases of farm animals. The other authors declare no competing interests.

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Nature Microbiology thanks Matthew Hepworth, Edna Ondari and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Distinct ILC response during early S. Typhimurium infection, (Related to Fig. 1).

a, Schematic depiction of experimental design of S. Typhimurium infection in mice. b and c, Percentages in the Lin (b) and absolute numbers (c) of the ILC2s in LI-LPLs of uninfected and infected WT mice. Compiled data from two independent experiments (n = 5 and 7 mice for uninfected and infected groups, respectively). d, FACS analysis of Ki-67, IL-5 and IL-13 expression in LI ILC2s of uninfected and infected WT mice on day 2 post infection (d.p.i 2). Representative data of two independent experiments. eh, FACS analysis of Eomes (e), IFN-γ (g) expression in SI ILC1s of uninfected and infected WT mice on d.p.i 2. Representative data of two independent experiments (e and g). Percentages of Eomes (ILC1) and Eomes+ (NK) cells in group 1 ILCs (f), and percentages of IFN-γ+ cells in NK and ILC1 cells (h). Compiled data from two independent experiments (n = 4 and 5 mice for uninfected and infected groups, respectively) (f and h). i and j, FACS analysis of GATA3 expression after gating on Lin in the SI of uninfected and infected WT mice on d.p.i 2. Representative data of two independent experiments (i). Percentages of ILC2s in Lin cells in SI LPLs. Compiled data from two independent experiments (n = 4 and 5 mice for uninfected and infected groups, respectively) (j). k, Representative FACS gating/sorting strategies used for identification of different immune cell populations in this study. Gating strategies indicated in this panel were applied to the following FACS data panels in the figures: group 1 ILCs (LinRORγtNKp46+NK1.1+): Figs. 1c, f; ILC3s for intracellular staining and gating (LinRORγt+): Figs. 2a,j,m, 3a,d,5a,d,6a; ILC3s for surface staining and sorting (LinCD90hiCD45lo): Figs. 3h, 4f-g, 6g; ILC2 (LinGATA3+): Fig. 1j.

Source data

Extended Data Fig. 2 IL-22+ ILC3s are specifically induced in early S. Typhimurium infection, (Related to Fig. 2).

a and b, FACS analysis of RORγt expression in SI Lin of uninfected and S. Typhimurium-infected WT mice on day 2 post infection (d.p.i 2). Representative data of two independent experiments (a). Percentages of SI ILC3s in Lin. Compiled data from two independent experiments (n = 4 and 5 mice for uninfected and infected groups, respectively) (b). c–h, IL-22 and IL-17 expression in LI ILC3s of uninfected and infected WT mice on d.p.i 2. Representative FACS data of three independent experiments (a). Percentages (d) and absolute numbers (e) of IL-17+ LI ILC3s of uninfected and infected WT mice on d.p.i 2. Compiled data from three independent experiments (n = 7 and 9 mice for uninfected and infected groups, respectively) (d and e). Percentages (f), absolute numbers (g) and MFI (h) of IL-22+ LI ILC3s of uninfected and infected WT mice on d.p.i 2. Compiled data from three independent experiments (n = 8 and 9 mice for uninfected and infected groups, respectively) (fh). ik, FACS analysis of IL-22 and IL-17 expression in SI ILC3s of uninfected and infected WT mice on d.p.i 2, with or without P/I stimulation. Representative data of two independent experiments (i). Percentages of IL-22+ ILC3s, without (j) or with (k) P/I stimulation. Compiled data from two independent experiments (n = 4 and 5 mice for uninfected and infected groups, respectively) (j and k). l–m, FACS analysis of IL-22 expression in LI ILC3s of uninfected and infected WT mice at 12 h and 24 h post infection. Percentages of IL-22 in LI ILC3s (l). Compiled data from two independent experiments (n = 4 and 5 mice for uninfected and infected groups, respectively) (m). n and o, FACS analysis of RORγt and GATA3 expression after gating on Lin cells in LI-LPLs of uninfected and infected WT mice at 12 h and 24 h post infection. Percentages of ILC3s in Lin cells in LI-LPLs. Representative data of two independent experiments (n). Compiled data from two independent experiments (n = 4 and 5 mice for uninfected and infected groups, respectively) (o). p, Percentages of Annexin V+ Aqua+ in total ILC3s from LI-LPLs of uninfected and infected WT mice at 12 h and 24 h post infection. Compiled data from two independent experiments (n = 4 and 5 mice for uninfected and infected groups, respectively). q and r, FACS analysis of IL-22 and IL-17 expression in LI CD4+ T cells of uninfected and infected WT mice on d.p.i 2. Representative data of two independent experiments (q). r, Absolute numbers of LI IL-22+ CD4+ T cells. Compiled data from two independent experiments (n = 4 and 6 mice for uninfected and infected groups, respectively). s, Expression value of Il22 normalized by DESeq2 in ILCs and myeloid cells from ImmGen database. t, FACS analysis of IL-22 expression in LI ILC3s of Il22+/+ and Il22Cre/Cre mice. Representative data of two independent experiments.

Source data

Extended Data Fig. 3 IL-22 was repressed by S. Typhimurium infection in late stage, (Related to Fig. 2).

a, LI of uninfected and S. Typhimurium-infected WT mice on d.p.i 8. bf, Analysis of RORγt, NKp46 and CD4 expression in LI Lin or LinNKp46 cells of uninfected and infected WT mice on d.p.i 8. Representative FACS data of two independent experiments (b). Percentages of ILC3s in LI Lin (c), NKp46+ ILC3s (NKp46+RORγt+ among Lin) (d), CD4+ ILC3s (CD4+RORγt+ among LinNKp46) (e), CD4 ILC3s (CD4RORγt+ among LinNKp46) (f) of uninfected and infected WT mice. Compiled data from two independent experiments (n = 5 and 6 mice for uninfected and infected groups, respectively). g and h, Analysis of T-bet and RORγt expression in LI Lin of uninfected and infected WT mice on d.p.i 8. Representative FACS data of two independent experiments (g). Percentages of ILC1s in Lin (h). Compiled data from two independent experiments (n = 5 and 6 mice for uninfected and infected groups, respectively). ik, Analysis of IFN-γ expression in LI CD4+ T cells or ILC1s of uninfected and infected WT mice on d.p.i 8. Representative FACS data of two independent experiments (i). Percentages of IFN-γ+ ILC1s (j) and CD4+ T cells (k). Compiled data from two independent experiments (n = 5 and 6 mice for uninfected and infected groups, respectively). lo, Analysis of IL-22 and IL-17 expression in LI ILC3s (l and m) or CD4+ T cells (n and o) of uninfected and infected WT mice on d.p.i 8. Representative FACS data of two independent experiments (l and n). Percentages of IL-22+ ILC3s (m) and IL-22+ and IL-17+ CD4+ T cells (o). Complied data from two independent experiments (n = 5 and 6 mice for uninfected and infected groups, respectively). p and q, WT and Rag1−/− mice were orally inoculated with 2 × 103 c.f.u. of S. Typhimurium. Body weight analysis using linear regression to compare slope difference (p) and survival curve with the log-rank test for statistical analysis (q). Complied data from two independent experiments (WT, n = 4 mice; Rag1−/−, n = 5 mice). rv, S. Typhimurium c.f.u. in feces (r), LI (s), liver (t), spleen (u) and mLNs (v) of Il22+/+ and Il22Cre/Cre mice on d.p.i 2 and 4. Compiled data from two independent experiments (n = 4 and 5 mice for d.p.i 2 and 4, respectively).

Source data

Extended Data Fig. 4 S. Typhimurium-secreted proteins enhance IL-22+ ILC3s in vitro, (Related to Fig. 3).

a, Schematic depiction of experimental design of in vitro LPL cultured with S. Typhimurium supernatant. bd, Analysis of IL-22 and IL-17 expression in WT LI-LPLs cultured with bacterial supernatant, with or without P/I stimulation. Representative FACS data of four independent experiments (b). Percentages of IL-22+ (c) and IL-17+ (d) ILC3s, with or without PMA and ionomycin (P/I) stimulation. Compiled data from four independent experiments (n = 9 repeats with independent mouse LPLs) (c and d). eg, Analysis of IL-22 and IL-17 expression in LI CD4+ T cells cultured with S. Typhimurium supernatant. Representative data of three independent experiments (e). Percentages of IL-17+ (f) and IL-22+ (g) CD4+ T cells. Compiled data from three independent experiments (n = 8 repeats with independent mouse LPLs) (f and g). h and i, Percentages of IFN-γ+ ILC1 and NK (h) and IFN-γ+ CD4+ T (i) cells. Compiled data from two independent experiments (n = 3 repeats with independent mouse LPLs). j and k, Il22 (j, n = 9 repeats with independent mouse LPLs) and Il17 (k, n = 3 repeats with independent mouse LPLs) mRNA determined by RT-qPCR in LI-LPLs of WT mice co-incubated with bacterial supernatant. Compiled data from two independent experiments. l, Silver staining of S. Typhimurium culture supernatant with or without proteinase K (PK) treatment. Representative data of two independent experiments. m and n, Analysis of IL-22 and IL-17 expression in ILC3s of LPLs cultured with S. Typhimurium supernatant with or without PK treatment (no P/I stimulation). Representative FACS data of two independent experiments (m). n, Percentages of IL-22+ ILC3s. Compiled data from two independent experiments (n = 3 repeats with independent mouse LPLs). o, Il22 mRNA determined by RT-qPCR in LI-LPLs co-incubated with S. Typhimurium culture supernatant with or without PK treatment. Representative data of two independent experiments (o) (n = 3 repeats with independent mouse LPLs). p and q, Analysis of IL-22 and IL-17 expression in ILC3s from LI-LPLs cultured with S. Typhimurium supernatant treated with or without heat (no P/I stimulation). Representative FACS data of two independent experiments (p). Percentages of IL-22+ ILC3s (q). Compiled data from two independent experiments (n = 3 repeats with independent mouse LPLs).

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Extended Data Fig. 5 S. Typhimurium flagellin protein promotes IL-22+ ILC3s but not IL-22+ CD4+ T cells, (Related to Fig. 3).

a and b, Analysis of IL-22 and IL-17 expression in ILC3s of LPLs cultured with bacterial supernatants of WT strain UK-1 or indicated S. Typhimurium mutants (no P/I stimulation). Representative FACS data of three independent experiments (a). b, Percentages of IL-22+ cells in ILC3s. Compiled data from three independent experiments (n = 3 repeats with independent mouse LPLs). 15 µl or 45 µl of S. Typhimurium culture supernatant was added, respectively. Empty medium (MgM) was included as a negative control. Fractionated supernatant fractions (< 3 kDa) were used in LPL culture as well. c and d, Analysis of IL-22 and IL-17 expression in ILC3s of LI-LPLs cultured with purified S. Typhimurium flagellin protein encoded by fljB (no P/I stimulation). Representative FACS data of two independent experiments (c). Percentages of IL-22+ cells in ILC3s (d). Compiled data from two independent experiments (n = 3 repeats with independent mouse LPLs). e and f, Analysis of IL-22 and IL-17 expression in sorted WT ILC3s (no P/I stimulation) co-cultured with purified S. Typhimurium flagellin proteins, with or without the addition of CD11c+ APCs from Tlr5+/+ or Tlr5−/− mice. Representative FACS data of two independent experiments (e). Percentages of IL-22+ cells in ILC3s (f). Compiled data from two independent experiments (n = 3 repeats with independently sorted LI cells from mice with indicated genotypes). g, FPKM of Tlr genes in sorted ILC3s from uninfected and infected WT mice (n = 3 mice per group). h, Expression value of Tlr5 normalized by DESeq2 in ILC3s and CD11c+ DCs from ImmGen database. i, Il23 (p19) mRNA determined by RT-qPCR in cell culture containing CD11c+ APCs sorted from the LI of Tlr5+/+ or Tlr5−/− mice, and sorted LI WT ILC3s (no P/I stimulation), stimulated with S. Typhimurium culture supernatant for 5 hours. Representative data of two independent experiments (n = 3 repeats with independently sorted LI cells from mice with indicated genotypes). 15 µl or 45 µl of S. Typhimurium culture supernatant was added, respectively. Empty medium (MgM) was included as a negative control.

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Extended Data Fig. 6 Stimulation of IL-22+ ILC3s by S. Typhimurium infection is mediated by CD11c+ APCs via TLR5-flagellin signaling, (Related to Fig. 3).

a, Schematic depiction of flagellin and its truncated mutant proteins. bd, Analysis of IL-22 and IL-17 expression in ILC3s of LI-LPLs cultured with S. Typhimurium flagellin proteins (no P/I stimulation). Representative FACS data of two independent experiments (b). Percentages of IL-22+ cells (c) and IL-22 MFI (d) in ILC3s. Compiled data from two independent experiments (n = 3 repeats with independently sorted LI cells from mice with indicated genotypes) (c and d). e, TLR5 activation by S. Typhimurium flagellin proteins as indicated by SEAP activities (OD650nm) in HEK-Blue-mTLR5 reporter cells. Representative data of two independent experiments (n = 3 independent wells per group). fh, Analysis of IL-22 and IL-17 expression in ILC3s from LI-LPLs of Myd88+/+ and Myd88−/− littermates, cultured with S. Typhimurium supernatant (no P/I stimulation). Representative FACS data of two independent experiments (f). Percentages of IL-22+ cells (g) and IL-22 MFI (h) in ILC3s. Compiled data from two independent experiments (n = 3 repeats with independently sorted LI cells from mice with indicated genotypes) (g and h). i and j, Analysis of IL-22 and IL-17 expression in sorted WT ILC3s (no P/I stimulation) co-cultured with S. Typhimurium flagellin, with or without the addition of CD11c+ APCs from Myd88+/+ or Myd88−/− mice. Representative FACS data of two independent experiments (i). Percentages of IL-22+ cells (j) in ILC3s. Compiled data from two independent experiments (n = 3 repeats with independently sorted LI cells from mice with indicated genotypes).

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Extended Data Fig. 7 Presence of ILC3s increases the susceptibility to S. Typhimurium infection in mice, (Related to Fig. 4).

a and b, Rorcgfp/+ and Rorcgfp/gfp mice were orally gavaged with 2 × 107 c.f.u. of S. Typhimurium. Body weight change (a) and survival curve (b) are compiled from two independent experiments (Rorcgfp/+, n = 10 mice; Rorcgfp/gfp, n = 10 mice). Linear regression for comparing slope difference in (a) and the log-rank test for statistical analysis of survival curve in (b). c, Bacterial c.f.u. in the feces of Rorcgfp/+ and Rorcgfp/gfp were measured on d.p.i 2 and 4. Compiled data from two independent experiments and presented as c.f.u. per mg of the feces (n = 9 mice per group). d, Rorcgfp/+Rag1−/− and Rorcgfp/gfpRag1−/− mice were orally inoculated with 2 × 107 c.f.u. of S. Typhimurium. Compiled data from two independent experiments (Rorcgfp/+Rag1−/−, n = 10 mice; Rorcgfp/gfpRag1−/−, n = 10 mice). Linear regression for comparing slope difference. e, Heatmap of all of the differentially expressed genes (fold change ≥ 1.5, q value ≤ 0.05) and pathway analysis in the RNA-seq of sorted ILC3s (LinCD90hiCD45lo) from LI of uninfected and infected WT mice (n = 3 mice per group). f, Top 10 pathways of the differentially expressed genes identified by RNA-seq in ILC3s (fold change ≥ 1.5, q value ≤ 0.05) from LI of uninfected and infected WT mice (n = 3 mice per group). g, GSEA showing enrichment of apoptosis-related genes in LI ILC3s from infected mice, compared with ILC3s from uninfected mice. h, FPKM of inflammasome genes in sorted ILC3s from uninfected or infected mice (n = 3 mice per group). i, Expression value of Gsdmd and Casp1 normalized by DESeq2 in ILC3s from ImmGen database.

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Extended Data Fig. 8 Pyroptosis in gut ILC3s was induced by S. Typhimurium, (Related to Fig. 5).

a and b, Analysis of Ki-67 and RORγt expression in ILC3s from LI-LPLs of uninfected and S. Typhimurium-infected WT mice on d.p.i 2. Representative FACS data of two independent experiments. Percentages of Ki-67+ cells in ILC3s (b). Compiled data from two independent experiments (n = 6 mice per group). c and d, Analysis of Aqua and IL-22 expression in LI ILC3s of uninfected and infected WT mice on d.p.i 2. Representative FACS data of two independent experiments (c). Percentages of Aqua+ cells in IL-22+ vs IL-22 ILC3s (d). Compiled data from two independent experiments (n = 5 and 7 mice for uninfected and infected groups, respectively). e, FACS analysis of active caspase-1 expression (Ac-YVAD-cmk, FLICA assay) in LI ILC3s of Casp1+/+ and Casp1−/− mice. Representative data of two independent experiments f, FACS analysis of active caspase-1 expression in LI ILC2s of uninfected and infected WT mice on d.p.i 2. Representative data of two independent experiments. g, FACS analysis of Aqua and Annexin V expression in LI ILC3s uninfected and infected with S. Typhimurium in vitro. Cells were pretreated with Ac-YVAD-cmk or Disulfiram as indicated. Representative data of two independent experiments. h, FACS analysis of IL-22 and IL-17 expression in MNK-3 cells, with or without P/I stimulation. Representative data of two independent experiments. i, Cell viability of MNK-3 cells infected with S. Typhimurium counted by FACS. Compiled data from three independent experiments (n = 6 independent wells). j, Western blot analysis to determine GSDMD in cell lysates of MNK-3 cells uninfected or infected with S. Typhimurium. Representative data of two independent experiments. k, Quantitative evaluation of mature IL-1β secreted in the culture supernatant of S. Typhimurium-infected MNK-3 cells using ELISA, with or without pretreatment of Ac-YVAD-cmk or Disulfiram. l, Quantitative evaluation of IL-22 secreted in the culture supernatant of S. Typhimurium-infected MNK-3 cells using ELISA, with or without pretreatment of Ac-YVAD-cmk or Disulfiram.

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Extended Data Fig. 9 Caspase-1 controls ILC3 cell death in a cell-intrinsic manner during S. Typhimurium infection, (Related to Fig. 6).

a and b, FACS analysis of RORγt and Lin expression (a), and Aqua and Annexin V expression (b) in LI-LPLs of S. Typhimurium-infected Casp1+/+ and Casp1−/− mice on d.p.i 2. Representative data of two independent experiments. c and d, Bone-marrow chimeric mice were orally gavaged with 2 × 107 c.f.u. of S. Typhimurium and assessed for survival following infection. Survival curve with the log-rank test for statistical analysis (c) and body weight change with linear regression to compare slope difference (d) are compiled from two independent experiments (Casp1WT: n = 5 mice; Casp1ΔILC3: n = 6 mice). e, Working model of ILC1 and ILC3 responses during S. Typhimurium infection, and pyroptotic cell death of ILC3s to defend the host against S. Typhimurium infection.

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Xiong, L., Wang, S., Dean, J.W. et al. Group 3 innate lymphoid cell pyroptosis represents a host defence mechanism against Salmonella infection. Nat Microbiol 7, 1087–1099 (2022). https://doi.org/10.1038/s41564-022-01142-8

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