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The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death

Abstract

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn’s disease, a major type of inflammatory bowel disease1,2,3,4,5,6,7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn’s disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte–epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

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Fig. 1: γδ IELs restore Paneth cells and improve the viability of ATG16L1-deficient intestinal organoids.
Fig. 2: API5 secreted from γδ IELs promotes viability of ATG16L1-deficient organoids.
Fig. 3: γδ IELs and API5 prevent Paneth cell loss and protect against intestinal injury in Atg16l1-mutant mice.
Fig. 4: API5 protects mouse and human Paneth cells against the detrimental effects of ATG16L1 mutation.

Data availability

Sequencing data were deposited to Gene Expression Omnibus (GEO) under the accession number GSE204822.

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Acknowledgements

We thank T. Shiomi and the Center for Biospecimen Research and Development, Histology and Immunohistochemistry laboratory (NYU Langone Health, NIH/NCI P30CA016087) for technical support in preparation of histology slides of human organoids; M. B. da Silva, Y. Shono and M. R. M. van den Brink for their support with TUNEL and cleaved caspase-3 staining; K. A. Lacey and V. J. Torres for their support in cytokine quantification; S. Y. Kim and Rodent Genetic Engineering Laboratory (RGEL) (NYU Langone Health, NIH/NCI P30CA016087) for generating Api5-knockout mice; the NYU Langone Health’s Cytometry and Cell Sorting Laboratory (NIH/NCI P30ACA016087) for their assistance with cell sorting; DART Microcopy Lab (NIH/NCI P30CA016087) for their assistance with microscopic analysis; Experimental Pathology Research Laboratory (NIH/NCI P30CA016087) for their assistance with preparation and staining of mouse intestine samples; and Genome Technology Center (NIH/NCI P30CA0167087) for their assistance with single-cell RNA-sequencing analysis. Cartoon images in Fig. 1a and Extended Data Figs. 1c, 4f and 8b are adapted from the mouse, organoid, T cell and intestine templates at BioRender.com. This work was supported in part by US National Institute of Health (NIH) grants HL123340 (K.C.), DK093668 (K.C.), AI140754 (K.C.), AI121244 (K.C.), AI130945 (K.C.), DK124336 (K.C.), and DK088199 (R.S.B.); Faculty Scholar grant from the Howard Hughes Medical Institute (K.C.), Synergy Award from the Kenneth Rainin Foundation (K.C.), Senior Research Award from the Crohn’s and Colitis Foundation (K.C.), Research Fellowship Award from Crohn’s and Colitis Foundation (Y.M.-I.), and a pilot award from the Takeda–Columbia–NYU Alliance (K.C. and S.K.).

Author information

Authors and Affiliations

Authors

Contributions

Y.M.-I., S.K. and K.C. formulated the original hypothesis, designed the study and analysed the results. Y.M.-I. contributed to all the experiments and data analysis. X.Y. and A.K. generated constructs for producing rAPI5 proteins and designed the Api5-knockout gene-targeting strategy. A.K. and S.K. produced rAPI5 proteins. B.M.U. performed and analysed LC–MS of supernatant samples. J.E.A. collected the human endoscopic samples. B.S.R., R.P. and D.M. performed the intravital imaging and whole-mount imaging, and provided technical support in FACS analyses of IELs. J.A.N. produced MNV stocks and performed the bacterial translocation assay. J.C.D. and K.V.R. performed analysis of scRNA-seq data. E.R. provided assistance with western blot and immunoprecipitation experiments. M.Z.D. contributed to the preparation of histology samples. M.C. assisted with microscopy. R.S.B. generated and provided the Defa6-cre mouse. Y.D. performed histopathology analyses. Y.M.-I., S.K. and K.C. wrote the manuscript, and all authors commented on the manuscript, data and conclusions.

Corresponding authors

Correspondence to Shohei Koide or Ken Cadwell.

Ethics declarations

Competing interests

K.C. has received research support from Pfizer, Takeda, Pacific Biosciences, Genentech and Abbvie. K.C. has consulted for or received honoraria from Puretech Health, Genentech, Abbvie, GentiBio and Synedgen. K.C. is an inventor on US patent 10,722,600 and provisional patent 62/935,035, and K.C., S.K., A.K. and Y.M.-I. are inventors on US patent 63/157,225. S.K. was a scientific advisory borard member of, held equity in and received consulting fees from Black Diamond Therapeutics, has received research support from Argenx BVBA, Black Diamond Therapeutics and Puretech Health and is a co-founder of Revalia Bio.

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Nature thanks Charles Bevins and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 IELs display altered motility and positioning in MNV-infected mice (related to supplementary videos 14).

a-b, TCRδGFP mice were anesthetized and prepped for intravital imaging analyses 18 h post MNV-infection. Naïve TCRδGFP mice were used as control (see supplementary movies 3 and 4). a, Unbiased computational quantification (mean and SEM) of flossing. Data analysis with two-sided unpaired Student’s t-test. Data points are the average flossing per animal, from 2 independent experiments. b, Unbiased computational quantification (mean and SEM) of cell speed. Each dot represents the average speed of each cell. Data pooled of 3 animals, from 2 independent experiments. c, d, Transversal segments of the ileum from MNV-infected (red) and naïve (blue) TCRδGFP mice were fixed in 4% PFA and imaged using multiphoton microscope. c, γδ T cell distribution along the villi, from crypt (0) to tip of the villus (100). d, Mean distribution of γδ T cell along the villi. Data points in (c) and (d) represent a segment. Bars represent means ± SEM.

Source data

Extended Data Fig. 2 Flow cytometric characterization of IELs in MNV-infected mice.

al, The IEL compartment of the small intestine from WT mice euthanized on day 10 post MNV infection were analyzed by flow cytometry and compared with naïve WT control mice. a, Representative flow cytometry plots showing gating strategy. b, Total number of live IELs harvested from whole small intestine. n = 40 (naïve) and 40 (MNV). cl, Absolute number and proportion of the indicated subpopulations in the small intestine. n = 10 (naïve) and 10 (MNV). Data analysis with two-sided unpaired Student’s t-test in (bl). Data points in (b)–(l) are individual mice. Bars represent means ± SEM, and at least two independent experiments were performed.

Source data

Extended Data Fig. 3 Single cell RNA sequencing of IELs in MNV-infected mice.

a-c, CD45+CD103+ IELs from the small intestines of WT mice euthanized on day 10 post MNV-infection or naïve mice were analyzed for single cell RNA sequencing (scRNA-seq). a, Representative flow cytometry plots showing the gating strategy used to sort CD45+CD103+ IELs. b,c, UMAP analysis of CD45+CD103+ IELs profiled by scRNA-Seq and colored by unbiased louvain clustering (b) and MNV infection status (c). n = 4 (naïve) and 4 (MNV).

Extended Data Fig. 4 Analyses of secreted products by γδ IELs.

a, Western blot analysis of supernatant (SN) harvested from TCRγδ+ IELs sorted from naïve WT mice either unstimulated (incubated in the medium for 4 h) or stimulated with anti-CD3/CD28 for 24 h. b-d, Representative images (b), viability (c), and area (d) of small intestinal organoids from ΔIEC mice cultured for 48 h with either unstimulated or stimulated γδ SN in (a). Scale bar 25 μm. e, Viability of small intestinal organoids from Atg16L1f/f (f/f) and Atg16L1ΔIEC (ΔIEC) mice cultured ± 10 ng/ml recombinant KGF. Intestinal crypts were harvested from 3 mice per genotype. f, Schematic representation for the preparation of IEL SN for LC-MS. IELs were harvested from small intestine of naïve WT mice, and cultured with serum free medium stimulated with anti-CD3/CD28 for 24 h. g, Quantification of the indicated cytokines in either γδ or αβ SN harvested from naïve WT mice (n = 5). An ANOVA with Tukey’s multiple-comparison test in (c, d). Two-sided unpaired Student’s t-test in (e, g). Data points in (c) represent organoid viability in each well, data points in (d) represent individual organoids, data points in (e) are mean of organoid viabilities performed in triplicate, and data points in (g) represent individual mice. Bars represent means ± SEM, and at least two independent experiments were performed.

Source data

Extended Data Fig. 5 Generation of recombinant API5 protein and effect of environmental triggers on the secretome of IELs.

a, Architecture of API5. The N and C termini, the HEAT and ARM-like domains, residues mutated in our study (Y8, Y11) are indicated. b, Size-exclusion chromatograms of Superdex200 (GE Healthcare) of wild-type and (Y8K:Y11K) rAPI5, indicating that they are predominantly monomeric. c, Thermal denaturation of API5 monitored using SYPRO Orange binding. The graph shows the first derivative of fluorescence intensity (n = 3). d, Viability of small intestinal organoids from Atg16L1ΔIEC (ΔIEC) mice cultured ± 50 nM and 200 nM wild-type or mutant (Y8K:Y11K) recombinant API5 for 48 h. e, Western blot analysis of SN samples harvested from TCRγδ+ IELs stimulated with anti-CD3/CD28 for 24 h corresponding to Fig. 2i. Total SN was equally divided into three; 1 left untreated (Pre-IP), and the other 2 were immunoprecipitated with anti-API5 antibody or control IgG antibody-conjugated magnetic beads for 24 h. f, g, Representative images (f) and viability (g) of small intestinal organoids from ΔIEC mice stimulated ± 0.5 ng/ml IFNγ for 48 h after pretreatment with 50 nM wild-type rAPI5 for 3 days. Scale bar 25 μm. h, i, Representative H&E images (h) and quantification (i) of Paneth cell and total IEC number per organoid stimulated ± 0.5 ng/ml IFNγ ± 50 nM wild-type rAPI5 for 24 h after pretreatment with 50 nM wild-type rAPI5 for 4 days. Arrowheads indicate Paneth cells. Scale bar 20 μm. j, Quantification of the indicated cytokines in SN of γδ IELs harvested either from naïve or MNV-infected WT mice (n = 5 per condition). k, Western blot analysis of SN from γδ IELs harvested from naïve, Salmonella-infected, or indomethacin-treated WT mice following stimulation with anti-CD3/CD28 for 24 h. l, Quantification of API5 normalized to PGRP-L by densitometric analyses of (k). Each value is divided by naïve. In organoid experiments, intestinal crypts were harvested from 3 mice per genotype. Two-sided unpaired Student’s t-test in (d, j). An ANOVA with Tukey’s multiple-comparison test in (g, i). Two-sided paired Student’s t-test in (l). Data points in (d) and (g) represent organoid viability in each well, data points in (i) represent individual organoids, data points in (j) represent individual mice, and data points in (l) represent API5/PGRP-L value in each western blot. Bars represent means ± SEM, and at least two independent experiments were performed.

Source data

Extended Data Fig. 6 Additional characterization of mice deficient in γδ T cells and ATG16L1 in the epithelium.

a, Colony forming units (CFU) of bacteria in mesenteric lymph nodes harvested from naïve Atg16L1f/f (f/f), Atg16L1ΔIEC (ΔIEC), Atg16L1f/f TCRδ−/− (f/f TCRδ−/−), and Atg16L1ΔIEC TCRδ−/− (ΔIEC TCRδ−/−) mice. n = 7 (f/f), 7 (ΔIEC), 7 (f/f TCRδ−/−), and 7 (ΔIEC TCRδ−/−). LOD; limit of detection. Data analysis with an ANOVA with Tukey’s multiple-comparison test. b, c, Representative images of periodic acid-Schiff (PAS)/Alcian blue staining (b) and quantification of goblet cells (c) in the small intestinal samples harvested from naïve f/f, ΔIEC, f/f TCRδ−/−, and ΔIEC TCRδ−/− mice. n = 11 (f/f), 11 (ΔIEC), 10 (f/f TCRδ−/−), and 10 (ΔIEC TCRδ−/−). Scale bar 100 μm. di, Absolute number and proportion of the indicated subpopulations in the small intestine. n = 9 (f/f), 6 (ΔIEC), 8 (f/f TCRδ−/−), and 7 (ΔIEC TCRδ−/−). j, Western blot analysis of SN harvested from TCRαβ+ IELs sorted from naïve f/f, ΔIEC, f/f TCRδ−/−, and ΔIEC TCRδ−/− mice. The IELs were stimulated with anti-CD3/CD28 for 24 h. Each cell was pooled from 3 mice per genotype. Data points in (a), (c)–(i) are individual mice. Bars represent means ± SEM, and at least two independent experiments were performed.

Source data

Extended Data Fig. 7 γδ IELs and rAPI5 protect Atg16L1ΔIEC mice against DSS-induced intestinal inflammation.

ae, Atg16L1f/f (f/f), Atg16L1ΔIEC (ΔIEC), Atg16L1f/f TCRδ−/− (f/f TCRδ−/−), and Atg16L1ΔIEC TCRδ−/− (ΔIEC TCRδ−/−) mice were treated with 5% DSS, and euthanized on day 5. n = 7 (f/f), 7 (ΔIEC), 7 (f/f TCRδ−/−), and 7 (ΔIEC TCRδ−/−). a, Quantification of the indicated cytokines in SN harvested from gut explants. b, c, d, e, Representative images of H&E staining (b and d) and quantification of villi length (c) and Paneth cells (e). Scale bar 100 μm (b) and 20 μm (e). f, g, Survival (f) and disease score on day 6 (g) of f/f TCRδ−/− and ΔIEC TCRδ−/− mice injected intravenously with 40 μg/mouse of wild-type or Y8K:Y11K rAPI5 protein on day 0, 3, and 6, while treated with 5% DSS for 6 days. n = 9 (f/f TCRδ−/− rAPI5WT), 8 (ΔIEC TCRδ−/− rAPI5WT), 6 (f/f TCRδ−/− rAPI5Y8K:Y11K), and 7 (ΔIEC TCRδ−/− rAPI5Y8K:Y11K). An ANOVA with Tukey’s multiple-comparison test in (a, c, e, g). Mantel-Cox log-rank test in (f). Data points in (a), (c), and (e) are individual mice, and data points in (g) are mean of disease scores of viable mice. Bars represent means ± SEM, and at least two independent experiments were performed.

Source data

Extended Data Fig. 8 Generation and additional characterization of Api5-knockout mice.

a, Schematic strategy for the generation and genotyping of Api5 knockout mouse. The CRISPR-Cas9 gene targeting mixture containing sgRNAs #1 and #2 targeting exon 1 of Api5 and Cas9 mRNA were injected into zygotes generated from Atg16L1f/f females impregnated by Atg16L1ΔIEC males. The 23 resulting chimeras were screened through a PAGE-based genotyping approach4 in which amplicons generated using the indicated primers and tail DNA from chimeras and wildtype mice as templates were annealed. Heteroduplexes signifying mismatches between the wildtype sequence and the disrupted locus were used to identify 12 candidate knockout mice. Three candidates were selected for further backcrossing and breeding, one of which successfully produced reproductively viable offspring. b, Representative genotyping gel images. Middle gel shows byproducts from first PCR reaction and annealing process using primers from (a). Since Api5+/+ and Api5−/− mice cannot be distinguished by this approach, their PCR products were denatured and annealed with wild-type B6 tail DNA in a second reaction shown in the right gel, which yields multiple bands in the presence of sequence mismatches in Api5−/− and Api5+/− mice but not Api5+/+ mice. c, Sequencing of the Api5 locus from Api5 mutant mice identified a dinucleotide AT insertion after the third codon that causes a frameshift leading to an early stop codon at amino acid position 30. d, Expected and observed number of offspring mice with indicated genotypes from Atg16L1 flox/flox villinCre+ Api5+/− and Atg16L1 flox/flox villinCre Api5+/− breeders. e, Proportion of the indicated IEL subpopulations of the small intestine from Api5+/+and Api5+/− mice. n = 3 (Api5+/+) and 3 (Api5+/−). f, g, Representative H&E images (f) and quantification (g) of Paneth cells per organoid from Atg16L1ΔIEC (ΔIEC) mice co-cultured with ~5 x 104 γδ or αβ IELs harvested from Api5+/+ or Api5+/− mice. n = 30 per condition. h, i, Representative images (h) and viability (i) of small intestinal organoids from ΔIEC mice. Scale bar 50 μm. In organoid experiments, intestinal crypts were harvested from 3 mice per genotype, and the viability assay was performed in triplicate. An ANOVA with Tukey’s multiple-comparison test in (g, i). Data points in (e) are individual mice, data points in (g) represent individual organoids, and data points in (i) represent organoid viability in each well. Bars represent means ± SEM. At least two independent experiments were performed.

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Extended Data Fig. 9 Additional characterization of mice deficient in ATG16L1 in Paneth cells.

a, Survival of Atg16L1f/f (f/f) and Atg16L1ΔPC (ΔPC) mice treated with 3% DSS ± MNV-infection. n = 13 (f/f), 14 (ΔPC), 18 (f/f +MNV) and 14 (ΔPC +MNV). b, c, Representative images of H&E (b) and quantification of Paneth cells (c) of indicated mice euthanized on day 10 post MNV-infection. Arrowheads indicate Paneth cells. n = 5 (f/f), 5 (ΔPC), 10 (f/f +MNV) and 12 (ΔPC +MNV). Scale bar 20 μm. d, e, Representative images (d) and quantification of lysozyme staining (e) of indicated mice euthanized on day 10 post MNV-infection. Arrowheads indicate Paneth cells. n = 6 (f/f), 5 (ΔPC), 5 (f/f +MNV) and 6 (ΔPC +MNV). Scale bar 20 μm. f, g, Representative images (f) and viability (g) of small intestinal organoids from f/f and ΔPC mice. Scale bar 400 μm. h, Viability of small intestinal organoids from ΔPC mice stimulated ± 20 ng/ml TNFα and/or 20 μM Necrostatin-1 (Nec-1) for 48 h. i, Viability of small intestinal organoids from ΔPC mice transduced with lentiviruses encoding shRNA targeting Mlkl or nonspecific control and stimulated ± 20 ng/ml TNFα for 48 h. j, Representative Western blot image of MLKL and β-actin in small intestinal organoids from ΔPC mice after transduction with lentiviruses encoding indicated shRNAs. k, Viability of small intestinal organoids co-cultured for 48 h with 1 x 105 IELs harvested from naïve WT mice. In organoid experiments, intestinal crypts were harvested from 3 mice per genotype. Mantel-Cox log-rank test in (a). An ANOVA with Tukey’s multiple-comparison test in (c, e, h). Two-sided unpaired Student’s t-test in (g, i, k). Data points in (c) and (e) represent individual mice, data points in (g) are mean of organoid viabilities performed in triplicate, data points in (h), (i), and (k) represent organoid viability in each well. Bars represent means ± SEM, and survival data in (a) are combined results of at least 3 experiments performed independently. At least two independent experiments were performed in (j).

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Extended Data Fig. 10 rAPI5 protects Atg16L1ΔPC mice against DSS-induced intestinal inflammation.

Atg16L1f/f (f/f) and Atg16L1ΔPC (ΔPC) mice were treated with 5% DSS with or without injection of rAPI5 (either rAPI5WT or rAPI5Y8K; Y11K) on day 0, 3, and 6, and euthanized on day 8. n = 5 (f/f), 5 (ΔPC), 7 (f/f rAPI5WT), and 6 (ΔPC rAPI5WT), 5 (f/f rAPI5Y8K; Y11K), and 5 (ΔPC rAPI5Y8K; Y11K). a, Quantification of TNFα in SN harvested from gut explants. b, c, d, e, Representative images of H&E staining (b and d) and quantification of villi length (c) and Paneth cells (e). Scale bar 100 μm (b) and 20 μm (e). An ANOVA with Tukey’s multiple-comparison test in (a, c, e). Data points in (b), (c), and (e) are individual mice. Bars represent means ± SEM, and at least two independent experiments were performed.

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Supplementary information

Supplementary Figure 1

Original source images for western blots and genotyping gels (raw data).

Reporting Summary

Peer Review File

Supplementary Methods Table

List of antibodies used in p-MLKL and lysozyme co-staining. Detailed information for the antibodies used in phospho (p)-MLKL and lysozyme co-staining of intestinal organoids in Fig. 2n.

Supplementary Table 1

List of protein candidates detected in IEL supernatant. List of proteins detected in LC/MS (corresponding to Fig. 2b). Total proteins (left tab), unique in γδ (middle tab) and αβ IEL (right tab). Proteins which displayed #PSM 5≤ were highlighted with bold.

Supplementary Table 2

Patient information for API5 and TCRγδ co-staining experiment. M; Male, F; Female, non-IBD; individuals without inflammatory bowel disease, CD; Crohn’s disease.

Supplementary Table 3

Patient information for human organoids. NR; non-risk, R; Risk, M; Male, F; Female, CD; Crohn’s disease.

Supplementary Video 1

Intravital Imaging analyses of IELs in MNV-infected mice. Intravital multiphoton microscopic imaging of naive E8Itomato reporter mice. In red (tomato) CD8+ T cells, and in blue (Hoechst), inter-epithelial cell nuclei.

Supplementary Video 2

Intravital Imaging analyses of IELs in MNV-infected mice. Intravital multiphoton microscopic imaging of MNV-infected E8Itomato reporter mice. In red (tomato) CD8+ T cells, and in blue (Hoechst), inter-epithelial cell nuclei.

Supplementary Video 3

Intravital multiphoton microscopic imaging of naive TCRδGFP mice. In green (GFP) TCRγδ+ cells, and in blue (Hoechst), inter-epithelial cell nuclei.

Supplementary Video 4

Intravital multiphoton microscopic imaging of MNV-infected TCRδGFP mice. In green (GFP) TCRγδ+ cells, and in blue (Hoechst), inter-epithelial cell nuclei.

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Matsuzawa-Ishimoto, Y., Yao, X., Koide, A. et al. The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death. Nature 610, 547–554 (2022). https://doi.org/10.1038/s41586-022-05259-y

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