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Cited by 190NF-κB Signaling PathwaysInflammatory Bowel DiseaseCell death mechanisms and regulation

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NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions

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Vlantis, Katerina, Wullaert, Andy, Polykratis, Apostolos, Kondylis, Vangelis, Dannappel, Marius, Schwarzer, Robin, Welz, Patrick, Corona, Teresa, Walczak, Henning, Weih, Falk, Klein, Ulf, Kelliher, Michelle, Pasparakis, Manolis·2016·Immunity
DOI 10.1016/j.immuni.2016.02.020PMC PMC4803910
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Abstract

Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

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Graphical abstract CC BY-NC-ND · PubMed Central
Figure 1 Colitis in NEMOIEC-KO Mice Is Driven by the Microbiota and Epithelial TNFR1 Signaling (A) Representative images of colon sections from NEMOIEC-KO and NemoFL littermates stained with H&E or immunostained for cleaved caspase-3 (cl. casp3). (B) Graph depicting scores for inflammation (infl), tissue damage (dam), and overall histopathology of colon sections from NEMOIEC-KO and NemoFL mice (n = 11–16 mice per genotype). (C) Graph depicting percentage of crypts with cl. casp. 3 stained cells on colon sections of NEMOIEC-KO and NemoFL mice (n = 5 or 6 mice per genotype). (D) Graphs depicting mRNA levels of the indicated genes normalized to Tbp in the colon of NEMOIEC-KO and NemoFL mice (n = 7 mice per genotype). (E) Representative images of colon sections from NEMOIEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL littermates. (F) Graph depicting histopathological scores of colon sections from NEMO IEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL mice (n = 4–6 mice per genotype). (G) Graph depicting percentage of crypts with cl. casp. 3 stained cells on colon sections of NEMOIEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL littermates (n = 4–6 mice per genotype). (H) Graphs depicting mRNA levels of the indicated genes in the colon of NEMOIEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL mice (n = 4–6 mice per genotype). (I) Representative images of colon sections from germ-free or germ-free NEMOIEC-KO and NemoFL mice 4 weeks after cohousing with SPF animals. (J–L) Graphs depicting histopathological scores of colon sections, percentage of crypts with cl. casp. 3 stained colon IECs, and mRNA levels of Tnf in colons from germ-free, conventionalized, and SPF housed NEMOIEC-KO and NemoFL mice (n = 3–11 mice per genotype). Scale bars represent 100 μm. See also Figure S1.
Figure 1 Colitis in NEMOIEC-KO Mice Is Driven by the Microbiota and Epithelial TNFR1 Signaling (A) Representative images of colon sections from NEMOIEC-KO and NemoFL littermates stained with H&E or immunostained for cleaved caspase-3 (cl. casp3). (B) Graph depicting scores for inflammation (infl), tissue damage (dam), and overall histopathology of colon sections from NEMOIEC-KO and NemoFL mice (n = 11–16 mice per genotype). (C) Graph depicting percentage of crypts with cl. casp. 3 stained cells on colon sections of NEMOIEC-KO and NemoFL mice (n = 5 or 6 mice per genotype). (D) Graphs depicting mRNA levels of the indicated genes normalized to Tbp in the colon of NEMOIEC-KO and NemoFL mice (n = 7 mice per genotype). (E) Representative images of colon sections from NEMOIEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL littermates. (F) Graph depicting histopathological scores of colon sections from NEMO IEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL mice (n = 4–6 mice per genotype). (G) Graph depicting percentage of crypts with cl. casp. 3 stained cells on colon sections of NEMOIEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL littermates (n = 4–6 mice per genotype). (H) Graphs depicting mRNA levels of the indicated genes in the colon of NEMOIEC-KO TNFR1IEC-KO, and NemoFLTnfr1FL mice (n = 4–6 mice per genotype). (I) Representative images of colon sections from germ-free or germ-free NEMOIEC-KO and NemoFL mice 4 weeks after cohousing with SPF animals. (J–L) Graphs depicting histopathological scores of colon sections, percentage of crypts with cl. casp. 3 stained colon IECs, and mRNA levels of Tnf in colons from germ-free, conventionalized, and SPF housed NEMOIEC-KO and NemoFL mice (n = 3–11 mice per genotype). Scale bars represent 100 μm. See also Figure S1.CC BY-NC-ND · PubMed Central
Figure 2 Reduced Paneth Cell Numbers and Increased IEC Apoptosis in Ileal Crypts of NEMOIEC-KO Mice (A and G) Representative images of ileal sections from NEMOIEC-KO and NemoFL, NEMOtamIEC-KO, and NemoFL littermates 8 days after tamoxifen administration stained with H&E or immunostained for cl. casp3 or lysozyme. (B and K) Graph depicting histopathological scores of ileal sections from NEMOIEC-KO and NemoFL, NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 6–9 mice per genotype). (C, F, J, and L) Graphs depicting mRNA levels of the indicated genes in the ileum of NEMOIEC-KO and NemoFL mice and NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 5–8 mice per genotype). (D and H) Graphs depicting percentage of crypts with cl. casp. 3 stained IECs on ileal sections from NEMOIEC-KO and NemoFL mice and NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 6–8 mice per genotype). (E and I) Graphs depicting Paneth cell scores of ileal sections from NEMOIEC-KO and NemoFL mice and NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 6–9 mice per genotype). Scale bars represent 100 μm. See also Figure S2.
Figure 2 Reduced Paneth Cell Numbers and Increased IEC Apoptosis in Ileal Crypts of NEMOIEC-KO Mice (A and G) Representative images of ileal sections from NEMOIEC-KO and NemoFL, NEMOtamIEC-KO, and NemoFL littermates 8 days after tamoxifen administration stained with H&E or immunostained for cl. casp3 or lysozyme. (B and K) Graph depicting histopathological scores of ileal sections from NEMOIEC-KO and NemoFL, NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 6–9 mice per genotype). (C, F, J, and L) Graphs depicting mRNA levels of the indicated genes in the ileum of NEMOIEC-KO and NemoFL mice and NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 5–8 mice per genotype). (D and H) Graphs depicting percentage of crypts with cl. casp. 3 stained IECs on ileal sections from NEMOIEC-KO and NemoFL mice and NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 6–8 mice per genotype). (E and I) Graphs depicting Paneth cell scores of ileal sections from NEMOIEC-KO and NemoFL mice and NEMOtamIEC-KO and NemoFL littermates 8 days after tamoxifen administration (n = 6–9 mice per genotype). Scale bars represent 100 μm. See also Figure S2.CC BY-NC-ND · PubMed Central
Figure 3 Paneth Cell Death in NEMOIEC-KO Mice Is Independent of the Microbiota and Epithelial Death Receptor Signaling (A and F) Representative images of ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice. (B and I) Graphs depicting Paneth cell scores of ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 5–9 mice per genotype). (C and J) Graphs depicting mRNA levels of the indicated genes in the ileum of germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 3–7 mice per genotype). (D and H) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 5–9 mice per genotype). (E and G) Graphs depicting histopathological scores of ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 5–9 mice per genotype). Scale bars represent 100 μm. See also Figure S3.
Figure 3 Paneth Cell Death in NEMOIEC-KO Mice Is Independent of the Microbiota and Epithelial Death Receptor Signaling (A and F) Representative images of ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice. (B and I) Graphs depicting Paneth cell scores of ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 5–9 mice per genotype). (C and J) Graphs depicting mRNA levels of the indicated genes in the ileum of germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 3–7 mice per genotype). (D and H) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 5–9 mice per genotype). (E and G) Graphs depicting histopathological scores of ileal sections from germ-free NEMOIEC-KO and NemoFL mice, and of SPF NEMOIEC-KO DRIEC-KO and NemoFL DRFL mice (n = 5–9 mice per genotype). Scale bars represent 100 μm. See also Figure S3.CC BY-NC-ND · PubMed Central
Figure 4 Combined Deficiency in FADD and RIPK3 Prevents IEC Death, Paneth Cell Loss, and Colitis in NEMOIEC-KO Mice (A and F) Representative images of colon and ileal sections from mice with the indicated genotypes. (B) Immunoblot analysis of protein extracts from primary small intestinal IECs from mice with the indicated genotypes and with the indicated antibodies. (C and G) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 5 or 6 mice per genotype). (D and I) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 5 or 6 mice per genotype). (E, J, and K) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 9 or 10 mice per genotype). (H) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 5 or 6 mice per genotype). Scale bars represent 100 μm.
Figure 4 Combined Deficiency in FADD and RIPK3 Prevents IEC Death, Paneth Cell Loss, and Colitis in NEMOIEC-KO Mice (A and F) Representative images of colon and ileal sections from mice with the indicated genotypes. (B) Immunoblot analysis of protein extracts from primary small intestinal IECs from mice with the indicated genotypes and with the indicated antibodies. (C and G) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 5 or 6 mice per genotype). (D and I) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 5 or 6 mice per genotype). (E, J, and K) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 9 or 10 mice per genotype). (H) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 5 or 6 mice per genotype). Scale bars represent 100 μm.CC BY-NC-ND · PubMed Central
Figure 5 RIPK3 Deficiency Ameliorates Colitis but Does Not Prevent Paneth Cell Loss in NEMOIEC-KO Mice (A and E) Representative images of colon and ileal sections from mice with the indicated genotypes. (B and F) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 12–17 mice per genotype). (C and G) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 11–16 mice per genotype). (D) Graphs depicting mRNA levels of the indicated genes in the colon of mice with the indicated genotypes (n = 8–12 mice per genotype). (H) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 12–17 mice per genotype). Scale bars represent 100 μm. See also Figure S4.
Figure 5 RIPK3 Deficiency Ameliorates Colitis but Does Not Prevent Paneth Cell Loss in NEMOIEC-KO Mice (A and E) Representative images of colon and ileal sections from mice with the indicated genotypes. (B and F) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 12–17 mice per genotype). (C and G) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 11–16 mice per genotype). (D) Graphs depicting mRNA levels of the indicated genes in the colon of mice with the indicated genotypes (n = 8–12 mice per genotype). (H) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 12–17 mice per genotype). Scale bars represent 100 μm. See also Figure S4.CC BY-NC-ND · PubMed Central
Figure 6 Lack of RIPK1 Kinase Activity Prevents Colitis Development and Paneth Cell Loss in NEMOIEC-KO Mice (A and E) Representative images of colon and ileal sections from mice with the indicated genotypes. (B and F) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 6–13 mice per genotype). (C and I) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 4–12 mice per genotype). (D, H) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 6 or 7 mice per genotype). (G) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 6–10 mice per genotype). (J) Immunoblot analysis of the indicated proteins co-immunoprecipitated with FADD from total colon lysates from mice with the indicated genotypes (one out of three independent experiments shown). Scale bars represent 100 μm.
Figure 6 Lack of RIPK1 Kinase Activity Prevents Colitis Development and Paneth Cell Loss in NEMOIEC-KO Mice (A and E) Representative images of colon and ileal sections from mice with the indicated genotypes. (B and F) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 6–13 mice per genotype). (C and I) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 4–12 mice per genotype). (D, H) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 6 or 7 mice per genotype). (G) Graph depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 6–10 mice per genotype). (J) Immunoblot analysis of the indicated proteins co-immunoprecipitated with FADD from total colon lysates from mice with the indicated genotypes (one out of three independent experiments shown). Scale bars represent 100 μm.CC BY-NC-ND · PubMed Central
Figure 7 Epithelial NF-κB Deficiency Does Not Trigger Colitis but Causes RIPK1 Kinase Dependent Paneth Cell Death (A, F, and J) Representative images of colon and ileal sections from mice with the indicated genotypes. (B, G, and K) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 5–18 mice per genotype). (C, H, and L) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 4–12 mice per genotype). (D and M) Graphs depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 4–12 mice per genotype). (E, I, and N) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 5–12 mice per genotype). Scale bars represent 100 μm. See also Figures S5 and S6.
Figure 7 Epithelial NF-κB Deficiency Does Not Trigger Colitis but Causes RIPK1 Kinase Dependent Paneth Cell Death (A, F, and J) Representative images of colon and ileal sections from mice with the indicated genotypes. (B, G, and K) Graphs depicting histopathological scores of colon and ileal sections from mice with the indicated genotypes (n = 5–18 mice per genotype). (C, H, and L) Graphs depicting the percentage of crypts with cl. casp. 3 stained cells on colon and ileal sections from littermates with the indicated genotypes (n = 4–12 mice per genotype). (D and M) Graphs depicting Paneth cell scores of ileal sections from mice with the indicated genotypes (n = 4–12 mice per genotype). (E, I, and N) Graphs depicting mRNA levels of the indicated genes in the colon and ileum of mice with the indicated genotypes (n = 5–12 mice per genotype). Scale bars represent 100 μm. See also Figures S5 and S6.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Vlantis, K., Wullaert, A., Polykratis, A., Kondylis, V., Dannappel, M., Schwarzer, R., Welz, P., Corona, T., Walczak, H., Weih, F., Klein, U., Kelliher, M., & Pasparakis, M.
Affiliations
  • University of Cologne (DE)
  • University College London (GB)
  • Leibniz Institute on Aging - Fritz Lipmann Institute (FLI) (DE)
  • Herbert Irving Comprehensive Cancer Center (US)
  • University of Massachusetts Chan Medical School (US)
Corresponding author
Pasparakis, Manolis
ORCID
Venue
Immunity
Publisher
Elsevier BV
Published
2016-03
Volume
44
Issue
3
Pages
553-567
ISSN
1074-7613 (ISSN-L)
DOI
10.1016/j.immuni.2016.02.020
PMC
PMC4803910
OpenAlex
W2299243013
Cited by
189
References
38
Citations by year
2026: 8 · 2025: 11 · 2024: 22 · 2023: 19 · 2022: 16 · 2021: 20 · 2020: 24 · 2019: 20 · 2018: 25 · 2017: 19 · 2016: 4
FWCI
9.46
Type
journal article
Language
en
Open access
Licence
Locations
  • Immunity (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Informationszentrum Lebenswissenschaften (ZB MED) (submittedVersion, cc-by-nc-nd)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion, other-oa)
Funders
  • European Commission (223151, 223404)
  • Deutsche Forschungsgemeinschaft (SPP-1656, SFB 670)
  • Universität Ulm
  • National Institutes of Health
  • Genentech
  • National Institute of Allergy and Infectious Diseases (R01AI075118)
  • FP7 Ideas: European Research Council (323040)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › NF-κB Signaling Pathways
  • Health Sciences › Medicine › Gastroenterology › Inflammatory Bowel Disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Cell death mechanisms and regulation
Keywords
NEMO, RIPK1, NF-κB, intestinal epithelial cells, epithelial cell death, chronic intestinal inflammation, colitis, inflammatory bowel disease, FADD, RIPK3, microbiota, RelA, c-Rel, RelB, RIPK1 inhibitors
MeSH terms
Animals, Cells, Cultured, Humans, Intestinal Mucosa, Mice, Inbred C57BL, Inflammatory Bowel Diseases, NF-kappa B, Apoptosis, Mice, Knockout, Paneth Cells, Proto-Oncogene Proteins c-rel, Intracellular Signaling Peptides and Proteins, Mice, Transcription Factor RelB, Transcription Factor RelA, Fas-Associated Death Domain Protein, Receptor-Interacting Protein Serine-Threonine Kinases

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