ORIGINAL RESEARCH RIPK1 Mediates TNF-Induced Intestinal Crypt Apoptosis During Chronic NF-kB Activation Jerry Wong,1,2,3,a Ricard Garcia-Carbonell,1,2,3,4,a Matija Zelic,5 Samuel B. Ho,6,7 Brigid S. Boland,6,8 Shih-Jing Yao,1,2,3 Shalin A. Desai,1,2,3 Soumita Das,3 Núria Planell,9 Philip A. Harris,10 Joan Font-Burgada,1,2,3 Koji Taniguchi,1,2,3 John Bertin,5 Azucena Salas,9 Manolis Pasparakis,11 Pete J. Gough,10 Michelle Kelliher,5 Michael Karin,1,2,3 and Monica Guma12,13 1 Laboratory of Gene Regulation and Signal Transduction, 2Department of Pharmacology, 3Department Pathology, 6Division of Gastroenterology, and 12Department Rheumatology, 8San Diego Digestive Disease Research Center, San Diego, California; 4 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain, 5 Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts; 10 Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania; 7Department of Medicine, VA San Diego Healthcare System, San Diego, California; 9Department of Gastroenterology, IDIBAPS, Hospital Clínic, CIBER-EHD, Barcelona, Spain; 11Institute for Genetics, Centre for Molecular Medicine and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany; 13Department of Medicine, Autonomous University of Barcelona, Plaça Cívica, Bellaterra, Barcelona, Spain METHODS: Human IBD specimens were stained for p65 and SUMMARY cleaved caspase-3. C57BL/6 mice with constitutively active IKKb in Here we describe how chronic nuclear factor–kB activation IEC (Ikkb(EE)IEC), Ripk1D138N/D138N knockin mice, and Ripk3-/- mice facilitates the formation of the ripoptosome, which enhances were injected with TNF or lipopolysaccharide. Enteroids were also tumor necrosis factor–induced apoptosis in intestinal isolated from these mice and challenged with TNF with or without epithelial cells. Blockade of the kinase activity of RIPK1 RIPK1 and RIPK3 inhibitors or butylated hydroxyanisole. prevents tumor necrosis factor’s destructive properties Ripoptosome-mediated caspase-8 activation was assessed by while preserving its survival and proliferative functions. immunoprecipitation. RESULTS: NF-kB activation in human IBD correlated with appearance of cleaved caspase-3. Congruently, unlike normal BACKGROUND AND AIMS: Tumor necrosis factor (TNF) is a mouse IECs that are TNF-resistant, IECs in Ikkb(EE)IEC mice major pathogenic effector and a therapeutic target in inflam- and enteroids were susceptible to TNF-dependent apoptosis, matory bowel disease (IBD), yet the basis for TNF-induced in- which depended on the protein kinase function of RIPK1. testinal epithelial cell (IEC) death is unknown, because TNF Constitutively active IKKb facilitated ripoptosome formation, does not kill normal IECs. Here, we investigated how chronic a RIPK1 signaling complex that mediates caspase-8 activa- nuclear factor (NF)- kB activation, which occurs in human IBD, tion by TNF. Butylated hydroxyanisole treatment and RIPK1 promotes TNF-dependent IEC death in mice. inhibitors attenuated TNF-induced and ripoptosome- 296 Wong et al Cellular and Molecular Gastroenterology and Hepatology Vol. 9, No. 2 mediated caspase-8 activation and IEC death in vitro and Ikkb(EE)IEC mice display severe TNF-dependent epithelial in vivo. layer destruction when challenged with TNF or various stimuli that induce TNF production.14 The mechanism by CONCLUSIONS: Contrary to common expectations, chronic NF- which constitutive IKKb/NF-kB activation renders mouse kB activation induced intestinal crypt apoptosis after TNF stimulation, resulting in severe mucosal erosion. RIPK1 kinase IEC susceptible to TNF-induced killing, rather than prevent inhibitors selectively inhibited TNF destructive properties while it, is unknown, but is likely to be relevant to the effect of preserving its survival and proliferative properties, which do not chronic NF-kB activation in IEC of active IBD lesions. We require RIPK1 kinase activity. RIPK1 kinase inhibition could be a have therefore investigated the mechanisms by which TNF potential treatment for IBD. (Cell Mol Gastroenterol Hepatol induces IEC death in Ikkb(EE)IEC mice. 2020;9:295–312; https://doi.org/10.1016/j.jcmgh.2019.10.002) We focused our studies on the function of RIPK1, a protein kinase that serves as a key regulator of life and death in TNF- exposed cells. Under conditions in which RIPK1 is subject to Keywords: IBD; RIPK1; Intestinal Epithelial Cell; Ripoptosome; Cell Death. K63-linked and linear ubiquitination, TNFR1 engagement in- duces cell survival, but when the RIPK1 ubiquitination pattern is altered, TNF induces 1 of 2 forms of programmed cell Inflammatory bowel disease (IBD), including ulcera- tive colitis (UC) and Crohn’s disease (CD), represent a major cause of morbidity and mortality, affecting approxi- death: necroptosis15,16 or noncanonical apoptosis that is not inhibited by NF-kB.17 The latter depends on formation of a RIPK1-dependent signaling complex that also contains FADD mately 1.4 million Americans.1 IBDs are characterized by and caspase-8, known as complex IIb or the ripoptosome.17 colon or small intestine inflammation and a relapsing course However, in cells that are completely deficient of RIPK1, with clinically quiescent periods followed by bouts of severe which is needed for NF-kB activation,18 TNF leads to a clas- and damaging inflammation associated with tissue sical apoptotic response that is NF-kB preventable.19–21 Add- destruction and mucosal erosion.1 Apoptotic and necrotic ing to the complexities of TNF-mediated cell death and its features are often found in the crypt compartment of the dependence on NF-kB inhibition or RIPK1 kinase activation, affected intestinal tissue.2 IBD etiology depends on genetic we found that elevated A20 expression facilitates ripopto- susceptibility and environmental triggers.3 Although most some formation and RIPK1 activation.13 Here we describe the research efforts have focused on immune cells, it is becoming role of RIPK1 in TNF-mediated IEC killing and mucosal increasingly clear that intestinal epithelial cells (IEC) are also erosion in Ikkb(EE)IEC mice. important players in IBD pathogenesis4 and their ability to regenerate and heal the injured mucosa is critical for long- Results term remission.5 Interestingly, single nucleotide poly- NF-kB and Caspase-3 Activation in Human IBD morphisms in ubiquitously expressed genes encoding nuclear We conducted immunohistochemistry (IHC) analysis of factor (NF)-kB-regulated molecules show strong association human tissue specimens from healthy individuals and with IBD3,6 and activated/nuclear NF-kB is present in both patients suffering with either ileal or colonic CD or UC to IEC and lamina propria macrophages of active disease areas.7 determine the correlation between NF-kB activation and NF-kB stimulates transcription of numerous genes implicated cell death. As previously described,13 we examined 10 in IBD pathogenesis, including TNF, which codes for the normal colon specimens, 10 samples with active UC, and prototypical inflammatory cytokine tumor necrosis factor 10 samples with colonic CD, as well as 4 active ileitis (TNF). TNF inhibition is one of the main therapeutic options in samples and 5 inactive ileal CD samples, all of which were IBD7 leading to reduced IEC apoptosis and enhanced mucosal stained for p65/RelA and cleaved caspase-3 (cC-3). In repair.8 Unlike other TNF-dependent inflammatory diseases, general, normal colonic or ileal specimens contained such as psoriasis, extensive epithelial cell death is a cardinal hardly any IEC that were positive for cC-3 or nuclear p65 feature of IBD, yet the basis for TNF-induced IEC death is (Figure 1A). As described,13 active IBD specimens con- unknown, because TNF is not cytotoxic to normal IEC unless tained more cC-3-positive cells than control samples. treated with protein synthesis inhibitors. Interestingly, the areas enriched for cC-3-positive cells In fact, in most cell types, IEC included, transient TNF signaling inhibits apoptosis because of IKKb-dependent NF- a Both authors contributed equally to this work. kB activation.9 Correspondingly, ablation of IKKb10,11 or its Abbreviations used in this paper: BHA, butylated hydroxyanisole; cC-3, regulatory subunit IKKg/NEMO12 renders IEC susceptible cleaved caspase-3; cC-8, cleaved caspase-8; CD, Crohn’s disease; to TNF-induced death. Elevated expression of A20, a protein CHX, cycloheximide; DPI, phenyleneiodonium; IB, immunoblotting; IBD, inflammatory bowel disease; IEC, intestinal epithelial cell; IHC, thought to be a negative regulator of NF-kB, also sensitizes immunohistochemistry; i.p., intraperitoneally; LPS, lipopolysaccharide; IEC to TNF-mediated killing but this effect is not due to NF- Nec-1, necrostatin-1; NF, nuclear factor; PBS, phosphate-buffered kB inhibition.13 Indeed, IKK or NF-kB deficiencies have not saline; qPCR, quantitative polymerase chain reaction; TNF, tumor necrosis factor; UC, ulcerative colitis; WT, wild type. been described in IBD. Most current article To determine whether persistent NF-kB activation in IBD7 has a pathogenic function, we generated Ikkb(EE)IEC © 2020 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND mice in which a constitutively active IKKb(EE) variant is license (http://creativecommons.org/licenses/by-nc-nd/4.0/). expressed in IEC from the villin promoter.14 Surprisingly, 2352-345X https://doi.org/10.1016/j.jcmgh.2019.10.002 instead of being resistant to TNF-induced mucosal erosion, 2020 RIPK1 Mediates Crypt Death Downstream to NF-kB Activation 297 Figure 1. Active IBD tissue displays spatially adjacent NF-kB and caspase-3 activation in intestinal epithelial cells. Human control (A), CD and UC colon sections (B) were stained with antibodies to cC-3 and p65(RelA). Magnification bars: 50 mm. Arrows show positive cells. Results are representative for 15 healthy, 14 CD, and 10 UC specimens. 298 Wong et al Cellular and Molecular Gastroenterology and Hepatology Vol. 9, No. 2 Table 1.Number of Samples and the Corresponding Percentages of Nuclear p65 and Cleaved Caspase 3 Expression Level in IEC of Control Tissue and Active IBD Specimens Expression level C ileum C colon CD ileum CD colon UC colon Nuclear p65 expression level 0 4 (80) 7 (70) 1 (25) 0 0 1 1 (20) 3 (30) 1 (25) 2 (20) 1 (10) 2 0 0 2 (50) 5 (50) 4 (40) 3 0 0 0 3 (30) 5 (50) Total 5 10 4 10 10 Caspase-3 expression level 0 2 (40) 5 (50) 1 (25) 0 0 1 3 (60) 5 (50) 1 (25) 3 (30) 2 (20) 2 0 0 2 (50) 5 (50) 4 (40) 3 0 0 0 2 (20) 4 (40) Total 5 10 4 10 10 C, control; CD, Crohn’s disease; IBD, inflammatory bowel disease; IEC, intestinal epithelial cells; UC, ulcerative colitis. correlated with areas harboring cells with nuclear of caspase activation (Figure 3B). As described,14 Ikk- p65 (Figure 1A and B). Active UC specimens contained b(EE)IEC/Tnf–/– mice remained hypersensitive to exogenous more cC-3- and p65-positive cells than CD specimens and TNF and showed caspase-3 and -8 activation after its hardly any differences were observed between colonic administration (6.99 ± 1.50 cC-3þ and 3.38 ± 0.59 cC-8þ and ileal CD (Table 1). Our finding of activated/nuclear cells per crypt in Ikkb(EE)IEC vs 5.07 ± 0.78 cC-3þ and 1.93 NF-kB in IEC is consistent with other reports.7,22 In ± 0.61 cC-8þ cells per crypt in Ikkb(EE)IEC/Tnf–/– mice; P ¼ addition, mining of publicly available data sets also .015 and P < .001) (Figure 3C). Similar results were ob- showed upregulation of numerous NF-kB target genes tained in TNF-treated Ikkb(EE)IEC/Myd88DIEC mice (5.72 ± including TNFAIP3, CXCL10, CXCL11, CCL2, CCL8, CSF1, 2.22 cC-3þ and 4.35 ± 1.12 cC-8þ cells per crypt in Ikk- CCL11, and CCL20 in active IBD areas that decreased after b(EE)IEC/Myd88F/F mice vs 8.18 ± 1.17 cC-3þ and 5.61 ± anti-TNF therapy (Figure 2A).23 0.76 cC-8þ cells per crypt in Ikkb(EE)IEC/Myd88DIEC mice; P ¼ .015 and P ¼ .02) (Figure 3D), suggesting an inherent ability of persistent IKKb activation to sensitize IEC to TNF- TNF-Induced Apoptosis in Ikkb(EE)IEC Mice induced apoptosis independently of IEC-autonomous toll- To determine the pathogenic function of persistent NF- like receptor signaling. kB activation we used Ikkb(EE)IEC mice, which instead of To further investigate this phenomenon and determine being resistant to TNF-induced mucosal erosion are highly whether or not it depends on microbiota and immune cells, sensitive to TNF.14 Of note, many of the genes found to be we established enteroid cultures.24 Whereas WT enteroids up-regulated in human IBD and described in our previous exhibited minimal TNF-induced death, Ikkb(EE)IEC and Ikk- work13 were also up-regulated in Ikkb(EE)IEC mice relative b(EE)IEC/Tnf–/– enteroids underwent extensive cell death, to the wild-type (WT) mouse epithelium (Figure 2B).14 especially within crypt domains, after incubation with TNF Because TNF can trigger either apoptosis or necroptosis, (1.9% ± 3.8% WT dead organoids vs 95.5% ± 5.4% dead in we used IHC with antibodies specific for activated/cleaved Ikkb(EE)IEC organoids; P < .001) (Figure 4A and B), leading caspase-8 (cC-8) or cC-3 to determine the type of cell death to their rapid disintegration. cC-3 and TUNEL immunofluo- affecting the Ikkb(EE)IEC small bowel epithelium after rescent staining (Figure 4C and D) and IB analysis of administration of TNF or lipopolysaccharide (LPS). Treat- caspases-3 and -8 (Figure 4E and F) correlated and ment of Ikkb(EE)IEC mice with either agent activated both confirmed that Ikkb(EE)IEC or Ikkb(EE)IEC/Tnf-/- enteroids caspases (Figure 3A and B). TNF-treated WT mice showed underwent TNF-induced apoptosis mainly within crypt do- only a mild and transient caspase-3 activation near the villi mains. Inducible expression of IKKb(EE) in WT enteroids tips, but not within the crypt compartment, and hardly any also conferred susceptibility to TNF-induced apoptosis caspase-8 activation, correlating with little or no mucosal (Figure 5A). Doxycycline, which was used to induce IKK- damage. TNF-treated Ikkb(EE)IEC mice, however, displayed b(EE) expression, did not cause cell death, neither did it activation of both caspases in villi and especially within sensitize WT enteroids to TNF-induced apoptosis crypt compartments, leading to cell shedding and tissue (Figure 5B). damage (0.02 ± 0.03 cC-3þ and 0.01 ± 0.02 cC-8þ cells per To determine whether the elevated susceptibility of crypt in WT vs 7.01 ± 1.15 cC-3þ and 4.35 ± 2.19 cC-8þ Ikkb(EE)IEC enteroids to TNF-induced death was NF-kB- cells per crypt in Ikkb(EE)IEC mice; P < .001 and P < .001). dependent, we transduced them with a lentivirus expressing Immunoblotting (IB) analysis of the intestinal crypt fraction IkBa super-repressor (IkBaSR). Enteroids containing of Ikkb(EE)IEC mice intraperitoneally (i.p.) injected with LPS IkBaSR no longer exhibited TNF-induced crypt apoptosis showed strong and sustained cleavage of caspases-3 and -8 (Figure 5C), but unexpectedly IkBaSR expression led to and delayed degradation of IKKb(EE) protein, likely because reduced IKKb(EE) expression (Figure 5D). 2020 RIPK1 Mediates Crypt Death Downstream to NF-kB Activation 299 Figure 2. Anti-TNF treatment decreases expression of NF-kB-related genes in human IBD tissue. (A) Heat map showing differentially expressed NF-kB-related genes between healthy control subjects and CD subjects, before and after anti-TNF therapy, in tissue biopsies described in array GSE52746.23 Fc_cdIw12_cdAw0 means fold-change between CD samples from inactive areas after 12 weeks of anti-TNF therapy and CD samples from active areas at time 0; fc_cdAtnf_cdAw0 means fold-change between CD samples from active areas after 12 weeks of anti-TNF therapy and CD samples from active areas at time 0; fc_cdAtnf_cdIw12 means fold change between CD samples from active areas after 12 weeks of anti-TNF therapy and CD samples from inactive areas after 12 weeks of anti-TNF therapy; fc_cdAtnf_control means fold change between CD samples from active areas after 12 weeks of anti-TNF therapy and healthy control samples; fc_cdAw0_control means fold change between CD samples from active areas at time 0 and healthy control samples; fc_cdAw12_control means fold change between CD samples from active areas after 12 weeks of anti-TNF therapy and healthy control samples. (B) Comparison between genes that are differentially expressed between WT and Ikkb(EE)IEC enterocytes14 and those that are differentially expressed between CD and normal human ileum (left).13 RIPK1 Kinase Activity Is Required for TNF- includes TRADD, RIPK1, TRAF2, and cIAP1/2, and is Induced Tissue Damage responsible for IKK and NF-kB activation and inhibition of TNFR1 engagement triggers assembly of several distinct apoptosis. The latter is mediated by induction of anti- signaling complexes, the first of which is complex I, which apoptotic genes, including those coding for c-FLIP, a 300 Wong et al Cellular and Molecular Gastroenterology and Hepatology Vol. 9, No. 2 caspase-8 inhibitor,25 and survivin, a caspase-3 inhibitor.26 Notably, and despite its susceptibility to TNF-induced Complex I formation and NF-kB activation depend on the apoptosis, the intestinal epithelium of Ikkb(EE)IEC mice scaffold functions of RIPK1 but not on its kinase activity.16 exhibited elevated expression of antiapoptotic molecules, 2020 RIPK1 Mediates Crypt Death Downstream to NF-kB Activation 301 including BIRC3, BCL2, and BCL2L1.14 Persistent TNFR1 cells per crypt in Ikkb(EE)IEC vs 1.1 ± 0.35 cC-3þ cells in engagement, however, results in receptor internalization, Ikkb(EE)IEC/Ripk1D138N/D138N mice, 4 hours after LPS which can promote cell death through either RIPK1 kinase- administration; P < .001), as assessed by IHC (Figure 8C). dependent apoptosis or necroptosis.16,27 To query the Furthermore, the specific RIPK1 inhibitor GSK’963 pre- involvement of RIPK1 kinase activity in the observed vented LPS-induced tissue damage, apoptosis (5.5 ± 0.3 cC- response of Ikkb(EE)IEC enteroids, we examined the ability 3þ cells per crypt in Ikkb(EE)IEC mice vs 3.2 ± 0.6 cC-3þ of necrostatin-1 (Nec-1), a RIPK1 inhibitor,28 to block TNF- cells in GSK’963-treated Ikkb(EE)IEC mice; P ¼ .002) and induced killing. Indeed, Nec-1 effectively blocked TNF- death in Ikkb(EE)IEC mice challenged with LPS (Figure 8B induced Ikkb(EE)IEC enteroid apoptosis (Figure 6A and B), and D). Curiously, within 1 hour after TNF or LPS admin- without any effect on crypt proliferation (Figure 6C and D). istration, WT mice contained a few cC-3þ IEC in their villi, Nec-1, however, can also inhibit other kinases, such as whose number was also reduced on RIPK1 inactivation PAK1.29 We therefore examined the effect of more specific (4.5 ± 0.8 cC-3þ cells per villus in WT mice vs 1.5 ± 0.5 cC- RIPK1 kinase inhibitors and inhibitors of RIPK3, a key 3þ cells per villus in Ripk1D138N/D138N mice; P ¼ .01) inducer of necroptosis.30,31 The RIPK1 inhibitors (GSK’963 (Figure 8C). and GSK’728 or Nec-1s, which is the active enantiomer of the optimized necrostatin 7-Cl-O-Nec-1), but not the RIPK3 Ripoptosome Formation and Protection by selective inhibitors (GSK’843 and GSK’872), prevented the Antioxidants TNF-induced death of Ikkb(EE)IEC intestinal enteroids and RIPK1 activation as a kinase and RIPK1-dependent blocked caspase-3 and -8 cleavage (95.5% ± 5.4% dead apoptosis depend on formation of the ripoptosome, or organoids after control treatment vs 2.8% ± 3.3% after Nec- complex IIb, which also contains FADD, A20, and caspase- 1, 3.0% ± 3.5% after GSK’963, 4.9% ± 0.5% after Nec-1s, 8.13,17,36 We have recently shown that A20 overexpression 95.7% ±5.8% after GSK’843, and 97.7% ± 4.5% after in IECs induces ripoptosome formation and activation in GSK’872 treatments) (Figure 6A and B). response to TNF.13 Because A20 expression is elevated in To further validate the role of RIPK1 in TNF-induced IKKb(EE)-expressing IEC (Figures 9A and 2B) we examined apoptosis and tissue damage in Ikkb(EE)IEC mice, we its role in TNF-induced Ikkb(EE)IEC enteroid death by crossed the latter to Ripk1D138N/D138N homozygous knockin crossing Ikkb(EE)IEC mice with A20DIEC mice, which spe- mice, which express a catalytically inactive version of cifically lack A20 in IEC.13 We then infected IKKb(EE)/ RIPK1 that retains its scaffold function.32 We also crossed A20DIEC enteroids with a doxycycline inducible vector Ikkb(EE)IEC mice with Rip3-/- mice33 to genetically rule out coding for either WT A20 or an A20 variant with a C764A/ a role for RIPK3-dependent necroptosis in TNF-induced C767A double substitution that impairs the linear ubiquitin mucosal erosion in our model. Notably, Ripk3 ablation binding ability of zinc finger 7 (A20ZNF7mut), which is did not prevent TNF-induced apoptosis (Figure 7A and B) needed for RIPK1 activation.13 As expected, TNF induced but RIPK1 kinase inactivation completely blocked TNF- caspase activation in IKKb(EE)/A20DIEC (29930103). Sur- induced crypt cell death and caspase-3 activation in Ikk- prisingly, expression of the protective A20ZNF7mut did not b(EE)IEC enteroids (95.5% ± 5.4% dead Ikkb(EE)IEC orga- prevent caspase-3 and -8 activation as effectively as WT noids vs 2.0% ± 4.0% dead Ikkb(EE)IEC/Ripk1D138N/D138N A20 (Figure 9B), demonstrating that A20 upregulation is organoids) (Figure 7C and D). As expected,34,35 Ripk3 gene not responsible for TNF-induced apoptosis in Ikkb(EE)IEC ablation prevented necroptosis in enteroids incubated with IEC. TNF þ cycloheximide (CHX) þ zVAD (5.59 ± 3.12 cC-3þ Previous studies have shown that RIPK1 activation in- cells per crypt in Ikkb(EE)IEC mice vs 6.79 ± 1.02 cC-3þ creases reactive oxygen species production after TNF cells per crypt in Ikkb(EE)IEC/Rip3-/- mice; P ¼ .91) stimulation37 to potentiate RIPK1-dependent cell (Figure 7E and F). death.38,39 We therefore examined the effect of the anti- Ikkb(EE)IEC mice are highly sensitive to TNF-induced oxidant butylated hydroxyanisole (BHA), which was shown mucosal damage and even a low dose of LPS, which in- to inhibit ripoptosome formation,38 on TNF-mediated duces TNF expression, causes their rapid death.14 Whereas apoptosis and caspase activation in Ikkb(EE)IEC enteroids. Ripk3 ablation did not decrease LPS-induced mortality, or Consistent with previous reports, BHA, but not dipheny- LPS-induced caspase-3 or -8 activation (not shown) in leneiodonium (DPI), a NOX inhibitor, fully prevented TNF- Ikkb(EE)IEC mice (Figures 4A and 5D), RIPK1 inactivation induced apoptosis and caspase activation (95.5% ± 5.4% was fully protective (Figure 8A and B). RIPK1 inactivation dead organoids after control treatment vs 23.7% ± 20.0% also inhibited TNF- and LPS-induced caspase-3 activation dead organoids after BHA or 97.9% ± 4.1% after DPI in Ikkb(EE)IEC intestinal villi and crypts (5.25 ± 1 cC-3þ treatment) (Figure 9C and D). BHA treatment also inhibited Figure 3. (See previous page). IKKb(EE)-expressing intestinal crypts are highly susceptible to TNF-induced apoptosis independently of toll-like receptor signaling. (A) WT and Ikkb(EE)IEC mice were analyzed 4 hours after TNF injection (2 mg intravenously). Jejunal sections were stained with either H&E or antibodies to cC-3 or cC-8. Magnification bars: 100 mm. (B) Lysates of WT and Ikkb(EE)IEC intestinal mucosa were analyzed at different times after LPS injection (5 mg/kg) by IB. (C) Ikkb(EE)IEC and Ikkb(EE)IEC/Tnf–/– mice were injected with TNF (2 mg intravenously). Jejunal sections were prepared 4 hours later and stained with antibodies to either cC-3 or cC-8. Magnification bars: 100 mm. (D) Ikkb(EE)IEC/Myd88DIEC and Ikkb(EE)IEC/ Myd88F/F mice were analyzed 4 hours after TNF injection as above. 302 Wong et al Cellular and Molecular Gastroenterology and Hepatology Vol. 9, No. 2 ripoptosome-mediated caspase-8 activation, assessed by apoptosis (5.85 ± 1.04 cC-3þ and 3.01 ± 0.83 cC-8þ cells caspase-8 coprecipitation with FADD, almost as effectively per crypt after control treatment vs 0.00 ± 0.00 cC-3þ and as Nec-1 (Figure 9E). Accordingly, BHA administration to 0.00 ± 0.00 cC-8þ cells per crypt in BHA-treated mice; P < Ikkb(EE)IEC mice abrogated LPS-induced mortality and IEC .001 and P < .001) (Figure 9F and G). 2020 RIPK1 Mediates Crypt Death Downstream to NF-kB Activation 303 Figure 5. Susceptibility of Ikkb(EE)IEC enteroids to TNF-induced death is NF- kB-dependent. (A) WT enteroids transduced with an inducible IKKb(EE) construct were pre- incubated with or without doxycycline as indicated, lysed, and IB analyzed 4 hours after TNF addition. (B) Control and inducible-IKK- b(EE) transduced WT enter- oids were incubated for 24 hours with doxycycline before TNF stimulation as indicated. Original magnifi- cation X200. (C) Control and IkBaSR-transduced Ikk- b(EE)IEC enteroids were incubated with TNF as indi- cated and photographed under bright field. Original magnification 200. (D) Control and IkBaSR-trans- duced Ikkb(EE)IEC enteroids incubated with or without TNF were lysed and IB analyzed. Discussion loci, some of them involving the NF-kB pathway, such as TNF is a major pathogenic factor in IBD and its TNFAIP3 and NFKB1,3,43–45 TNF is a poor killer of normal blockade represents one of the most effective therapeutic IEC and can promote antiapoptotic NF-kB activation.11 Not options for these diseases. However, how TNF triggers IEC surprising, NF-kB-deficient IEC are highly susceptible to killing and mucosal erosion has remained a mystery. TNF-induced killing, which depends on conventional, cas- Although certain genetic mutations associated with IBD, pase-8-dependent, apoptosis.10–12,46 However, rather un- such as ATGL1T300A, increase IEC susceptibility to expectedly, we found that excessive IKKb-mediated NF-kB TNF3,40–42 and several studies have identified, in different activation reduces the threshold needed for induction of ethnic cohorts, 235 genetic markers in 200 susceptibility TNF-induced IEC death.14 Here we show that the TNF Figure 4. (See previous page). TNF induces apoptosis in IKKb(EE)-expressing intestinal enteroids. (A) WT and Ikkb(EE)IEC enteroids were photographed under brightfield 4 hours after TNF stimulation (40 ng/mL). Original magnification 200. (B) Ikkb(EE)IEC and Ikkb(EE)IEC/Tnf–/– enteroids were photographed under brightfield before and after incubation with TNF (40 ng/mL). Original magnification 200. (C) WT and Ikkb(EE)IEC enteroids were stained with cC-8 antibody and visualized by confocal microscopy at the indicated times after TNF (40 ng/mL) addition. Magnification bars: 100 mm. (D) WT and Ikkb(EE)IEC enteroids were TUNEL stained and analyzed by confocal microscopy before and after incubation with TNF. Apoptotic cells are bright green. Magnification bars: 100 mm. (E) Lysates of WT and Ikkb(EE)IEC enteroids were IB analyzed at different times after TNF addition. (F) Lysates of Tnf-/- and Ikkb(EE)IEC/Tnf–/– enteroids incubated with or without TNF were analyzed for caspase activation by IB. 304 Wong et al Cellular and Molecular Gastroenterology and Hepatology Vol. 9, No. 2 Figure 6. RIPK1 kinase activity is required for TNF-induced enteroid death. (A) Ikkb(EE)IEC enteroids were incubated with TNF (40 ng/mL) in the absence or presence of RIPK1 or RIPK3 inhibitors and photographed 4 hours later. Original magnification 200. (B) Lysates of Ikkb(EE)IEC enteroids treated as above were subjected to IB anal- ysis after 4 hours. (C) WT enteroids were treated for 24 hours with interleukin- 22 (10 ng/mL) in the absence or presence of Nec-1. Two hours before fixation, EdU was added to the culture medium. ****P  .0001. (D) Percentages of EdU positive cells per crypt are shown. IL, interleukin; ns, not significant. induced death of IKKb(EE)-expressing IEC depends on activated in active human IBD. Indeed, in previous studies RIPK1 kinase activation and presumably ripoptosome we found that Ikkb(EE)IEC mice exhibit higher levels of formation. oxidative DNA damage, which facilitates loss of APC heter- TNF-induced ripoptosome formation in IEC and RIPK1- ozygocity to cause that formation of colonic adenomas and dependent apoptosis are also facilitated by elevated premalignant aberrant crypt foci,48 a pathologic process expression of the deubiquitinase A20.13 Since expression of that also occurs in patients with IBD.49,50 It is also plausible TNFAIP3, the gene coding for A20, is stimulated by NF-kB that chronic NF-kB signaling affects Paneth cell differenti- and is elevated in Ikkb(EE)IEC mice, a likely explanation to ation or alters autophagy signaling, thereby increasing the the reduced threshold for TNF-induced mucosal erosion susceptibility to TNF-induced cell death, in a similar and IEC death driven by IKKb(EE) is A20. Although A20 manner to the effect of the ATG16L1 or NOD2 gene muta- ablation abrogated the TNF-induced death of IKKb(EE)- tions.51 Overall, our results suggest that RIPK1 inhibition expressing IEC, reconstitution of A20 expression with a and treatment with antioxidants may attenuate these pa- variant, A20ZNF7mut, that no longer enhances TNF-induced thologies, especially in conjunction with anti-TNF drugs. ripoptosome formation13 led to full restoration of TNF- induced apoptosis. These results suggest that IKKb(EE) expression overcomes the requirement for linear Materials and Methods ubiquitin binding by the seventh ZnF of A20.13 Exactly how Mice IKKb(EE) expression does that is not entirely clear, but our Ikkb(EE)IEC and Ripk1D138N mice were described.14,32 results show that its ability to reduce the threshold for TNF- Ripk3-/- mice were obtained from Dr. Vishva Dixit (Gen- induced death is NF-kB mediated. Because TNF-induced entech).52 A20F/F mice were obtained from Dr. Avril Ma.53 ripoptosome formation and RIPK1-mediated apoptosis in Ikkb(EE)IEC mice were crossed to Tnf-/-, Ripk3-/-, and IKKb (EE)-expressing IEC is inhibited by BHA, it is plausible Ripk1D138N/D138N mice, and maintained in the C57BL/6J that NF-kB activation may promote the accumulation of background originally acquired from Jackson Laboratories. reactive oxygen species or other pro-oxidants. Curiously, Mice used in this work were 8–12 weeks old and were oxidative stress is also thought to be involved in IBD maintained under specific pathogen free (SPF) conditions at pathogenesis,47 and our results show that NF-kB is a University of California San Diego facility, accredited by 2020 RIPK1 Mediates Crypt Death Downstream to NF-kB Activation 305 Figure 7. TNF-induced mucosal damage and IEC death is RIPK3-independent. (A) Ikkb(EE)IEC and Ikkb(EE)IEC/Rip3-/- enteroids were photographed under brightfield before and 4 hours after incubation with TNF (40 ng/mL). Original magnification 200. (B) WT, Ikkb(EE)IEC, Rip3-/- and Ikkb(EE)IEC/Rip3-/- enteroids were incubated with or without TNF, lysed, and IB analyzed 5 hours later. (C) Ikkb(EE)IEC and Ikkb(EE)IEC/Ripk1D138N/D138N enteroids were photographed under brightfield before and 5 hours after TNF addition. Original magnification 200. (D) WT, Ikkb(EE)IEC, Ripk1D138N/D138N, and Ikkb(EE)IEC/ Ripk1D138N/D138N enteroids were incubated with or without TNF, lysed, and IB analyzed 5 hours later. (E) Ikkb(EE)IEC and Ikkb(EE)IEC/Rip3-/- enteroids were incubated with TNF (40 ng/mL), CHX (25 mg/mL), and zVAD (10 mg/mL) for 4 hours, stained with Hoechst 33342 and propidium iodide and examined by confocal microscopy. Original magnification 200. (F) WT, Ikkb(EE)IEC, Rip3-/-, and Ikkb(EE)IEC/Rip3-/- mice were analyzed 4 hours after LPS (0.5 mg/g i.p.) injection. Jejunal sections were stained with antibody to cC-3 and photographed. Magnification bars: 100 mm. the American Association for Accreditation of Laboratory Reagents Animal Care. All animal protocols were approved by the LPS (Escherichia coli O111:B4) was purchased from institutional review board, following National Institutes of Sigma (St. Louis, MO) and was i.p. injected at 0.5 mg/kg, Health guidelines. Mice were fed autoclaved standard chow unless otherwise indicated. TNF was purchased from R&D and all of the different strains were cohoused to minimize systems (Minneapolis, MN) and injected at 2 mg per mouse microbiome fluctuations. unless otherwise indicated. In vitro, TNF was used at 40 ng/ 306 Wong et al Cellular and Molecular Gastroenterology and Hepatology Vol. 9, No. 2 Figure 8. RIPK1 kinase activity is required for TNF-induced death in intestinal crypts. (A) Ikkb(EE)IEC, Ikkb(EE)IEC/ Ripk1D138N/D138N, and Ikkb(EE)IEC/Ripk3-/- mice were injected with LPS (0.5 mg/kg, Escherichia coli O111:B4) and their survival was monitored over a 36-hour period (n ¼ 5 per group). (B) Ikkb(EE)IEC mice were injected with LPS in the absence or presence of GSK’963 (50 mg/kg) and their survival analyzed over a 36-hour period and compared with that of LPS-injected Ikkb(EE)IEC/ Ripk1D138N/D138N mice (n ¼ 5 per group). (C) The strains listed above along with WT mice were injected with TNF (2 mg intravenously) or LPS (0.5 mg/kg i.p.). Jejunal sections were collected at indicated times and stained for cC-3. Magnification bars: 100 mm. (D) Ikkb(EE)IEC were injected with LPS (0.5 mg/kg i.p) in the absence or presence of GSK’963 (50 mg/kg). Jejunal sections were collected after 4 hours of LPS injection and stained for H&E or cC-3. Magnification bars: 100 mm. mL. CHX (25 mg/mL), zVAD-FMK (50 mM), BHA (10 mM), (843: 5 mM, 872: 5 mM) inhibitors were synthesized at GSK Nec-1 (100 mM), and DPI (5 mM) were purchased from and purified before use at the previously determined Sigma. RIPK1 (Nec-1s: 50 nM and 963: 50 nM) and RIPK3 doses.30,31 For in vivo studies, BHA (20 mg/kg) and the 2020 RIPK1 Mediates Crypt Death Downstream to NF-kB Activation 307 308 Wong et al Cellular and Molecular Gastroenterology and Hepatology Vol. 9, No. 2 RIPK1 inhibitor GSK’963 (50 mg/kg) were dissolved in sa- should be viewed in this light. Where comparisons are made line with 5% dimethyl sulfoxide and 6% Cavitron and were between expression patterns of human and the mouse i.p. injected. model, we used the Homologene database to translate mouse genes into their human homologues, if such a ho- mologue exists, and compared mouse expression data with Human Samples their human counterparts. Archived specimens of ileum and colon for IHC analyses were obtained from patients who had undergone routine colonoscopy for clinical indications. Prospective ileum and Real-Time Quantitative Polymerase Chain colon specimens used for RNA analysis were obtained from Reaction patients with CD or UC and healthy control subjects under- Intestinal human tissue from healthy control subjects going colonoscopy as part of routine medical care at UCSD. and patients with CD was collected by colonoscopy and total The recruited participants had a diagnosis of CD or UC and RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) were older than 18 years. Healthy control subjects were and reverse-transcribed with random hexamers and Su- undergoing colonoscopy as part of routine colon cancer perscript II Kit (Invitrogen). Quantitative polymerase chain screening. Healthy control subjects were not included if they reaction (qPCR) was performed with SYBR Green PCR reported gastrointestinal symptoms. The clinical status of the Master Mix Kit (Applied Biosystems, Foster City, CA). The patients was verified by chart review of all medical records, relative amounts of each transcript were compared with laboratory and pathologic data, and all specimens were dei- those of GAPDH and normalized to untreated samples by the DD dentified before use. All participants provided written Ct method. Primers are available on request. informed consent. The institutional review boards at UCSD and at VA San Diego Healthcare System approved the study. Histologic Analysis and IHC Intestines were removed, opened longitudinally, cleaned, RNAseq and Bioinformatics processed as "Swiss rolls," and fixed in 10% phosphate- Total RNA was isolated from human intestinal tissues buffered formalin for 24 hours. Fixed tissues were and its quality was assessed on an Agilent Bioanalyzer embedded in paraffin, and 5 mm sections were prepared and (Santa Clara, CA). Samples that were determined to have an stained with H&E. For IHC, sections from murine and human RNA Integrity Number of 7 or greater were used to generate samples were incubated overnight at 4 C with anti-p65/ RNA libraries using Illumina TruSeq Stranded Total RNA RelA, anti-cC-3, or anti-cC-8 antibodies (1697, 9661, and Sample Prep Kit starting with 100 ng of RNA. Libraries were 8592, respectively; Cell Signaling, Danvers, MA) at a 1:200 then sequenced using the Illumina Hiseq2500 sequencer dilution. Antigen retrieval was with citrate buffer pH 6.0 at (San Diego, CA). Ikkb(EE)IEC expression array profiling was 96 C for 20 minutes. All sections were counterstained with published14 and submitted to GEO (GSE29701). The fastq hematoxylin and photographed using an Axioplan 200 mi- files were aligned to human and mouse transcriptomes of croscope with AxioVision Release 4.5 software (Zeiss, the Genome Reference Consortium GRCh.37/hg19 and Oberkochen, Germany). GRCm.38/mm10, respectively, using the STAR aligner.54 Transcript-level counts were calculated with an expecta- Enteroid Isolation and Culture tion maximization algorithm RSEM.55 Transcript-level Small intestinal organoids (enteroids) were cultured as summaries were processed into gene-level summaries by described.24 Briefly, crypts were collected from small in- combining all transcript counts from the same gene. Gene testines of mice after 30-minute incubation in counts from different samples were normalized and phosphate-buffered saline (PBS; pH 7.4) containing 2-mM analyzed for differential expression using DESeq.56 Because EDTA in 6 C. Enteroids were plated in Matrigel (BD at least 1 of the samples does not have biologic replicates, Bioscience, San Jose, CA) and maintained in DMEM/F12 we used an ansatz in which dispersion of gene counts is (Life Technologies, Carlsbad, CA) containing B27 and N2 estimated from all available samples as if they were repli- supplements (Life Technologies), 1.25 mM N-acetyl L- cates. All presented measures of statistical significance cysteine (Sigma), 100 ng/mL noggin (GoldBio, St. Louis, Figure 9. (See previous page). TNF-induced apoptosis of IKKb(EE)-expressing IEC involves ripoptosome formation and can be inhibited by BHA. (A) WT and Ikkb(EE)IEC enteroids were IB analyzed with antibodies to the indicated proteins. (B) Ikkb(EE)IEC/A20DIEC enteroids infected with the indicated A20 constructs and preincubated with doxycycline were stimu- lated without or with TNF (40 mg/mL) for 4 hours and IB analyzed with the indicated antibodies. (C) Ikkb(EE)IEC enteroids were incubated with dimethyl sulfoxide, BHA (10 mM), or diphenylene iodonium (5 mM) together with TNF (40 ng/mL) and photo- graphed under brightfield 4 hours later. Original magnification 200. (D) Lysates of Ikkb(EE)IEC enteroids treated with BHA and/ or TNF as indicated were IB analyzed after 4 hours. (E) WT and Ikkb(EE)IEC enteroids were stimulated without (C) or with TNF together with zVAD (10 mg/mL) (TZ), BHA (TZB), or Nec-1 (50 nM) (TZN) for 2 hours. Complex IIb was isolated by FADD immunoprecipitation and IB analyzed with the indicated antibodies. (F) Ikkb(EE)IEC mice were treated with or without BHA (20 mg/kg), challenged with LPS (0.5 mg/kg Escherichia coli O111:B4), and their survival was monitored over a 36-hour period (n ¼ 5 mice per group). (G) Ikkb(EE)IEC mice treated as above were analyzed 4 hours after LPS injection. Jejunal sections were stained with either H&E or antibodies to cC-8 or cC-3. Magnification bars: 100 mm. DMSO, dimethyl sulfoxide; DPI, diphe- nylene iodonium 2020 RIPK1 Mediates Crypt Death Downstream to NF-kB Activation 309 MO), 50 ng/mL mEGF (Biosource, San Diego, CA), and 10% Constructs and Lentivirus Transduction of Rspo1-Fc-conditioned medium (the Rspo1-Fc-expressing Enteroids cell line was a generous gift from Dr. Calvin Kuo, The inducible-IKKb(EE) lentiviral expression constructs Stanford). were made by subcloning PCR amplified attB-flanked IKK- b(EE),57 or A2058 and the different A20 mutants59 cDNAs were PCR amplified and cloned into pINDUCER2060 using Immunoblotting and Immunoprecipitation the Gateway Clonase II system (Life Technologies). All Whole cell extracts were obtained by lysing enteroids constructs were verified by sequencing. in ice-cold RIPA lysis buffer (Cell Signaling) containing 20 The lentiviral constructs were transfected along with mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM EDTA, 1% pMD2.G (Addgene, Plasmid 12259) and psPAX2 (Addgene, Triton X-100, and 1% deoxycholate, supplemented with a Plasmid 12260) into 293T cells to produce viral particles protease inhibitor cocktail (Roche, Basel, Switzerland). used to infect enteroids as described.61 Briefly, 2 days Proteins were separated by sodium dodecyl before virus infection, enteroids were supplemented with sulfate–polyacrylamide gel electrophoresis and transferred 50% Wnt3a conditioned medium (the Wnt3a-expressing to nitrocellulose membranes that were incubated with cell line was a generous gift from Dr. Karl Willert, antibodies against IKKb (Millipore, Billerica, MA), A20 UCSD) and 10 mM nicotinamide. Single cells were ob- (Thermofisher, Waltham, MA), IkBa, p65, (Santa Cruz tained by digesting the enteroids with TrypLE (Life Biotechnology Inc, Santa Cruz, CA), RIPK1, IKKg, and TNF Technologies) for 5 minutes at 37 C. Cells were mixed (R&D Systems, Minneapolis, MN), RIPK3 (eBioscience, with high-titer lentivirus plus polybrene (8 mg/mL, Santa Waltham, MA), cC-3, cC-8, A20, and ERK1/2 (Cell Cruz) and Y-27632 (10 mM, Sigma) in a 48-well culture Signaling), actin, and tubulin (Sigma). For immunoprecip- plate and centrifuged at 600  g at 32 C for 60 minutes, itation, whole cell extracts were incubated with the indi- followed by 5 hours incubation at 37 C. Cells were then cated antibodies and Protein G Dynabeads (Life plated in Matrigel and cultured in media with 50% Wnt3a, Technologies) overnight at 4 C. Immunocomplexes were nicotinamide, and Y-27632 for 2 days, followed by selec- washed with lysis buffer and analyzed by IB. 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Newton K, Dugger DL, Wickliffe KE, Kapoor N, de Almagro MC, Vucic D, Komuves L, Ferrando RE, Correspondence Address correspondence to: Monica Guma, MD, PhD, Department of French DM, Webster J, Roose-Girma M, Warming S, Rheumatology, University of California San Diego, San Diego Digestive Dixit VM. Activity of protein kinase RIPK3 determines Disease Research Center, 9500 Gilman Drive, San Diego, California 92093. whether cells die by necroptosis or apoptosis. Science e-mail: mguma@ucsd.edu; fax: (858) 534-8158; and Michael Karin, Department of Pharmacology, University of California San Diego, San Diego, 2014;343:1357–1360. CA 92093. e-mail: karinoffice@ucsd.edu. 53. Rott HD, Fahsold R. Tuberous sclerosis in two sibs of Acknowledgements normal parents. Clin Genet 1991;39:306–308. The authors acknowledge Genentech and Dr. Vishva Dixit for the provision of 54. Dobin A, Davis CA, Schlesinger F, Drenkow J, Rip3-/- mice. All authors had access to the study data and had reviewed and approved the final manuscript. Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinfor- Author contributions matics 2013;29:15–21. Jerry Wong, Monica Guma, and Michael Karin designed the study. Jerry Wong, Monica Guma, Ricard Garcia-Carbonell, Matija Zelic, Shih-Jing Yao, and Shalin 55. Li B, Dewey CN. RSEM: accurate transcript quantifica- A. Desai performed research. Philip A. Harris, Brigid S Boland, Joan Font- tion from RNA-Seq data with or without a reference Burgada, Koji Taniguchi, Soumita Das, Núria Planell, Azucena Salas, John Bertin, Manolis Pasparakis, Pete J. Gough, and Michelle Kelliher contributed genome. BMC Bioinformatics 2011;12:323. new constructs, chemical inhibitors, enteroid isolation, mice and human 56. Anders S, Huber W. Differential expression analysis inflammatory bowel disease samples, and transcriptome data. Jerry Wong, for sequence count data. Genome Biol 2010;11:R106. Monica Guma, Ricard Garcia-Carbonell, and Michael Karin analyzed data. Jerry Wong, Ricard Garcia-Carbonell, Monica Guma, and Michael Karin 57. Delhase M, Hayakawa M, Chen Y, Karin M. Positive and wrote the paper. negative regulation of IkappaB kinase activity through Conflicts of interest IKKbeta subunit phosphorylation. Science 1999; These authors disclose the following: Philip A. Harris, John Bertin, and Pete J. 284:309–313. Gough are employees and shareholders of GlaxoSmithKline. The remaining authors disclose no conflicts. 58. Wertz IE, O’Rourke KM, Zhou H, Eby M, Aravind L, Seshagiri S, Wu P, Wiesmann C, Baker R, Boone DL, Funding Ma A, Koonin EV, Dixit VM. De-ubiquitination and ubiq- Supported by fellowships from the Canadian Institutes of Health Research (J.W.) and Boehringer-Ingelheim Fonds (R.G.-C.); the National Institutes of uitin ligase domains of A20 downregulate NF-kappaB Health 1K08AR064834 (M.G.), R01AI075118 (M. Kelliher), and T32DK007202 signalling. Nature 2004;430:694–699. (B.S.B.); M.P. was funded by the ERC (2012-ADG_20120314), DFG (SFB670, SFB829, SPP1656), European Commission FP7 grants 223404 59. Lu TT, Onizawa M, Hammer GE, Turer EE, Yin Q, (Masterswitch) and 223151 (InflaCare), the Deutsche Krebshilfe (Grant Damko E, Agelidis A, Shifrin N, Advincula R, Barrera J, 110302), and the Helmholtz Alliance Preclinical Comprehensive Cancer Malynn BA, Wu H, Ma A. Dimerization and ubiquitin Center; M.K. was supported by NIH R01AI043477, is an American Cancer Society Research Professor, and holds the Ben and Wanda Hildyard Chair mediated recruitment of A20, a complex deubiquitinating for Mitochondrial and Metabolic Diseases. M.G. and M. Karin were also enzyme. Immunity 2013;38:896–905. supported by GlaxoSmithKline.