Center for Molecular Medicine Cologne

Pasparakis, Manolis - B 07

Role of FADD and caspase-8 in the regulation of intestinal epithelial cell death and inflammation

Introduction

Inflammatory bowel diseases (IBD) are chronic relapsing inflammatory disorders of the gastrointestinal tract with increasing incidence over the past 50 years. Despite a wide variety of possible triggers, inappropriate immune responses to the commensal microbiota are generally considered a central underlying theme in IBD. The intestinal epithelium forms a structural barrier between the commensal bacteria residing in the intestinal lumen and the mucosal immune cells and plays a critical role for the maintenance of gut immune homeostasis.
We showed previously that mice lacking FADD in intestinal epithelial cells (FADDIEC-KO) develop chronic ileitis and colitis due to RIPK3-mediated death of IECs. Moreover, mice with IEC-specific knockout of caspase-8 (Casp8IEC‑KO) were reported to develop ileitis but not colitis, suggesting that FADD and caspase-8 may have different functions in the regulation of intestinal epithelial homeostasis and inflammation.
The main aim of thhis project is to dissect the mechanisms by which FADD and caspase-8 regulate intestinal inflammation using genetic mouse models. The recent discovery that human patients with caspase-8 mutations develop severe intestinal inflammation suggests that the FADD/Casp8-dependent pathways regulating intestinal homeostasis and inflammation are conserved between humans and mice.

Therefore, our project is relevant for the pathogenesis of intestinal inflammation in humans and may lead to the identification of new therapeutic targets for patients with IBD.

Our Aims

  1. Dissect the upstream pathways that regulate the development of colitis and ileitis in FADDIEC-KO and CASP8IEC-KO  mice

  2. Unravel the mechanisms by which FADD or Caspase-8 act in intestinal epithelial cells to regulate immune homeostasis and prevent mucosal inflammation

  3. Study the interplay of FADD/Casp8 with other pathways that are important for the pathogenesis of inflammatory bowel diseases

Results

Mice with IEC-specific knockout of FADD or caspase-8 were previously shown to develop necroptosis-mediated colitis that depended on TNFR1 signalling and ileitis that was independent of TNFR1. We found that in addition to TNFR1, also Z-DNA Binding Protein 1 (ZBP1) acts as an upstream inducer of IEC death and inflammation in FADDIEC-KO mice. Our results showed that TNFR1 and ZBP1 exhibit redundant functions inducing necroptosis and ileitis in FADDIEC-KO mice, while both receptors are important for colitis development (Schwarzer et al, 2020). Moreover, we could show that ZBP1 activation in intestinal epithelial cells of FADDIEC-KO mice depends on sensing endogenous Z-nucleic acids via its Zα domains (Jiao et al, 2020) . These results revealed a new role of ZBP1, acting as sensor of endogenous Z-nucleic acids to induce necroptosis and intestinal inflammation.

Previous studies showed that RIPK3 knockout prevented both colitis and ileitis in FADDIEC-KO mice (Welz et al, 2011), suggesting that necroptosis of IECs drives the pathology in both the small and large intestine. Surprisingly, we found that, whereas MLKL deficiency prevented both colitis and ileitis in CASP8IEC-KO mice, MLKL knockout fully prevented colitis but only partially ameliorated ileitis in FADDIEC-KO mice, suggesting that necroptosis-independent mechanisms contribute to small intestinal inflammation in mice lacking FADD, but not Caspase-8, in IECs.
Our results showed that MLKL-independent ileitis in FADDIEC-KO mice is driven by caspase-8, which activates GSDMD-dependent pyroptosis-like death of IECs to induce small intestinal inflammation (Schwarz et al, 2020).

Figure 1

Collectively, our studies revealed an important role of ZBP1 acting as a sensor of endogenous Z-nucleic acids to induce cell death and inflammation in the skin and the intestine. In particular, our work in genetic mouse models provided experimental evidence that ZBP1 cooperates with TNFR1 to drive intestinal inflammation by triggering necroptosis of intestinal epithelial cells when caspase-8 is inhibited. TNF is a key pathogenic factor in inflammatory bowel disease with anti-TNF antibodies constituting a front-line and highly effective therapy in both Crohn’s Disease and Ulcerative Colitis. However, a subset of patients does not respond to anti-TNF therapy whereas a considerable percentage of patients respond initially but show secondary loss of response to anti-TNF, therefore TNF-independent mechanisms drive intestinal inflammation. Based on our results, we hypothesize that ZBP1 may act as a pathogenic factor in IBD to induce TNF-independent inflammation. These results warrant further studies examining whether ZBP1 contributes to IBD and other inflammatory pathologies in humans.

  • Jiao, H., Wachsmuth, L., Kumari, S., Schwarzer, R., Lin, J., Eren, R.O., Fisher, A., Lane, R., Young, G.R., Kassiotis, G., Kaiser, W.J., and Pasparakis, M. (2020). Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation. Nature 580, 391-395.
  • Schwarzer, R., Jiao, H., Wachsmuth, L., Tresch, A., and Pasparakis, M. (2020). FADD and Caspase-8 Regulate Gut Homeostasis and Inflammation by Controlling MLKL- and GSDMD-Mediated Death of Intestinal Epithelial Cells. Immunity 52, 978-993 e976.
  • Vlantis, K., Wullaert, A., Polykratis, A., Kondylis, V., Dannappel, M., Schwarzer, R., Welz, P., Corona, T., Walczak, H., Weih, F., Klein, U., Kelliher, M., and Pasparakis, M. (2016). NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-kappaB-Dependent and -Independent Functions. Immunity 44, 553-567.
  • Welz, P.S., Wullaert, A., Vlantis, K., Kondylis, V., Fernandez-Majada, V., Ermolaeva, M., Kirsch, P., Sterner-Kock, A., van Loo, G., and Pasparakis, M. (2011). FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation. Nature 477, 330-334.
  • Jiao, H., Wachsmuth, L., Kumari, S., Schwarzer, R., Lin, J., Eren, R.O., Fisher, A., Lane, R., Young, G.R., Kassiotis, G., Kaiser, W.J., and Pasparakis, M. (2020). Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation. Nature 580, 391-395.
  • Imai, T., Van, T.M., Pasparakis, M., and Polykratis, A. (2022). Smooth muscle cell specific NEMO deficiency inhibits atherosclerosis in ApoE(-/-) mice. Sci Rep 12, 12538. 
  • Kondylis V, Schneider F, Schorn F, Oikonomou N, Straub BK, Werner S, Rosenstiel P, and Pasparakis M (2022). p62 Promotes Survival and Hepatocarcinogenesis in Mice with Liver-Specific NEMO Ablation. Cancers (Basel)14. doi:10.3390/cancers14102436.
  • Kumari S, Van TM, Preukschat D, Schuenke H, Basic M, Bleich A, Klein U, and Pasparakis M (2021). NF-kappaB inhibition in keratinocytes causes RIPK1-mediated necroptosis and skin inflammation. Life Sci Alliance4. doi:10.26508/lsa.202000956.
  • Oikonomou N, Schuijs MJ, Chatzigiagkos A, Androulidaki A, Aidinis V, Hammad H, Lambrecht BN, and Pasparakis M (2021). Airway epithelial cell necroptosis contributes to asthma exacerbation in a mouse model of house dust mite-induced allergic inflammation. Mucosal Immunol14, 1160-1171. doi:10.1038/s41385-021-00415-5.
  • Schunke H, Gobel U, Dikic I, and Pasparakis M (2021). OTULIN inhibits RIPK1-mediated keratinocyte necroptosis to prevent skin inflammation in mice. Nat Commun12, 5912. doi:10.1038/s41467-021-25945-1.
  • Theobald SJ, Grab J, Fritsch M, Suarez I, Eisfeld HS, Winter S, Koch M, Holscher C, Pasparakis M, Kashkar H, and Rybniker J (2021). Gasdermin D mediates host cell death but not interleukin-1beta secretion in Mycobacterium tuberculosis-infected macrophages. Cell Death Discov7, 327. doi:10.1038/s41420-021-00716-5.
  • Jiao H, Wachsmuth L, Kumari S, Schwarzer R, Lin J, Eren RO, Fisher A, Lane R, Young GR, Kassiotis G, Kaiser WJ, and Pasparakis M (2020b). Z-nucleic-acid sensing triggers ZBP1-dependent necroptosis and inflammation. Nature 580, 391-5.
     
  • Lalaoui N, Boyden SE, Oda H, Wood GM, Stone DL, Chau D, Liu L, Stoffels M, Kratina T, Lawlor KE, Zaal KJM, Hoffmann PM, Etemadi N, Shield-Artin K, Biben C, Tsai WL, Blake MD, Kuehn HS, Yang D, Anderton H, Silke N, Wachsmuth L, Zheng L, Moura NS, Beck DB, Gutierrez-Cruz G, Ombrello AK, Pinto-Patarroyo GP, Kueh AJ, Herold MJ, Hall C, Wang H, Chae JJ, Dmitrieva NI, McKenzie M, Light A, Barham BK, Jones A, Romeo TM, Zhou Q, Aksentijevich I, Mullikin JC, Gross AJ, Shum AK, Hawkins ED, Masters SL, Lenardo MJ, Boehm M, Rosenzweig SD, Pasparakis M, Voss AK, Gadina M, Kastner DL, and Silke J (2020). Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease. Nature 577, 103-8.
     
  • Laurien L, Nagata M, Schunke H, Delanghe T, Wiederstein JL, Kumari S, Schwarzer R, Corona T, Kruger M, Bertrand MJM, Kondylis V, and Pasparakis M (2020). Autophosphorylation at serine 166 regulates RIP kinase 1-mediated cell death and inflammation. Nat Commun 11, 1747.
  • Schiffmann LM, Werthenbach JP, Heintges-Kleinhofer F, Seeger JM, Fritsch M, Gunther SD, Willenborg S, Brodesser S, Lucas C, Jungst C, Albert MC, Schorn F, Witt A, Moraes CT, Bruns CJ, Pasparakis M, Kronke M, Eming SA, Coutelle O, and Kashkar H (2020). Mitochondrial respiration controls neoangiogenesis during wound healing and tumour growth. Nat Commun 11.
  • Verboom L, Martens A, Priem D, Hoste E, Sze M, Vikkula H, Van Hove L, Voet S, Roels J, Maelfait J, Bongiovanni L, de Bruin A, Scott CL, Saeys Y, Pasparakis M, Bertrand MJM, and van Loo G (2020). OTULIN Prevents Liver Inflammation and Hepatocellular Carcinoma by Inhibiting FADD- and RIPK1 Kinase-Mediated Hepatocyte Apoptosis. Cell Rep 30, 2237-47 e6.
     
  • Wong J, Garcia-Carbonell R, Zelic M, Ho SB, Boland BS, Yao SJ, Desai SA, Das S, Planell N, Harris PA, Font-Burgada J, Taniguchi K, Bertin J, Salas A, Pasparakis M, Gough PJ, Kelliher M, Karin M, and Guma M (2020). RIPK1 Mediates TNF-Induced Intestinal Crypt Apoptosis During Chronic NF-kappaB Activation. Cell Mol Gastroenterol Hepatol 9, 295-312.
  • Schwarzer R, Laurien L, and Pasparakis M (2020). New insights into the regulation of apoptosis, necroptosis, and pyroptosis by receptor interacting protein kinase 1 and caspase-8. Curr Opin Cell Biol63, 186-193. doi:10.1016/j.ceb.2020.02.004.
  • Schwarzer, R., Jiao, H., Wachsmuth, L., Tresch, A. & Pasparakis, M. FADD and Caspase-8 Regulate Gut Homeostasis and Inflammation by Controlling MLKL- and GSDMD-Mediated Death of Intestinal Epithelial Cells. Immunity 52, 978-993.e6 (2020).
Prof. Dr. Manolis Pasparakis CMMC Cologne
Prof. Dr. Manolis Pasparakis

Institute for Genetics - CECAD Research Center

CMMC - PI - B 07

+49 221 478 84351

+49 221 478 6360

Institute for Genetics - CECAD Research Center

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50931 Cologne

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