Intestinal epithelial Caspase-8 signaling is essential to prevent necroptosis during Salmonella Typhimurium induced enteritis

Published online:


Although induction of host cell death is a pivotal step during bacteria-induced gastroenteritis, the molecular regulation remains to be fully characterized. To expand our knowledge, we investigated the role of the central cell death regulator Caspase-8 in response to Salmonella Typhimurium. Here, we uncovered that intestinal salmonellosis was associated with strong upregulation of members of the host cell death machinery in intestinal epithelial cells (IECs) as an early event, suggesting that elimination of infected IECs represents a host defense strategy. Indeed, Casp8∆IEC mice displayed severe tissue damage and high lethality after infection. Additional deletion of Ripk3 or Mlkl rescued epithelial cell death and lethality of Casp8∆IEC mice, demonstrating the crucial role of Caspase-8 as a negative regulator of necroptosis. While Casp8∆IECTnfr1−/− mice showed improved survival after infection, tissue destruction was similar to Casp8∆IEC mice, indicating that necroptosis partially depends on TNF-α signaling. Although there was no impairment in antimicrobial peptide secretion during the early phase of infection, functional Caspase-8 seems to be required to control pathogen colonization. Collectively, these results demonstrate that Caspase-8 is essential to prevent Salmonella Typhimurium induced enteritis and to ensure host survival by two different mechanisms: maintenance of intestinal barrier function and restriction of pathogen colonization.

  • Subscribe to Mucosal Immunology for full access:



Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.


  1. 1.

    Kirk, M. D. et al. World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis. PLoS Med. 12, e1001921 (2015).

  2. 2.

    Kaiser, P., Diard, M., Stecher, B. & Hardt, W. D. The streptomycin mouse model for Salmonella diarrhea: functional analysis of the microbiota, the pathogen’s virulence factors, and the host’s mucosal immune response. Immunol. Rev. 245, 56–83 (2012).

  3. 3.

    Santos, R. L., Tsolis, R. M., Baumler, A. J. & Adams, L. G. Pathogenesis of Salmonella-induced enteritis. Braz. J. Med. Biol. Res. 36, 3–12 (2003).

  4. 4.

    Stecher, B. & Hardt, W. D. Mechanisms controlling pathogen colonization of the gut. Curr. Opin. Microbiol. 14, 82–91 (2011).

  5. 5.

    Carter, P. B. & Collins, F. M. The route of enteric infection in normal mice. J. Exp. Med. 139, 1189–1203 (1974).

  6. 6.

    Darwin, K. H. & Miller, V. L. Molecular basis of the interaction of Salmonella with the intestinal mucosa. Clin. Microbiol. Rev. 12, 405–428 (1999).

  7. 7.

    Fink, S. L. & Cookson, B. T. Pyroptosis and host cell death responses during Salmonella infection. Cell. Microbiol. 9, 2562–2570 (2007).

  8. 8.

    Liss, V. et al. Salmonella entericaremodels the host cell endosomal system for efficient intravacuolar nutrition. Cell Host Microbe 21, 390–402 (2017).

  9. 9.

    Knodler, L. A. et al. Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proc. Natl Acad. Sci. USA 107, 17733–17738 (2010).

  10. 10.

    Hybiske, K. & Stephens, R. S. Exit strategies of intracellular pathogens. Nat. Rev. Microbiol. 6, 99–110 (2008).

  11. 11.

    Friedrich, N., Hagedorn, M., Soldati-Favre, D. & Soldati, T. Prison break: pathogens’ strategies to egress from host cells. Microbiol. Mol. Biol. Rev. 76, 707–720 (2012).

  12. 12.

    Watson, K. G. & Holden, D. W. Dynamics of growth and dissemination of Salmonella in vivo. Cell. Microbiol. 12, 1389–1397 (2010).

  13. 13.

    Kim, J. M. et al. Apoptosis of human intestinal epithelial cells after bacterial invasion. J. Clin. Investig. 102, 1815–1823 (1998).

  14. 14.

    Sellin, M. E. et al. Epithelium-intrinsic NAIP/NLRC4 inflammasome drives infected enterocyte expulsion to restrict Salmonella replication in the intestinal mucosa. Cell Host Microbe 16, 237–248 (2014).

  15. 15.

    Becker, C., Watson, A. J. & Neurath, M. F. Complex roles of caspases in the pathogenesis of inflammatory bowel disease. Gastroenterology 144, 283–293 (2013).

  16. 16.

    Jorgensen, I. & Miao, E. A. Pyroptotic cell death defends against intracellular pathogens. Immunol. Rev. 265, 130–142 (2015).

  17. 17.

    Antonopoulos, C. et al. Caspase-8 as an effector and regulator of NLRP3 inflammasome signaling. J. Biol. Chem. 290, 20167–20184 (2015).

  18. 18.

    Gurung, P. & Kanneganti, T. D. Novel roles for caspase-8 in IL-1beta and inflammasome regulation. Am. J. Pathol. 185, 17–25 (2015).

  19. 19.

    Monie, T. P. & Bryant, C. E. Caspase-8 functions as a key mediator of inflammation and pro-IL-1beta processing via both canonical and non-canonical pathways. Immunol. Rev. 265, 181–193 (2015).

  20. 20.

    Rauch, I. et al. NAIP-NLRC4 inflammasomes coordinate intestinal epithelial cell expulsion with eicosanoid and IL-18 release via activation of caspase-1 and -8. Immunity 46, 649–659 (2017).

  21. 21.

    Robinson, N. et al. Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium. Nat. Immunol. 13, 954–962 (2012).

  22. 22.

    Shutinoski, B. et al. K45A mutation of RIPK1 results in poor necroptosis and cytokine signaling in macrophages, which impacts inflammatory responses in vivo. Cell Death Differ. 23, 1628–1637 (2016).

  23. 23.

    Gunther, C. et al. The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis. J. Clin. Investig. 126, 4346–4360 (2016).

  24. 24.

    Jung, H. C. et al. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J. Clin. Investig. 95, 55–65 (1995).

  25. 25.

    Gunther, C. et al. Caspase-8 controls the gut response to microbial challenges by Tnf-alpha-dependent and independent pathways. Gut 64, 601–610 (2015).

  26. 26.

    Gunther, C. et al. Caspase-8 regulates TNF-alpha-induced epithelial necroptosis and terminal ileitis. Nature 477, 335–339 (2011).

  27. 27.

    Sotolongo, J., Ruiz, J. & Fukata, M. The role of innate immunity in the host defense against intestinal bacterial pathogens. Curr. Infect. Dis. Rep. 14, 15–23 (2012).

  28. 28.

    Grassl, G. A., Valdez, Y., Bergstrom, K. S., Vallance, B. A. & Finlay, B. B. Chronic enteric salmonella infection in mice leads to severe and persistent intestinal fibrosis. Gastroenterology 134, 768–780 (2008).

  29. 29.

    Hoiseth, S. K. & Stocker, B. A. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981).

  30. 30.

    Zarepour, M. et al. The mucin Muc2 limits pathogen burdens and epithelial barrier dysfunction during Salmonella enterica serovar Typhimurium colitis. Infect. Immun. 81, 3672–3683 (2013).

  31. 31.

    Zhang, K. et al. Age-dependent enterocyte invasion and microcolony formation by Salmonella. PLoS Pathog. 10, e1004385 (2014).

  32. 32.

    Man, S. M. et al. Salmonella infection induces recruitment of Caspase-8 to the inflammasome to modulate IL-1beta production. J. Immunol. 191, 5239–5246 (2013).

  33. 33.

    Feltham, R., Vince, J. E. & Lawlor, K. E. Caspase-8: not so silently deadly. Clin. Transl. Immunol. 6, e124 (2017).

  34. 34.

    Gunther, C., Neumann, H., Neurath, M. F. & Becker, C. Apoptosis, necrosis and necroptosis: cell death regulation in the intestinal epithelium. Gut 62, 1062–1071 (2013).

  35. 35.

    Wittkopf, N. et al. Cellular FLICE-like inhibitory protein secures intestinal epithelial cell survival and immune homeostasis by regulating caspase-8. Gastroenterology 145, 1369–1379 (2013).

  36. 36.

    Jorgensen, I., Rayamajhi, M. & Miao, E. A. Programmed cell death as a defence against infection. Nat. Rev. Immunol. 17, 151–164 (2017).

  37. 37.

    Gurung, P. et al. FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes. J. Immunol. 192, 1835–1846 (2014).

  38. 38.

    Kitur, K. et al. Toxin-induced necroptosis is a major mechanism of Staphylococcus aureus lung damage. PLoS Pathog. 11, e1004820 (2015).

  39. 39.

    Kitur, K. et al. Necroptosis promotes Staphylococcus aureus clearance by inhibiting excessive inflammatory signaling. Cell Rep. 16, 2219–2230 (2016).

  40. 40.

    Gonzalez-Juarbe, N. et al. Pore-forming toxins induce macrophage necroptosis during acute bacterial pneumonia. PLoS Pathog. 11, e1005337 (2015).

  41. 41.

    Weng, D. et al. Caspase-8 and RIP kinases regulate bacteria-induced innate immune responses and cell death. Proc. Natl Acad. Sci. USA 111, 7391–7396 (2014).

  42. 42.

    Watson, A. J. & Hughes, K. R. TNF-alpha-induced intestinal epithelial cell shedding: implications for intestinal barrier function. Ann. N. Y. Acad. Sci. 1258, 1–8 (2012).

  43. 43.

    Gunther, C., Buchen, B., Neurath, M. F. & Becker, C. Regulation and pathophysiological role of epithelial turnover in the gut. Semin. Cell Dev. Biol. 35, 40–50 (2014).

  44. 44.

    Williams, J. M. et al. A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide. Dis. Models Mech. 6, 1388–1399 (2013).

  45. 45.

    Newton, K., Sun, X. & Dixit, V. M. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4. Mol. Cell. Biol. 24, 1464–1469 (2004).

  46. 46.

    Riedel, C. U. et al. Construction of p16Slux, a novel vector for improved bioluminescent labeling of gram-negative bacteria. Appl. Environ. Microbiol. 73, 7092–7095 (2007).

  47. 47.

    Valdivia, R. H. & Falkow, S. Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction. Mol. Microbiol. 22, 367–378 (1996).

  48. 48.

    Hansson, G. C. & Johansson, M. E. The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria. Gut Microbes 1, 51–54 (2010).

Download references


The authors thank H. Dorner, M. Zeitler, S. Gößwein, S. Wallmüller and V. Thonn for excellent technical assistance. The authors thank James Murphy (Associated Professor at the Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia) for providing us with Mlkl-/- mice. This research has received funding from DFG projects within SPP1656 and SFB1181-A08. Further support was given by the Interdisciplinary Center for Clinical Research (IZKF) of the University Erlangen-Nuremberg.

Author information


  1. Department of Medicine 1, University of Erlangen-Nuremberg, Erlangen, Germany

    • Manuela Hefele
    • , Iris Stolzer
    • , Barbara Ruder
    • , Gui-Wei He
    • , Mousumi Mahapatro
    • , Stefan Wirtz
    • , Markus F. Neurath
    •  & Claudia Günther


  1. Search for Manuela Hefele in:

  2. Search for Iris Stolzer in:

  3. Search for Barbara Ruder in:

  4. Search for Gui-Wei He in:

  5. Search for Mousumi Mahapatro in:

  6. Search for Stefan Wirtz in:

  7. Search for Markus F. Neurath in:

  8. Search for Claudia Günther in:


C.G. and M.H. designed the research. M.H., I.S., C.G., and B.R. performed the experiments. M.M., SW, G.W.H., M.F.N. provided material that made this study possible. M.H., M.F.N. and C.G. analysed the data and wrote the paper.

Competing interest

The authors declare no competing financial interests.

Corresponding author

Correspondence to Claudia Günther.

Electronic supplementary material