Molecular Medicine

, Volume 23, Issue 1, pp 155–165 | Cite as

Myosin Light Chain Kinase Knockout Improves Gut Barrier Function and Confers a Survival Advantage in Polymicrobial Sepsis

  • C. Adam Lorentz
  • Zhe Liang
  • Mei Meng
  • Ching-Wen Chen
  • Benyam P. Yoseph
  • Elise R. Breed
  • Rohit Mittal
  • Nathan J. Klingensmith
  • Alton B. Farris
  • Eileen M. Burd
  • Michael Koval
  • Mandy L. Ford
  • Craig M. Coopersmith
Research Article


Sepsis-induced intestinal hyperpermeability is mediated by disruption of the epithelial tight junction, which is closely associated with the perijunctional actin-myosin ring. Myosin light chain kinase (MLCK) phosphorylates the myosin regulatory light chain, resulting in increased permeability. The purpose of this study was to determine whether genetic deletion of MLCK would alter gut barrier function and survival from sepsis. MLCK−/− and wild-type (WT) mice were subjected to cecal ligation and puncture and assayed for both survival and mechanistic studies. Survival was significantly increased in MLCK−/− mice (95% versus 24%, p < 0.0001). Intestinal permeability increased in septic WT mice compared with unmanipulated mice. In contrast, permeability in septic MLCK−/− mice was similar to that seen in unmanipulated animals. Improved gut barrier function in MLCK−/− mice was associated with increases in the tight junction mediators ZO-1 and claudin 15 without alterations in claudin 1, 2, 3, 4, 5, 7, 8 and 13, occludin or JAM-A. Other components of intestinal integrity (apoptosis, proliferation and villus length) were unaffected by MLCK deletion, as were local peritoneal inflammation and distant lung injury. Systemic IL-10 was decreased greater than 10-fold in MLCK−/− mice; however, survival was similar between septic MLCK−/− mice given exogenous IL-10 or vehicle. These data demonstrate that deletion of MLCK improves survival following sepsis, associated with normalization of intestinal permeability and selected tight junction proteins.



This work was supported by funding from the National Institutes of Health (GM072808, GM095442, GM104323, GM109779, GM113228, HL116958).

Supplementary material

10020_2017_2301155_MOESM1_ESM.pdf (160 kb)
Supplementary material, approximately 160 KB.


  1. 1.
    Singer M, et al. (2016) The third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 315:801–10.CrossRefGoogle Scholar
  2. 2.
    Shankar-Hari M, et al. (2016) Developing a new definition and assessing new clinical criteria for septic shock: for the third international consensus definitions for sepsis and septic shock (Sepsis-3). JAMA. 315:775–87.CrossRefGoogle Scholar
  3. 3.
    Gaieski DF, Edwards JM, Kallan MJ, Carr BG. (2013) Benchmarking the incidence and mortality of severe sepsis in the United States. Crit. Care Med. 41:1167–74.CrossRefGoogle Scholar
  4. 4.
    Rhodes A, et al. (2017) Surviving sepsis campaign: international guidelines for management of sepsis and septic shock: 2016. Crit. Care Med. 45:486–552.CrossRefGoogle Scholar
  5. 5.
    Mittal R, Coopersmith CM. (2014) Redefining the gut as the motor of critical illness. Trends Mol. Med. 20:214–23.CrossRefGoogle Scholar
  6. 6.
    Klingensmith NJ, Coopersmith CM. (2016) The gut as the motor of multiple organ dysfunction in critical illness. Crit Care Clin. 32:203–12.CrossRefGoogle Scholar
  7. 7.
    Carrico CJ, Meakins JL, Marshall JC, Fry D, Maier RV. (1986) Multiple-organ-failure syndrome. The gastrointestinal tract: the “motor” of MOF. Arch. Surg. 121:196–208.CrossRefGoogle Scholar
  8. 8.
    Sertaridou E, Papaioannou V, Kolios G, Pneumatikos I. (2015) Gut failure in critical care: old school versus new school. Ann. Gastroenterol. 28:309–22.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Clark JA, Coopersmith CM. (2007) Intestinal crosstalk: a new paradigm for understanding the gut as the “motor” of critical illness. Shock. 28:384–93.CrossRefGoogle Scholar
  10. 10.
    Fay KT, Ford ML, Coopersmith CM. (2017) The intestinal microenvironment in sepsis. Biochim. Biophys. Acta.Google Scholar
  11. 11.
    Lyons JD, Coopersmith CM. (2017) Pathophysiology of the Gut and the Microbiome in the Host Response. Pediatr. Crit. Care Med. 18:S46–49.CrossRefGoogle Scholar
  12. 12.
    Clark JA, Clark AT, Hotchkiss RS, Buchman TG, Coopersmith CM. (2008) Epidermal growth factor treatment decreases mortality and is associated with improved gut integrity in sepsis. Shock. 30:36–42.CrossRefGoogle Scholar
  13. 13.
    Clark JA, Gan H, Samocha AJ, Fox AC, Buchman TG, Coopersmith CM. (2009) Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis. Am. J. Physiol Gastrointest. Liver Physiol 297: G471–79.CrossRefGoogle Scholar
  14. 14.
    Coopersmith CM, et al. (2002) Overexpression of Bcl-2 in the intestinal epithelium improves survival in septic mice. Crit. Care Med. 30:195–201.CrossRefGoogle Scholar
  15. 15.
    Coopersmith CM, et al. (2003) Sepsis from Pseudomonas aeruginosa pneumonia decreases intestinal proliferation and induces gut epithelial cell cycle arrest. Crit. Care Med. 31:1630–37.CrossRefGoogle Scholar
  16. 16.
    Dominguez JA, et al. (2013) Inhibition of IKKbeta in enterocytes exacerbates sepsis-induced intestinal injury and worsens mortality. Crit. Care Med. 41: e275–85.CrossRefGoogle Scholar
  17. 17.
    Liang Z, et al. (2014) Intestine-specific deletion of microsomal triglyceride transfer protein increases mortality in aged mice. PLoS. ONE. 9:e101828.CrossRefGoogle Scholar
  18. 18.
    Yoseph BP, et al. (2016) Mechanisms of intestinal barrier dysfunction in sepsis. Shock. In press.Google Scholar
  19. 19.
    Fink MP. (2003) Intestinal epithelial hyperpermeability: update on the pathogenesis of gut mucosal barrier dysfunction in critical illness. Curr. Opin. Crit Care 9:143–51.CrossRefGoogle Scholar
  20. 20.
    Klaus DA, et al. (2013) Increased plasma zonulin in patients with sepsis. Biochem. Med. (Zagreb.) 23:107–11.CrossRefGoogle Scholar
  21. 21.
    Li Q, Zhang Q, Wang C, Liu X, Li N, Li J. (2009) Disruption of tight junctions during polymicrobial sepsis in vivo. J. Pathol. 218:210–21.CrossRefGoogle Scholar
  22. 22.
    Moore FA, et al. (1991) Gut bacterial translocation via the portal vein: a clinical perspective with major torso trauma. J. Trauma 31:629–36.CrossRefGoogle Scholar
  23. 23.
    Alverdy JC, Laughlin RS, Wu L. (2003) Influence of the critically ill state on host-pathogen interactions within the intestine: gut-derived sepsis redefined. Crit. Care Med. 31:598–607.CrossRefGoogle Scholar
  24. 24.
    Krezalek MA, Defazio J, Zaborina O, Zaborin A, Alverdy JC. (2016) The shift of an intestinal “microbiome” to a “pathobiome” governs the course and outcome of sepsis gollowing surgical injury. Shock. 45:475–82.CrossRefGoogle Scholar
  25. 25.
    Odenwald MA, Turner JR. (2013) Intestinal permeability defects: is it time to treat? Clin. Gastroenterol. Hepatol. 11:1075–83.CrossRefGoogle Scholar
  26. 26.
    Nalle SC. (2014) Recipient NK cell inactivation and intestinal barrier loss are required for MHC-matched graft-versus-host disease. Sci. Transl. Med. 6:243ra87.CrossRefGoogle Scholar
  27. 27.
    Turner JR. (2009) Intestinal mucosal barrier function in health and disease. Nat. Rev. Immunol. 9:799–809.CrossRefGoogle Scholar
  28. 28.
    Dominguez JA, et al. (2011) Epidermal growth factor improves survival and prevents intestinal injury in a murine model of pseudomonas aeruginosa pneumonia. Shock. 36:381–89.CrossRefGoogle Scholar
  29. 29.
    Chen C, Wang P, Su Q, Wang S, Wang F. (2012) Myosin light chain kinase mediates intestinal barrier disruption following burn injury. PLoS ONE. 7:e34946.CrossRefGoogle Scholar
  30. 30.
    Barreau F, Hugot JP. (2014) Intestinal barrier dysfunction triggered by invasive bacteria. Curr. Opin. Microbiol. 17:91–98.CrossRefGoogle Scholar
  31. 31.
    Herrmann JR, Turner JR. (2016) Beyond Ussing’s chambers: contemporary thoughts on integration of transepithelial transport. Am. J. Physiol. Cell Physiol 310: C423–31.CrossRefGoogle Scholar
  32. 32.
    Khounlotham M, et al. (2012) Compromised intestinal epithelial barrier induces adaptive immune compensation that protects from colitis. Immunity. 37:563–73.CrossRefGoogle Scholar
  33. 33.
    Laukoetter MG, et al. (2007) JAM-A regulates permeability and inflammation in the intestine in vivo. J. Exp. Med. 204:3067–76.CrossRefGoogle Scholar
  34. 34.
    Shen L, Weber CR, Raleigh DR, Yu D, Turner JR. (2011) Tight junction pore and leak pathways: a dynamic duo. Annu. Rev. Physiol 73:283–309.CrossRefGoogle Scholar
  35. 35.
    Cunningham KE, Turner JR. (2012) Myosin light chain kinase: pulling the strings of epithelial tight junction function. Ann. N.Y. Acad. Sci. 1258:34–42.CrossRefGoogle Scholar
  36. 36.
    Jung C, et al. (2012) Yersinia pseudotuberculosis disrupts intestinal barrier integrity through hematopoietic TLR-2 signaling. J. Clin. Invest 122:2239–51.CrossRefGoogle Scholar
  37. 37.
    Meinzer U, et al. (2012) Yersinia pseudotuberculosis effector YopJ subverts the Nod2/RICK/TAK1 pathway and activates caspase-1 to induce intestinal barrier dysfunction. Cell Host Microbe. 11:337–51.CrossRefGoogle Scholar
  38. 38.
    Zahs A, et al. (2012) Inhibition of long myosin light-chain kinase activation alleviates intestinal damage after binge ethanol exposure and burn injury. Am. J. Physiol Gastrointest. Liver Physiol. 303:G705–12.CrossRefGoogle Scholar
  39. 39.
    Shen L. (2012) Tight junctions on the move: molecular mechanisms for epithelial barrier regulation. Ann. N.Y. Acad. Sci. 1258:9–18.CrossRefGoogle Scholar
  40. 40.
    Yu D, et al. (2010) MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function. Proc. Natl. Acad. Sci. U.S.A. 107:8237–41.CrossRefGoogle Scholar
  41. 41.
    Zolotarevsky Y, et al. (2002) A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease. Gastroenterology. 123:163–72.CrossRefGoogle Scholar
  42. 42.
    Yuhan R, Koutsouris A, Savkovic SD, Hecht G. (1997) Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability. Gastroenterology. 113:1873–82.CrossRefGoogle Scholar
  43. 43.
    Clayburgh DR, et al. (2005) Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J. Clin. Invest. 115:2702–15.CrossRefGoogle Scholar
  44. 44.
    Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA. (2009) Immunodesign of experimental sepsis by cecal ligation and puncture. Nat. Protoc. 4:31–36.CrossRefGoogle Scholar
  45. 45.
    Dellinger RP, et al. (2013) Surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. Crit. Care Med. 41:580–637.CrossRefGoogle Scholar
  46. 46.
    Vyas D, et al. (2007) Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine. Histol. Histopathol. 22:623–30.PubMedPubMedCentralGoogle Scholar
  47. 47.
    Lyons JD, et al. (2016) Murine lung cancer increases CD4+ T cell apoptosis and decreases gut proliferative capacity in sepsis. PLoS ONE. 11:e0149069.CrossRefGoogle Scholar
  48. 48.
    Noto MJ, Becker KW, Boyd KL, Schmidt AM, Skaar EP. (2017) RAGE-mediated suppression of interleukin-10 results in enhanced mortality in a murine model of cinetobacter baumannii sepsis. Infect. Immun. 85:e00954–16.CrossRefGoogle Scholar
  49. 49.
    Hotchkiss RS, Sherwood ER. (2015) Immunology. Getting sepsis therapy right. Science. 347:1201–02.CrossRefGoogle Scholar
  50. 50.
    Bruewer M, et al. (2003) Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J. Immunol. 171:6164–72.CrossRefGoogle Scholar
  51. 51.
    Abreu MT, Palladino AA, Arnold ET, Kwon RS, McRoberts JA. (2000) Modulation of barrier function during Fas-mediated apoptosis in human intestinal epithelial cells. Gastroenterology. 119:1524–36.CrossRefGoogle Scholar
  52. 52.
    Chin AC, Flynn AN, Fedwick JP, Buret AG. (2006) The role of caspase-3 in lipopolysaccharidemediated disruption of intestinal epithelial tight junctions. Can. J. Physiol. Pharmacol. 84:1043–50.CrossRefGoogle Scholar
  53. 53.
    Xiao YT, Yan WH, Cao Y, Yan JK, Cai W. (2016) Neutralization of IL-6 and TNF-alpha ameliorates intestinal permeability in DSS-induced colitis. Cytokine. 83:189–92.CrossRefGoogle Scholar
  54. 54.
    McDonald D, et al. (2016) Extreme dysbiosis of the microbiome in critical illness. mSphere. 1(4):e00199–16.CrossRefGoogle Scholar
  55. 55.
    Tamura A, et al. (2011) Loss of claudin-15, but not claudin-2, causes Na+ deficiency and glucose malabsorption in mouse small intestine. Gastroenterology. 140:913–23.CrossRefGoogle Scholar
  56. 56.
    Suzuki H, et al. (2014) Crystal structure of a claudin provides insight into the architecture of tight junctions. Science. 344:304–07.CrossRefGoogle Scholar
  57. 57.
    Feng L, et al. (2016) Deficiency of dietary niacin impaired intestinal mucosal immune function via regulating intestinal NF-kappaB, Nrf2 and MLCK signaling pathways in young grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol. 49:177–93.CrossRefGoogle Scholar
  58. 58.
    Du J, et al. (2015) 1,25-Dihydroxy vitamin D protects intestinal epithelial barrier by regulating the myosin light chain kinase signaling pathway. Inflamm. Bowel. Dis. 21:2495–06.CrossRefGoogle Scholar
  59. 59.
    Deitch EA. (2010) Gut lymph and lymphatics: a source of factors leading to organ injury and dysfunction. Ann. N.Y. Acad. Sci. 1207(Suppl 1):E103–11.CrossRefGoogle Scholar
  60. 60.
    Remick DG, et al. (1998) Exogenous interleukin-10 fails to decrease the mortality or morbidity of sepsis. Crit. Care Med. 26:895–904.CrossRefGoogle Scholar
  61. 61.
    Montoya-Ruiz C, et al. (2016) Variants in LTA, TNF, IL1B and IL10 genes associated with the clinical course of sepsis. Immunol. Res. 64:1168–78.CrossRefGoogle Scholar
  62. 62.
    Rajan S, et al. (2007) Intestine-specific overexpression of IL-10 improves survival in polymicrobial sepsis. Shock. 45:486–522.Google Scholar
  63. 63.
    Deng M, et al. (2016) Toll-like receptor 4 signaling on dendritic cells suppresses polymorphonuclear leukocyte CXCR2 expression and trafficking via interleukin 10 during intra-abdominal sepsis. J. Infect. Dis. 213:1280–88.CrossRefGoogle Scholar
  64. 64.
    van der Poll T, et al. (1995) Endogenous IL–10 protects mice from death during septic peritonitis. J. Immunol. 155:5397–5401.PubMedGoogle Scholar
  65. 65.
    Murphey ED, Sherwood ER. (2006) Bacterial clearance and mortality are not improved by a combination of IL-10 neutralization and IFN-gamma administration in a murine model of post-CLP immunosuppression. Shock. 26:417–24.CrossRefGoogle Scholar
  66. 66.
    Ralay RH, et al. (2007) Protection against endotoxic shock as a consequence of reduced nitrosative stress in MLCK210-null mice. Am. J. Pathol. 170:439–46.CrossRefGoogle Scholar
  67. 67.
    Alverdy JC, Krezalek MA. (2017) Collapse of the microbiome, emergence of the pathobiome, and the immunopathology of sepsis. Crit. Care Med. 45:337–47.CrossRefGoogle Scholar
  68. 68.
    Su L, et al. (2009) Targeted epithelial tight junction dysfunction causes immune activation and contributes to development of experimental colitis. Gastroenterology. 136:551–63.CrossRefGoogle Scholar
  69. 69.
    Zheng W, Kou Y, Gao FL, Ouyang XH. (2016) Enzymatic changes in myosin regulatory proteins may explain vasoplegia in terminally ill patients with sepsis. Biosci. Rep. 36(2):e00305.CrossRefGoogle Scholar
  70. 70.
    Cohen TS, DiPaolo BC, Lawrence GG, Margulies SS. 2012. Sepsis enhances epithelial permeability with stretch in an actin dependent manner. PLoS ONE. 7:e38748.CrossRefGoogle Scholar

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Authors and Affiliations

  • C. Adam Lorentz
    • 1
  • Zhe Liang
    • 2
  • Mei Meng
    • 3
  • Ching-Wen Chen
    • 2
  • Benyam P. Yoseph
    • 2
  • Elise R. Breed
    • 2
  • Rohit Mittal
    • 2
  • Nathan J. Klingensmith
    • 2
  • Alton B. Farris
    • 4
  • Eileen M. Burd
    • 4
  • Michael Koval
    • 5
  • Mandy L. Ford
    • 6
  • Craig M. Coopersmith
    • 2
    • 7
  1. 1.Department of UrologyEmory University School of MedicineAtlantaUSA
  2. 2.Department of Surgery and Emory Critical Care CenterEmory University School of MedicineAtlantaUSA
  3. 3.Department of Critical Care MedicineShandong Provincial Hospital Affiliated Shandong UniversityJinanChina
  4. 4.Department of Pathology and Laboratory MedicineEmory University School of MedicineAtlantaUSA
  5. 5.Department of Internal Medicine and Emory Alcohol and Lung Biology CenterEmory University School of MedicineAtlantaUSA
  6. 6.Department of Surgery and Emory Transplant CenterEmory University School of MedicineAtlantaUSA
  7. 7.AtlantaUSA

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