Acute Crit Care.  2018 Aug;33(3):154-161. 10.4266/acc.2018.00619.

Anti-inflammatory Role of Mesenchymal Stem Cells in an Acute Lung Injury Mouse Model

• Huh JW
• Kim WY
• Park YY
• Lim CM
• Koh Y
• Kim MJ
• Hong SB

• KMID: 2436327
• DOI: http://doi.org/10.4266/acc.2018.00619

Abstract

BACKGROUND
Mesenchymal stem cells (MSCs) attenuate injury in various lung injury models through paracrine effects. We hypothesized that intratracheal transplantation of allogenic MSCs could attenuate lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice, mediated by anti-inflammatory responses.
METHODS
Six-week-old male mice were randomized to either the control or the ALI group. ALI was induced by intratracheal LPS instillation. Four hours after LPS instillation, MSCs or phosphate-buffered saline was randomly intratracheally administered. Neutrophil count and protein concentration in bronchoalveolar lavage fluid (BALF); lung histology; levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein-2; and the expression of proliferation cell nuclear antigen (PCNA), caspase-3, and caspase-9 were evaluated at 48 hours after injury.
RESULTS
Treatment with MSCs attenuated lung injury in ALI mice by decreasing protein level and neutrophil recruitment into the BALF and improving the histologic change. MSCs also decreased the protein levels of proinflammatory cytokines including IL-1β, IL-6, and TNF-α, but had little effect on the protein expression of PCNA, caspase-3, and caspase-9.
CONCLUSIONS
Intratracheal injection of bone marrow-derived allogenic MSCs attenuates LPSinduced ALI via immunomodulatory effects.

Keyword

acute lung injury; cytokines; immunomodulation; inflammation; lipopolysaccharides

MeSH Terms

Acute Lung Injury*
Animals
Bronchoalveolar Lavage Fluid
Caspase 3
Caspase 9
Cytokines
Humans
Immunomodulation
Inflammation
Interleukin-6
Interleukins
Lipopolysaccharides
Lung
Lung Injury
Macrophages
Male
Mesenchymal Stromal Cells*
Mice*
Neutrophil Infiltration
Neutrophils
Proliferating Cell Nuclear Antigen
Tumor Necrosis Factor-alpha
Caspase 3
Caspase 9
Cytokines
Interleukin-6
Interleukins
Lipopolysaccharides
Proliferating Cell Nuclear Antigen
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1. Mesenchymal stem cells attenuate lung injury in lipopolysaccharide-induced acute lung injury. (A) Total cell counts of bronchoalveolar lavage fluid (BALF). (B) Percentage of neutrophils in BALF. (C) Total protein level in BALF. (D) Myeloperoxidase activity in serum. CTL: control group; MSC: mesenchymal stem cell injected group; LPS: lipopolysaccharide-induced acute lung injury group; LPS/MSC: MSC injection following LPS induced acute lung injury group. *P<0.05 compared to the CTL group; **P<0.05 compared to the LPS group.

• 

  Figure 2. Light microscopy findings of the lung. (A, B) In the control (CTL) group, the normal parenchymal architecture, including the alveolar septa, alveolar lumen, and capillaries were well preserved. The infiltration of inflammatory cells was not observed. (C, D) In the mesenchymal stem cell (MSC) injected group, the normal pulmonary architecture was well preserved similar to that in the CTL group. (E, F) In the lipopolysaccharide (LPS)-induced acute lung injury group, the pulmonary architecture, including the alveolar septa and alveolar lumen were not preserved. Perivascular edema (asterisk) and intrapulmonary hemorrhage (arrow heads) were observed. The infiltration of inflammatory cells, perivascular cuffing (black arrows), and floating of phagocytes in the alveolar lumen (white arrows) were observed. (G, H) In the MSC injection following LPS induced acute lung injury (LPS/MSC) group, the alveolar septa and alveolar lumen were relatively well preserved. Intrapulmonary hemorrhage and perivascular cuffing was less prominent compared to those of the lungs in the LPS group. The infiltration of inflammatory cells was not observed. (H&E; magnification, A, C, E, G: ×200; B, D, F, H: ×400).

• 

  Figure 3. Lung injury scores. CTL: control group; MSC: mesenchymal stem cell injected group; LPS: lipopolysaccharide-induced acute lung injury group; LPS/MSC: MSC injection following LPS induced acute lung injury group. *P<0.05, compared to the CTL group; **P<0.05, compared to the LPS group.

• 

  Figure 4. Mesenchymal stem cells attenuate lipopolysaccharide-induced acute lung injury by anti-inflammatory effects. (A) Tumor necrosis factor (TNF)-α, (B) interleukin (IL)-1β, (C) IL-6, and (D) macrophage inflammatory protein (MIP)-2 in bronchoalveolar lavage fluid (BALF). CTL: control group; MSC: mesenchymal stem cell injected group; LPS: lipopolysaccharide-induced acute lung injury group; LPS/MSC: MSC injection following LPS induced acute lung injury group. *P<0.05, compared to the CTL group; **P<0.05, compared to the LPS group.

• 

  Figure 5. The effect of mesenchymal stem cells on proliferation and cell death. (A) Western blot of proliferation cell nuclear antigen (PCNA), caspase-9 and cleaved-caspase-9, caspase-3, and β-actin. (B) Relative protein level (PCNA/β-actin ratio). CTL: control group; MSC: mesenchymal stem cell injected group; LPS: lipopolysaccharide-induced acute lung injury group; LPS/MSC: MSC injection following LPS induced acute lung injury group.

Reference

1. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. N Engl J Med. 2005; 353:1685–93.

2. Bellani G, Laffey JG, Pham T, Fan E, Brochard L, Esteban A, et al. Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. JAMA. 2016; 315:788–800.

3. Zambon M, Vincent JL. Mortality rates for patients with acute lung injury/ARDS have decreased over time. Chest. 2008; 133:1120–7.

4. Amato MB, Barbas CS, Medeiros DM, Magaldi RB, Schettino GP, Lorenzi-Filho G, et al. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med. 1998; 338:347–54.

5. Guérin C, Reignier J, Richard JC, Beuret P, Gacouin A, Boulain T, et al. Prone positioning in severe acute respiratory distress syndrome. N Engl J Med. 2013; 368:2159–68.

6. Peek GJ, Mugford M, Tiruvoipati R, Wilson A, Allen E, Thalanany MM, et al. Efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (CESAR): a multicentre randomised controlled trial. Lancet. 2009; 374:1351–63.

7. Hudson LD, Milberg JA, Anardi D, Maunder RJ. Clinical risks for development of the acute respiratory distress syndrome. Am J Respir Crit Care Med. 1995; 151:293–301.

8. Doyle RL, Szaflarski N, Modin GW, Wiener-Kronish JP, Matthay MA. Identification of patients with acute lung injury. Predictors of mortality. Am J Respir Crit Care Med. 1995; 152:1818–24.

9. Matthay MA, Zimmerman GA. Acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management. Am J Respir Cell Mol Biol. 2005; 33:319–27.

10. Chollet-Martin S. Polymorphonuclear neutrophil activation during the acute respiratory distress syndrome. Intensive Care Med. 2000; 26:1575–7.

11. Abraham E. Neutrophils and acute lung injury. Crit Care Med. 2003; 31(4 Suppl):S195–9.

12. Stephens KE, Ishizaka A, Wu ZH, Larrick JW, Raffin TA. Granulocyte depletion prevents tumor necrosis factor-mediated acute lung injury in guinea pigs. Am Rev Respir Dis. 1988; 138:1300–7.

13. Perkins GD, Nathani N, McAuley DF, Gao F, Thickett DR. In vitro and in vivo effects of salbutamol on neutrophil function in acute lung injury. Thorax. 2007; 62:36–42.

14. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999; 284:143–7.

15. Li TS, Hayashi M, Ito H, Furutani A, Murata T, Matsuzaki M, et al. Regeneration of infarcted myocardium by intramyocardial implantation of ex vivo transforming growth factor-betapreprogrammed bone marrow stem cells. Circulation. 2005; 111:2438–45.

16. Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 2006; 12:459–65.

17. Lee RH, Pulin AA, Seo MJ, Kota DJ, Ylostalo J, Larson BL, et al. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell. 2009; 5:54–63.

18. Parekkadan B, van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, et al. Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure. PLoS One. 2007; 2:e941.

19. Mei SH, Haitsma JJ, Dos Santos CC, Deng Y, Lai PF, Slutsky AS, et al. Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis. Am J Respir Crit Care Med. 2010; 182:1047–57.

20. Ortiz LA, Gambelli F, McBride C, Gaupp D, Baddoo M, Kaminski N, et al. Mesenchymal stem cell engraftment in lung is enhanced in response to bleomycin exposure and ameliorates its fibrotic effects. Proc Natl Acad Sci U S A. 2003; 100:8407–11.

21. Rojas M, Woods CR, Mora AL, Xu J, Brigham KL. Endotoxininduced lung injury in mice: structural, functional, and biochemical responses. Am J Physiol Lung Cell Mol Physiol. 2005; 288:L333–41.

22. Gupta N, Su X, Popov B, Lee JW, Serikov V, Matthay MA. Intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxininduced acute lung injury in mice. J Immunol. 2007; 179:1855–63.

23. van Helden HP, Kuijpers WC, Steenvoorden D, Go C, Bruijnzeel PL, van Eijk M, et al. Intratracheal aerosolization of endotoxin (LPS) in the rat: a comprehensive animal model to study adult (acute) respiratory distress syndrome. Exp Lung Res. 1997; 23:297–316.

24. Kim ES, Chang YS, Choi SJ, Kim JK, Yoo HS, Ahn SY, et al. Intratracheal transplantation of human umbilical cord bloodderived mesenchymal stem cells attenuates Escherichia coliinduced acute lung injury in mice. Respir Res. 2011; 12:108.

25. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006; 8:315–7.

26. Everhart MB, Han W, Sherrill TP, Arutiunov M, Polosukhin VV, Burke JR, et al. Duration and intensity of NF-kappaB activity determine the severity of endotoxin-induced acute lung injury. J Immunol. 2006; 176:4995–5005.

27. Hong SB, Koh Y, Lee IC, Kim MJ, Kim WS, Kim DS, et al. Induced hypothermia as a new approach to lung rest for the acutely injured lung. Crit Care Med. 2005; 33:2049–55.

28. Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med. 2000; 342:1334–49.

29. Pugin J, Ricou B, Steinberg KP, Suter PM, Martin TR. Proinflammatory activity in bronchoalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1. Am J Respir Crit Care Med. 1996; 153:1850–6.

30. Koh Y, Lee YM, Lim CM, Lee SS, Shim TS, Lee SD, et al. Effects of heat pretreatment on histopathology, cytokine production, and surfactant in endotoxin-induced acute lung injury. Inflammation. 2001; 25:187–96.

31. Schütte H, Lohmeyer J, Rosseau S, Ziegler S, Siebert C, Kielisch H, et al. Bronchoalveolar and systemic cytokine profiles in patients with ARDS, severe pneumonia and cardiogenic pulmonary oedema. Eur Respir J. 1996; 9:1858–67.

32. Suter PM, Suter S, Girardin E, Roux-Lombard P, Grau GE, Dayer JM. High bronchoalveolar levels of tumor necrosis factor and its inhibitors, interleukin-1, interferon, and elastase, in patients with adult respiratory distress syndrome after trauma, shock, or sepsis. Am Rev Respir Dis. 1992; 145:1016–22.

33. Koh Y, Hybertson BM, Jepson EK, Cho OJ, Repine JE. Cytokine-induced neutrophil chemoattractant is necessary for interleukin-1-induced lung leak in rats. J Appl Physiol. 1995; 79:472–8.

34. Kawamura H, Kawamura T, Kanda Y, Kobayashi T, Abo T. Extracellular ATP-stimulated macrophages produce macrophage inflammatory protein-2 which is important for neutrophil migration. Immunology. 2012; 136:448–58.