J Vet Sci.  2016 Dec;17(4):539-548. 10.4142/jvs.2016.17.4.539.

Canine adipose tissue-derived mesenchymal stem cells ameliorate severe acute pancreatitis by regulating T cells in rats

• Kim HW
• Song WJ
• Li Q
• Han SM
• Jeon KO
• Park SC
• Ryu MO
• Chae HK
• Kyeong K
• Youn HY

• KMID: 2412610
• DOI: http://doi.org/10.4142/jvs.2016.17.4.539

Abstract

Severe acute pancreatitis (SAP) is associated with systemic complications and high mortality rate in dogs. Mesenchymal stem cells (MSCs) have been investigated for their therapeutic potential in several inflammation models. In the present study, the effects of canine adipose tissue-derived (cAT)-MSCs in a rat model of SAP induced by retrograde injection of 3% sodium taurocholate solution into the pancreatic duct were investigated. cAT-MSCs labeled with dioctadecyl-3,3,3"²-tetramethylindo-carbocyanine perchlorate (1 × 10⁷ cells/kg) were systemically administered to rats and pancreatic tissue was collected three days later for histopathological, quantitative real-time polymerase chain reaction, and immunocytochemical analyses. Greater numbers of infused cAT-MSCs were detected in the pancreas of SAP relative to sham-operated rats. cAT-MSC infusion reduced pancreatic edema, inflammatory cell infiltration, and acinar cell necrosis, and decreased pancreatic expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, -6, -12, -17, and -23 and interferon-γ, while stimulating expression of the anti-inflammatory cytokines IL-4 and IL-10 in SAP rats. Moreover, cAT-MSCs decreased the number of clusters of differentiation 3-positive T cells and increased that of forkhead box P3-positive T cells in the injured pancreas. These results indicate that cAT-MSCs can be effective as a cell-based therapeutic strategy for treatment of SAP in dogs.

Keyword

acute pancreatitis; anti-inflammatory agents; dogs; mesenchymal stromal cell; regulatory T-lymphocytes

MeSH Terms

Acute Disease
Adipose Tissue/cytology
Animals
Dogs
*Immunity, Innate
Immunohistochemistry
Male
*Mesenchymal Stem Cell Transplantation
Mesenchymal Stromal Cells/*cytology
Pancreatitis/immunology/*therapy
Random Allocation
Rats
Rats, Sprague-Dawley
T-Lymphocytes/*immunology

Figure

• 

  Fig. 1 Identification of mesenchymal stem cells (MSCs) isolated from canine adipose tissue. (A) Immunophenotypic analysis by flow cytometry. (B) Adipogenic, osteogenic, and chondrogenic differentiation of canine adipose tissue-derived (cAT)-MSCs. 200×. Scale bars = 20 µm (B).

• 

  Fig. 2 Therapeutic effects of cAT-MSCs in rats with severe acute pancreatitis (SAP). (A) Histopathological analysis. (B) Serum amylase activities (U/L). (C) Lipase activities (U/L). (D) Pancreas to body weight ratio. Data are shown as the means ± standard deviation (SD). *p < 0.05, ***p < 0.001. Scale bars = 20 µm (A).

• 

  Fig. 3 Tracking of infused cAT-MSCs. (A) Pancreatic tissue sections following systemic administration of CM-DiI-labeled cAT-MSCs in rats with or without SAP. (B) PCR amplification of cRPS5 in pancreatic tissue. Lanes 1 and 2, SHAM+PBS; Lanes 3 and 4, SHAM+MSC; Lanes 5 and 6, SAP+PBS; Lanes 7 and 8; SAP+MSC; Lane 9, canine DNA. 200× (A).

• 

  Fig. 4 Effect of cAT-MSCs on inflammatory cytokines levels. (A) mRNA expression of pro- and anti-inflammatory cytokines in pancreas tissue. (B) Serum levels of IFN-γ and IL-10. Data are shown as the means ± SD. *p < 0.05, **p < 0.01.

• 

  Fig. 5 T cell regulation by cAT-MSCs. cAT-MSCs suppressed the proliferation of cPBMCs (A) and rat splenocytes (B) stimulated with ConA. Detection of CD3+ T cells (C) and FoxP3+ regulatory T cells (D) in pancreatic tissue. Percentages of CD3+ or FoxP3+ cells are shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. 200× (C and D).

Reference

1. Augello A, Tasso R, Negrini SM, Cancedda R, Pennesi G. Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis. Arthritis Rheum. 2007; 56:1175–1186.

2. Baek SJ, Kang SK, Ra JC. In vitro migration capacity of human adipose tissue-derived mesenchymal stem cells reflects their expression of receptors for chemokines and growth factors. Exp Mol Med. 2011; 43:596–603.

3. Beeton C, Chandy KG. Preparing T cell growth factor from rat splenocytes. J Vis Exp. 2007; 402.

4. Bhatia M, Brady M, Shokuhi S, Christmas S, Neoptolemos JP, Slavin J. Inflammatory mediators in acute pancreatitis. J Pathol. 2000; 190:117–125.

5. Brinkhof B, Spee B, Rothuizen J, Penning LC. Development and evaluation of canine reference genes for accurate quantification of gene expression. Anal Biochem. 2006; 356:36–43.

6. Carrade DD, Borjesson DL. Immunomodulation by mesenchymal stem cells in veterinary species. Comp Med. 2013; 63:207–217.

7. Chamberlain G, Fox J, Ashton B, Middleton J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells. 2007; 25:2739–2749.

8. Cook A, Breitschwerdt EB, Levine JF, Bunch SE, Linn LO. Risk factors associated with acute pancreatitis in dogs: 101 cases (1985-1990). J Am Vet Med Assoc. 1993; 203:673–679.

9. English K, Ryan JM, Tobin L, Murphy MJ, Barry FP, Mahon BP. Cell contact, prostaglandin E₂ and transforming growth factor beta 1 play non-redundant roles in human mesenchymal stem cell induction of CD4⁺CD25^Highforkhead box P3⁺ regulatory T cells. Clin Exp Immunol. 2009; 156:149–160.

10. François M, Romieu-Mourez R, Li M, Galipeau J. Human MSC suppression correlates with cytokine induction of indoleamine 2,3-dioxygenase and bystander M2 macrophage differentiation. Mol Ther. 2012; 20:187–195.

11. González MA, Gonzalez-Rey E, Rico L, Büscher D, Delgado M. Adipose-derived mesenchymal stem cells alleviate experimental colitis by inhibiting inflammatory and autoimmune responses. Gastroenterology. 2009; 136:978–989.

12. Gonzalez-Rey E, Anderson P, González MA, Rico L, Büscher D, Delgado M. Human adult stem cells derived from adipose tissue protect against experimental colitis and sepsis. Gut. 2009; 58:929–939.

13. Hess RS, Saunders HM, Van Winkle TJ, Shofer FS, Washabau RJ. Clinical, clinicopathologic, radiographic, and ultrasonographic abnormalities in dogs with fatal acute pancreatitis: 70 cases (1986-1995). J Am Vet Med Assoc. 1998; 213:665–670.

14. Hsieh CS, Macatonia SE, Tripp CS, Wolf SF, O'Garra A, Murphy KM. Development of T_H1 CD4⁺ T cells through IL-12 produced by Listeria-induced macrophages. Science. 1993; 260:547–549.

15. Hu J, Zhang L, Wang N, Ding R, Cui S, Zhu F, Xie Y, Sun X, Wu D, Hong Q, Li Q, Hi S, Liu X, Chen X. Mesenchymal stem cells attenuate ischemic acute kidney injury by inducing regulatory T cells through splenocyte interactions. Kidney Int. 2013; 84:521–531.

16. Hua J, He ZG, Qian DH, Lin SP, Gong J, Meng HB, Yang TS, Sun W, Xu B, Zhou B, Song ZS. Angiopoietin-1 gene-modified human mesenchymal stem cells promote angiogenesis and reduce acute pancreatitis in rats. Int J Clin Exp Pathol. 2014; 7:3580–3595.

17. Whitcomb DC, Gorry MC, Preston RA, Furey W, Sossenheimer MJ, Ulrich CD, Martin SP, Gates LK Jr, Amann ST, Toskes PP, Liddle R, McGrath K, Uomo G, Post JC, Ehrlich GD. Hereditary pancreatitis is caused by a mutation in the cationic trypsinogen gene. Nat Genet. 1996; 14:141–145.

18. Jung KH, Shin HP, Lee S, Lim YJ, Hwang SH, Han H, Park HK, Chung JH, Yim SV. Effect of human umbilical cord blood-derived mesenchymal stem cells in a cirrhotic rat model. Liver Int. 2009; 29:898–909.

19. Jung KH, Song SU, Yi T, Jeon MS, Hong SW, Zheng HM, Lee HS, Choi MJ, Lee DH, Hong SS. Human bone marrow-derived clonal mesenchymal stem cells inhibit inflammation and reduce acute pancreatitis in rats. Gastroenterology. 2011; 140:998–1008.

20. Jung KH, Yi T, Son MK, Song SU, Hong SS. Therapeutic effect of human clonal bone marrow-derived mesenchymal stem cells in severe acute pancreatitis. Arch Pharm Res. 2015; 38:742–751.

21. Kallmeyer K, Pepper MS. Homing properties of mesenchymal stromal cells. Expert Opin Biol Ther. 2015; 15:477–479.

22. Kang JW, Kang KS, Koo HC, Park JR, Choi EW, Park YH. Soluble factors-mediated immunomodulatory effects of canine adipose tissue-derived mesenchymal stem cells. Stem Cells Dev. 2008; 17:681–693.

23. Kehoe O, Cartwright A, Askari A, El Haj AJ, Middleton J. Intra-articular injection of mesenchymal stem cells leads to reduced inflammation and cartilage damage in murine antigen-induced arthritis. J Transl Med. 2014; 12:157.

24. Kota DJ, Wiggins LL, Yoon N, Lee RH. TSG-6 produced by hMSCs delays the onset of autoimmune diabetes by suppressing Th1 development and enhancing tolerogenicity. Diabetes. 2013; 62:2048–2058.

25. Kusske AM, Rongione AJ, Ashley SW, McFadden DW, Reber HA. Interleukin-10 prevents death in lethal necrotizing pancreatitis in mice. Surgery. 1996; 120:284–289.

26. Li H, Yan F, Lei L, Li Y, Xiao Y. Application of autologous cryopreserved bone marrow mesenchymal stem cells for periodontal regeneration in dogs. Cells Tissues Organs. 2009; 190:94–101.

27. Mansfield C. Acute pancreatitis in dogs: advances in understanding, diagnostics, and treatment. Top Companion Anim Med. 2012; 27:123–132.

28. Meng HB, Gong J, Zhou B, Hua J, Yao L, Song ZS. Therapeutic effect of human umbilical cord-derived mesenchymal stem cells in rat severe acute pancreatitis. Int J Clin Exp Pathol. 2013; 6:2703–2712.

29. Moodley Y, Atienza D, Manuelpillai U, Samuel CS, Tchongue J, Ilancheran S, Boyd R, Trounson A. Human umbilical cord mesenchymal stem cells reduce fibrosis of bleomycin-induced lung injury. Am J Pathol. 2009; 175:303–313.

30. Norman J. The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg. 1998; 175:76–83.

31. Pandiyan P, Zheng L, Ishihara S, Reed J, Lenardo MJ. CD4⁺CD25⁺Foxp3⁺ regulatory T cells induce cytokine deprivation–mediated apoptosis of effector CD4⁺ T cells. Nat Immunol. 2007; 8:1353–1362.

32. Penha EM, Meira CS, Guimarães ET, Mendonça MV, Gravely FA, Pinheiro CM, Pinheiro TM, Barrouin-Melo SM, Ribeiro-Dos-Santos R, Soares MB. Use of autologous mesenchymal stem cells derived from bone marrow for the treatment of naturally injured spinal cord in dogs. Stem Cells Int. 2014; 2014:437521.

33. Pittenger M. Sleuthing the source of regeneration by MSCs. Cell Stem Cell. 2009; 5:8–10.

34. Tsuda H, Yamahara K, Otani K, Okumi M, Yazawa K, Kaimori JY, Taguchi A, Kangawa K, Ikeda T, Takahara S, Isaka Y. Transplantation of allogenic fetal membrane-derived mesenchymal stem cells protects against ischemia/reperfusion-induced acute kidney injury. Cell Transplant. 2014; 23:889–899.

35. Van Laethem JL, Marchant A, Delvaux A, Goldman M, Robberecht P, Velu T, Devière J. Interleukin 10 prevents necrosis in murine experimental acute pancreatitis. Gastroenterology. 1995; 108:1917–1922.

36. Vieira NM, Brandalise V, Zucconi E, Secco M, Strauss BE, Zatz M. Isolation, characterization, and differentiation potential of canine adipose-derived stem cells. Cell Transplant. 2010; 19:279–289.

37. Yang B, Bai B, Liu CX, Wang SQ, Jiang X, Zhu CL, Zhao QC. Effect of umbilical cord mesenchymal stem cells on treatment of severe acute pancreatitis in rats. Cytotherapy. 2013; 15:154–162.

38. Yu X, Lu C, Liu H, Rao S, Cai J, Liu S, Kriegel AJ, Greene AS, Liang M, Ding X. Hypoxic preconditioning with cobalt of bone marrow mesenchymal stem cells improves cell migration and enhances therapy for treatment of ischemic acute kidney injury. PloS One. 2013; 8:e62703.

39. Zappia E, Casazza S, Pedemonte E, Benvenuto F, Bonanni I, Gerdoni E, Giunti D, Ceravolo A, Cazzanti F, Frassoni F, Mancardi G, Uccelli A. Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood. 2005; 106:1755–1761.

40. Zyromski N, Murr MM. Evolving concepts in the pathophysiology of acute pancreatitis. Surgery. 2003; 133:235–237.