Download PDF
Planta Med 2022; 88(03/04): 262-273
DOI: 10.1055/a-1510-5802
Biological and Pharmacological Activity
Original Papers

Establishment of an In Vitro Co-Culture Model of the Piglet Gut to Study Inflammatory Response and Barrier Integrity

Theresa Schott
1   BIOMIN Research Center, Tulln an der Donau, Austria
,
Nicole Reisinger
1   BIOMIN Research Center, Tulln an der Donau, Austria
,
Klaus Teichmann
1   BIOMIN Research Center, Tulln an der Donau, Austria
,
Jürgen König
2   Department of Nutritional Science, University of Vienna, Vienna, Austria
,
Andrea Ladinig
3   Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria
,
Elisabeth Mayer
1   BIOMIN Research Center, Tulln an der Donau, Austria
› Author AffiliationsSupported by: Österreichische Forschungsförderungsgesellschaft 866384
› Further Information
    Permissions and Reprints

Abstract

In intensive farming, piglets are exposed to various challenges that activate intestinal inflammatory processes, negatively affecting animal health and leading to economic losses. To study the role of the inflammatory response on epithelial barrier integrity, co-culture systems that mimic in vivo complexity are more and more preferred over cell monocultures. In this study, an in vitro gut co-culture model consisting of intestinal porcine epithelial cells and porcine peripheral blood mononuclear cells was established. The model provides an appropriate tool to study the role of the inflammatory response on epithelial barrier integrity and to screen for feed and food components, exerting beneficial effects on gut health. In the established model, inflammation-like reactions and damage of the epithelial barrier, indicated by a decrease of transepithelial electrical resistance, were elicited by activation of peripheral blood mononuclear cells via one of 3 stimuli: lipopolysaccharide, lipoteichoic acid, or concanavalin A. Two phytogenic substances that are commonly used as feed additives, licorice extract and oregano oil, have been shown to counteract the drop in transepithelial electrical resistance values in the gut co-culture model. The established co-culture model provides a powerful in vitro tool to study the role of intestinal inflammation on epithelial barrier integrity. As it consists of porcine epithelial and porcine blood cells it perfectly mimics in vivo conditions and imitates the inter-organ communication of the piglet gut. The developed model is useful to screen for nutritional components or drugs, having the potential to balance intestinal inflammation and strengthen the epithelial barrier integrity in piglets.

Key words

gut health - intestinal inflammation - intestinal porcine epithelial cells - porcine blood cells - oregano oil - licorice extract

Supporting Information

    This section consists of 4 figures and is available as Supporting Information. The effect of different cultivation media on PBMCs, the effect of LTA/LPS stimulation on cell viability of IPEC-J2 alone or in co-culture with PBMCs, and the cytokine analyses after stimulation of indirect co-cultures with ConA, LPS, or LTA measured by Luminex are included.

  • Supporting Information


Publication History

Received: 24 February 2021

Accepted after revision: 14 May 2021

Article published online:
18 June 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Liu Y. Fatty acids, inflammation and intestinal health in pigs. J Anim Sci Biotechnol 2015; 6: 1-9 DOI: 10.1186/s40104-015-0040-1.
    CrossrefPubMedGoogle Scholar
  • 2 Shin D, Chang SY, Bogere P, Won KH, Choi JY, Choi YJ, Lee HK, Hur J, Park BY, Kim Y, Heo J. Beneficial roles of probiotics on the modulation of gut microbiota and immune response in pigs. PLoS One 2019; 14: 1-23 DOI: 10.1371/journal.pone.0220843.
    CrossrefPubMedGoogle Scholar
  • 3 Valenzuela-Grijalva NV, Pinelli-Saavedra A, Muhlia-Almazan A, Domínguez-Díaz D, González-Ríos H. Dietary inclusion effects of phytochemicals as growth promoters in animal production. J Anim Sci Technol 2017; 59: 1-17 DOI: 10.1186/s40781-017-0133-9.
    CrossrefPubMedGoogle Scholar
  • 4 Wang T, Yao W, Li J, Shao Y, He Q, Xia J, Huang F. Dietary garcinol supplementation improves diarrhea and intestinal barrier function associated with its modulation of gut microbiota in weaned piglets. J Anim Sci Biotechnol 2020; 11: 1-13 DOI: 10.1186/s40104-020-0426-6.
    CrossrefPubMedGoogle Scholar
  • 5 Wang K, Chen G, Cao G, Xu Y, Wang Y, Yang C. Effects of Clostridium butyricum and Enterococcus faecalis on growth performance, intestinal structure, and inflammation in lipopolysaccharide-challenged weaned piglets. J Anim Sci 2019; 97: 4140-4151 DOI: 10.1093/jas/skz235.
    CrossrefPubMedGoogle Scholar
  • 6 Chen J, Yu B, Chen D, Huang Z, Mao X, Zheng P, Yu J, Luo J, He J. Chlorogenic acid improves intestinal barrier functions by suppressing mucosa inflammation and improving antioxidant capacity in weaned pigs. J Nutr Biochem 2018; 59: 84-92 DOI: 10.1016/j.jnutbio.2018.06.005.
    CrossrefPubMedGoogle Scholar
  • 7 Cencič A, Langerholc T. Functional cell models of the gut and their applications in food microbiology–a review. Int J Food Microbiol 2010; 141: S4 DOI: 10.1016/j.ijfoodmicro.2010.03.026.
    CrossrefPubMedGoogle Scholar
  • 8 Duell BL, Cripps AW, Schembri MA, Ulett GC. Epithelial cell coculture models for studying infectious diseases: benefits and limitations. J Biomed Biotechnol 2011; 2011: 852419 DOI: 10.1155/2011/852419.
    CrossrefPubMedGoogle Scholar
  • 9 Ponce de León-Rodríguez MC, Guyot JP, Laurent-Babot C. Intestinal in vitro cell culture models and their potential to study the effect of food components on intestinal inflammation. Crit Rev Food Sci Nutr 2019; 59: 3648-3666 DOI: 10.1080/10408398.2018.1506734.
    CrossrefPubMedGoogle Scholar
  • 10 Gu MJ, Song SK, Lee IK, Ko S, Han SE, Bae S, Ji SY, Park BC, Song KD, Lee HK, Han SH, Yun CH. Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol. Vet Res 2016; 47: 1-11 DOI: 10.1186/s13567-016-0309-1.
    CrossrefPubMedGoogle Scholar
  • 11 Gu MJ, Han SE, Hwang K, Mayer E, Reisinger N, Schatzmayr D, Park BC, Han SH, Yun CH. Hydrolyzed fumonisin B 1 induces less inflammatory responses than fumonisin B 1 in the co-culture model of porcine intestinal epithelial and immune cells. Toxicol Lett 2019; 305: 110-116 DOI: 10.1016/j.toxlet.2019.01.013.
    CrossrefPubMedGoogle Scholar
  • 12 Dehlink E, Domig KJ, Loibichler C, Kampl E, Eiwegger T, Georgopoulos A, Kneifel W, Urbanek R, Szépfalusi Z. Heat- and formalin-inactivated probiotic bacteria induce comparable cytokine patterns in intestinal epithelial cell-leucocyte cocultures. J Food Prot 2007; 70: 2417-2421 DOI: 10.4315/0362-028X-70.10.2417.
    CrossrefPubMedGoogle Scholar
  • 13 Haller D, Bode C, Hammes WP, Pfeifer AMA, Schiffrin EJ, Blum S. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures. Gut 2000; 47: 79-87 DOI: 10.1136/gut.47.1.79.
    CrossrefPubMedGoogle Scholar
  • 14 Loss H, Aschenbach JR, Ebner F, Tedin K, Lodemann U. Inflammatory responses of porcine MoDC and intestinal epithelial cells in a direct-contact co-culture system following a bacterial challenge. Inflammation 2020; 43: 552-567 DOI: 10.1007/s10753-019-01137-4.
    CrossrefPubMedGoogle Scholar
  • 15 Katial RK, Sachanandani D, Pinney C, Lieberman MM. Cytokine production in cell culture by peripheral blood mononuclear cells from immunocompetent hosts. Clin Diagn Lab Immunol 1998; 5: 78-81 DOI: 10.1128/cdli.5.1.78-81.1998.
    CrossrefPubMedGoogle Scholar
  • 16 Verfaillie T, Cox E, To LT, Vanrompay D, Bouchaut H, Buys N, Goddeeris BM. Comparative analysis of porcine cytokine production by mRNA and protein detection. Vet Immunol Immunopathol 2001; 81: 97-112 DOI: 10.1016/S0165-2427(01)00339-7.
    CrossrefPubMedGoogle Scholar
  • 17 Guo S, Al-Sadi R, Said HM, Ma TY. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14. Am J Pathol 2013; 182: 375-387 DOI: 10.1016/j.ajpath.2012.10.014.
    CrossrefPubMedGoogle Scholar
  • 18 Liu CH, Chaung HC, Chang HL, Peng YT, Chung WB. Expression of Toll-like receptor mRNA and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus. Vet Microbiol 2009; 136: 266-276 DOI: 10.1016/j.vetmic.2008.11.016.
    CrossrefPubMedGoogle Scholar
  • 19 Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA. BMC Res Notes 2013; 6: 1 DOI: 10.1186/1756-0500-6-56.
    CrossrefPubMedGoogle Scholar
  • 20 Mani V, Weber TE, Baumgard LH, Gabler NK. Growth and development symposium: Endotoxin, inflammation, and intestinal function in livestock. J Anim Sci 2012; 90: 1452-1465 DOI: 10.2527/jas.2011-4627.
    CrossrefPubMedGoogle Scholar
  • 21 Islam MA, Pröll M, Hölker M, Tholen E, Tesfaye D, Looft C, Schellander K, Cinar MU. Alveolar macrophage phagocytic activity is enhanced with LPS priming, and combined stimulation of LPS and lipoteichoic acid synergistically induce pro-inflammatory cytokines in pigs. Innate Immun 2013; 19: 631-643 DOI: 10.1177/1753425913477166.
    CrossrefPubMedGoogle Scholar
  • 22 Leonard F, Collnot EM, Lehr CM. A three-dimensional coculture of enterocytes, monocytes and dendritic cells to model inflamed intestinal mucosa in vitro . Mol Pharm 2010; 7: 2103-2119 DOI: 10.1021/mp1000795.
    CrossrefPubMedGoogle Scholar
  • 23 Ou G, Baranov V, Lundmark E, Hammarström S, Hammarström ML. Contribution of intestinal epithelial cells to innate immunity of the human gut–studies on polarized monolayers of colon carcinoma cells. Scand J Immunol 2009; 69: 150-161 DOI: 10.1111/j.1365-3083.2008.02208.x.
    CrossrefPubMedGoogle Scholar
  • 24 Parlesak A, Haller D, Brinz S, Baeuerlein A, Bode C. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria. Scand J Immunol 2004; 60: 477-485 DOI: 10.1111/j.0300-9475.2004.01495.x.
    CrossrefPubMedGoogle Scholar
  • 25 Liu Y, Chen F, Odle J, Lin X, Jacobi SK, Zhu H, Wu Z, Hou Y. Fish oil enhances intestinal integrity and inhibits TLR4 and NOD2 signaling pathways in weaned pigs after LPS challenge. J Nutr 2012; 142: 2017-2024 DOI: 10.3945/jn.112.164947.
    CrossrefPubMedGoogle Scholar
  • 26 Bruserud Ø, Wendelbo Ø, Paulsen K. Lipoteichoic acid derived from Enterococcus faecalis modulates the functional characteristics of both normal peripheral blood leukocytes and native human acute myelogenous leukemia blasts. Eur J Haematol 2004; 73: 340-350 DOI: 10.1111/j.1600-0609.2004.00307.x.
    CrossrefPubMedGoogle Scholar
  • 27 Nossol C, Diesing AK, Walk N, Faber-Zuschratter H, Hartig R, Post A, Kluess J, Rothkötter HJ, Kahlert S. Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochem Cell Biol 2011; 136: 103-115 DOI: 10.1007/s00418-011-0826-y.
    CrossrefPubMedGoogle Scholar
  • 28 Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewsksa E, Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clinical Investig 1993; 95: 55-65
    CrossrefPubMedGoogle Scholar
  • 29 Kämpfer AAM, Urbán P, Gioria S, Kanase N, Stone V, Kinsner-Ovaskainen A. Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state. Toxicol Vitr 2017; 45: 31-43 DOI: 10.1016/j.tiv.2017.08.011.
    CrossrefPubMedGoogle Scholar
  • 30 Vatzia E, Pierron A, Saalmüller A, Mayer E, Gerner W. Deoxynivalenol affects proliferation and expression of activation-related molecules in major porcine T-cell subsets. Toxins (Basel) 2019; 11: 644 DOI: 10.3390/toxins11110644.
    CrossrefPubMedGoogle Scholar
  • 31 Pié S, Lallès JP, Blazy F, Laffitte J, Sève B, Oswald IP. Weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets. J Nutr 2004; 134: 641-647 DOI: 10.1093/jn/134.3.641.
    CrossrefPubMedGoogle Scholar
  • 32 Koch L, Frommhold D, Buschmann K, Kuss N, Poeschl J, Ruef P. LPS- and LTA-induced expression of IL-6 and TNF- in neonatal and adult blood: role of MAPKs and NF-B. Mediators Inflamm 2014; 2014: 283126 DOI: 10.1155/2014/283126.
    CrossrefPubMedGoogle Scholar
  • 33 Liu X, Hu X, Zhang X, Li Z, Lu H. Role of rheum polysaccharide in the cytokines produced by peripheral blood monocytes in TLR4 mediated HLA-B27 associated AAU. Biomed Res Int 2013; 2013: 431232 DOI: 10.1155/2013/431232.
    CrossrefPubMedGoogle Scholar
  • 34 Vatzia E, Pierron A, Hoog AM, Saalmüller A, Mayer E, Gerner W. Deoxynivalenol Has the capacity to increase transcription factor expression and cytokine production in porcine T cells. Front Immunol 2020; 11: 1-17 DOI: 10.3389/fimmu.2020.02009.
    CrossrefPubMedGoogle Scholar
  • 35 Windisch W, Schedle K, Plitzner C, Kroismayr A. Use of phytogenic products as feed additives for swine and poultry. J Anim Sci 2008; 86: E140-E148 DOI: 10.2527/jas.2007-0459.
    CrossrefPubMedGoogle Scholar
  • 36 Lallès JP, Bosi P, Smidt H, Stokes CR. Nutritional management of gut health in pigs around weaning. Proc Nutr Soc 2007; 66: 260-268 DOI: 10.1017/S0029665107005484.
    CrossrefPubMedGoogle Scholar
  • 37 Dong GZ, Pluske JR. The low feed intake in newly-weaned pigs: problems and possible solutions. Asian-Australasian J Anim Sci 2007; 20: 440-452 DOI: 10.5713/ajas.2007.440.
    CrossrefPubMedGoogle Scholar
  • 38 Ayrle H, Mevissen M, Kaske M, Nathues H, Gruetzner N, Melzig M, Walkenhorst M. Medicinal plants–prophylactic and therapeutic options for gastrointestinal and respiratory diseases in calves and piglets? A systematic review. BMC Vet Res 2016; 12: 89 DOI: 10.1186/S12917-016-0714-8.
    CrossrefPubMedGoogle Scholar
  • 39 Kaschubek T, Mayer E, Rzesnik S, Grenier B, Bachinger D, Schieder C, König J, Teichmann K. Effects of phytogenic feed additives on cellular oxidative stress and inflammatory reactions in intestinal porcine epithelial cells. J Anim Sci 2018; 96: 3657-3669 DOI: 10.1093/jas/sky263.
    CrossrefPubMedGoogle Scholar
  • 40 Pu J, Chen D, Tian G, He J, Zheng P, Mao X, Yu J, Huang Z, Zhu L, Luo J, Luo Y, Yu B. Protective effects of benzoic acid, bacillus coagulans, and oregano oil on intestinal injury caused by enterotoxigenic escherichia coli in weaned piglets. Biomed Res Int 2018; 2018: 1829632 DOI: 10.1155/2018/1829632.
    CrossrefPubMedGoogle Scholar
  • 41 Zou Y, Xiang Q, Wang J, Peng J, Wei H. Oregano Essential oil improves intestinal morphology and expression of tight junction proteins associated with modulation of selected intestinal bacteria and immune status in a pig model. Biomed Res Int 2016; 2016: 5436738 DOI: 10.1155/2016/5436738.
    CrossrefPubMedGoogle Scholar
  • 42 Bachinger D, Mayer E, Kaschubek T, Schieder C, König J, Teichmann K. Influence of phytogenics on recovery of the barrier function of intestinal porcine epithelial cells after a calcium switch. J Anim Physiol Anim Nutr (Berl) 2019; 103: 210-220 DOI: 10.1111/jpn.12997.
    CrossrefPubMedGoogle Scholar
  • 43 Gallois M, Rothkötter HJ, Bailey M, Stokes CR, Oswald IP. Natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role?. Animal 2009; 3: 1644-1661 DOI: 10.1017/S1751731109004236.
    CrossrefPubMedGoogle Scholar
  • 44 Szépfalusi Z, Nentwich I, Gerstmayr M, Jost E, Todoran L, Gratzl R, Herkner K, Urbanek R. Prenatal allergen contact with milk proteins. Clin Exp Allergy 1997; 27: 28-35
    CrossrefPubMedGoogle Scholar
  • 45 Springler A, Hessenberger S, Schatzmayr G, Mayer E. Early activation of MAPK p44/42 is partially involved in DON-induced disruption of the intestinal barrier function and tight junction network. Toxins (Basel) 2016; 8: 264 DOI: 10.3390/toxins8090264.
    CrossrefPubMedGoogle Scholar
  • 46 Ladinig A, Lunney JK, Souza CJH, Ashley C, Plastow G, Harding JCS. Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome. Vet Res 2014; 45: 1-10 DOI: 10.1186/s13567-0140113-8.
    CrossrefPubMedGoogle Scholar