Immunomodulatory nutrients as the omega-3 polyunsaturated fatty acids (n-3 PUFA) and citrus pectins (CPn) are reported to beneficially affect the host intestinal immunity. However so far, the biological pathways modulated by these nutrients in intestinal inflammation at the level of intestinal epithelial layer (IEL) remains elusive. To bridge this knowledge gap, the aim of our present study was set in the direction to delineate the effects of n-3 PUFA in porcine IPEC-J2 cell line under (LPS) stress conditions underpinning pig nutrition, combining the state-of-the-art cell-based assays and bioinformatic analysis. The second part of our study was directed towards establishing a primary intestinal epithelial cell (IEC) culture from chicken embryos for the assessment of CPn against LPS stress, underpinning poultry nutrition. Utilizing different cell-based assays, we have successfully demonstrated the proliferative effects of n-3 PUFAs as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the IPEC-J2 cells. Besides, n-3 PUFA pre-treatment (DHA:EPA, 1:2, 10 µM, 24 h) was shown to counteract the cellular damage elicited by different stress factors as LPS, hydrogen-peroxide (H2O2) and dextran sulphate sodium (DSS). In addition, through system biology and multi-omics integration (transcriptomics and proteomics), we have demonstrated the cytoprotective properties of n-3 PUFA in IPEC-J2 cells against LPS-induced inflammatory and metabolic damage. Specifically, in these cells, we have identified that n-3 PUFA regulate the biological process as, (i) Axon guidance for developmental process; (ii) Defensin and interferon-mediated antimicrobial defense response for homeostasis; (iii) Cell junction assembly under stress-related cell proliferation; (iv) Amelioration of TLR/MyD88 and cytokine signaling in innate immune response; (v) Fatty acid storage in lipid droplets for lipid homeostasis; (vi) Recovery of central carbon metabolic process from dysregulation under infection; (vii) Lipolysis of fatty acids stored in lipid droplets for prevention of cell starvation during infection. To the best of the knowledge, this is the first study to comprehensively map the bioactivity of n-3 PUFA in enterocytes using multi-omics approach. The outcome of the present study, will enable us to better understand the role of n-3 PUFA in intestinal barrier for planning nutritional or therapeutic strategies. Further in the chicken in vitro study, we have preliminarily demonstrated a simplified method of cell isolation and establishment of primary IEC culture from chicken embryos using mechanical tissue disruptions method. We have also shown that the response of chicken cells is in accordance with the reference IPEC-J2 line using a dose-response study with CPn and LPS. This primary IEC culture model can further be utilized as a starting point for setting up poultry in vitro studies on intestinal barrier.

ESTABLISHING IN VITRO INTESTINAL EPITHELIAL CELL MODELS IN TRANSLATIONAL ANIMAL NUTRITION / T.s. Sundaram ; tutor: A. Baldi, PhD University of Milan (Italy) ; co-tutor: J. Pistl, PhD. The University of Veterinary Medicine and Pharmacy in Košice (Slovakia), M. F. Addis. - : . Dipartimento di Medicina Veterinaria e Scienze Animali, 2022. ((34. ciclo, Anno Accademico 2021.

ESTABLISHING IN VITRO INTESTINAL EPITHELIAL CELL MODELS IN TRANSLATIONAL ANIMAL NUTRITION

T.S. Sundaram
2022

Abstract

Immunomodulatory nutrients as the omega-3 polyunsaturated fatty acids (n-3 PUFA) and citrus pectins (CPn) are reported to beneficially affect the host intestinal immunity. However so far, the biological pathways modulated by these nutrients in intestinal inflammation at the level of intestinal epithelial layer (IEL) remains elusive. To bridge this knowledge gap, the aim of our present study was set in the direction to delineate the effects of n-3 PUFA in porcine IPEC-J2 cell line under (LPS) stress conditions underpinning pig nutrition, combining the state-of-the-art cell-based assays and bioinformatic analysis. The second part of our study was directed towards establishing a primary intestinal epithelial cell (IEC) culture from chicken embryos for the assessment of CPn against LPS stress, underpinning poultry nutrition. Utilizing different cell-based assays, we have successfully demonstrated the proliferative effects of n-3 PUFAs as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the IPEC-J2 cells. Besides, n-3 PUFA pre-treatment (DHA:EPA, 1:2, 10 µM, 24 h) was shown to counteract the cellular damage elicited by different stress factors as LPS, hydrogen-peroxide (H2O2) and dextran sulphate sodium (DSS). In addition, through system biology and multi-omics integration (transcriptomics and proteomics), we have demonstrated the cytoprotective properties of n-3 PUFA in IPEC-J2 cells against LPS-induced inflammatory and metabolic damage. Specifically, in these cells, we have identified that n-3 PUFA regulate the biological process as, (i) Axon guidance for developmental process; (ii) Defensin and interferon-mediated antimicrobial defense response for homeostasis; (iii) Cell junction assembly under stress-related cell proliferation; (iv) Amelioration of TLR/MyD88 and cytokine signaling in innate immune response; (v) Fatty acid storage in lipid droplets for lipid homeostasis; (vi) Recovery of central carbon metabolic process from dysregulation under infection; (vii) Lipolysis of fatty acids stored in lipid droplets for prevention of cell starvation during infection. To the best of the knowledge, this is the first study to comprehensively map the bioactivity of n-3 PUFA in enterocytes using multi-omics approach. The outcome of the present study, will enable us to better understand the role of n-3 PUFA in intestinal barrier for planning nutritional or therapeutic strategies. Further in the chicken in vitro study, we have preliminarily demonstrated a simplified method of cell isolation and establishment of primary IEC culture from chicken embryos using mechanical tissue disruptions method. We have also shown that the response of chicken cells is in accordance with the reference IPEC-J2 line using a dose-response study with CPn and LPS. This primary IEC culture model can further be utilized as a starting point for setting up poultry in vitro studies on intestinal barrier.
BALDI, ANTONELLA
BALDI, ANTONELLA
ADDIS, MARIA FILIPPA
CECILIANI, FABRIZIO
Omega-3 polyunsaturated fatty acids; Citrus pectin; Lipopolysaccharides; Intestinal epithelial layer; Cell-based assays; Bioinformatic analysis
Settore AGR/18 - Nutrizione e Alimentazione Animale
ESTABLISHING IN VITRO INTESTINAL EPITHELIAL CELL MODELS IN TRANSLATIONAL ANIMAL NUTRITION / T.s. Sundaram ; tutor: A. Baldi, PhD University of Milan (Italy) ; co-tutor: J. Pistl, PhD. The University of Veterinary Medicine and Pharmacy in Košice (Slovakia), M. F. Addis. - : . Dipartimento di Medicina Veterinaria e Scienze Animali, 2022. ((34. ciclo, Anno Accademico 2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/944348
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