Browsing by Author "Perry, Famatta"
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Item A Comparison of the Immunometabolic Effect of Antibiotics and Plant Extracts in a Chicken Macrophage-like Cell Line during a Salmonella Enteritidis Challenge(Antibiotics, 2023-02-08) Giovagnoni, Giulia; Perry, Famatta; Tugnoli, Benedetta; Piva, Andrea; Grilli, Ester; Arsenault, Ryan J.Immunometabolic modulation of macrophages can play an important role in the innate immune response of chickens triggered with a multiplicity of insults. In this study, the immunometabolic role of two antibiotics (oxytetracycline and gentamicin) and four plant extracts (thyme essential oil, grape seed extract, garlic oil, and capsicum oleoresin) were investigated on a chicken macrophage-like cell line (HD11) during a Salmonella Enteritidis infection. To study the effect of these substances, kinome peptide array analysis, Seahorse metabolic assay, and gene expression techniques were employed. Oxytetracycline, to which the bacterial strain was resistant, thyme essential oil, and capsicum oleoresin did not show any noteworthy immunometabolic effect. Garlic oil affected glycolysis, but this change was not detected by the kinome analysis. Gentamicin and grape seed extract showed the best immunometabolic profile among treatments, being able to both help the host with the activation of immune response pathways and with maintaining a less inflammatory status from a metabolic point of view.Item Chicken intestinal organoids: a novel method to measure the mode of action of feed additives(Frontiers in Immunology, 2024-05-20) Mitchell, Jordan; Sutton, Kate; Elango, Jeyashree Nathan; Borowska, Dominika; Perry, Famatta; Lahaye, Ludovic; Santin, Elizabeth; Arsenault, Ryan J.; Vervelde, LonnekeThere is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or “mini-guts” has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challenge model. To mimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EO for 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treated with OA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses.Item The immunometabolic responses Salmonella Enteritidis and Salmonella Heidelberg induce in chicken macrophages(University of Delaware, 2019) Perry, FamattaSalmonella is a burden to the agriculture and health sectors as a result of the high number of illnesses, food contamination, and recalls. Salmonella Enteritidis (S. Enteritidis) is one of the most prevalent serotypes isolated from poultry. Salmonella Heidelberg (S. Heidelberg), which is becoming more prevalent than S. Enteritidis, is one of the five most isolated serotypes. Many animals including poultry are carriers of Salmonella but do not show any symptoms. Thus, it is more difficult for producers to avoid the processing and the distribution of contaminated products especially due to the restriction of antibiotic use in food animals. Salmonella invades host cells and exploits host mechanisms for its own benefits. For example, Salmonellae are capable of surviving in macrophages whose role is to kill pathogenic bacteria. Understanding the mechanism by which Salmonella infects and creates a suitable niche in hosts will reveal a potential target for the treatment and prevention of Salmonella contamination without the use of antibiotics. Although S. Enteritidis and S. Heidelberg are almost genetically identical, they both are capable of inducing different immune and metabolic responses in host cells to successfully establish an infection. Kinome peptide array data and available literature showed significant changes in the phosphorylation states of mTOR and AMPK peptides in chickens during Salmonella infections. Therefore, focusing on the AMPK-mTOR signaling cascade, we demonstrated that S. Enteritidis and S. Heidelberg infections induced differential kinase activities in metabolic and immune related peptides of HD11 chicken macrophages. Metabolic flux assays measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) demonstrated that, i) S. Enteritidis at 30 minutes post infection increased glucose metabolism ii) S. Heidelberg at 30 minutes post infection decreased glucose metabolism iii) Both Salmonella infections induce increased oxygen metabolism. Gentamicin protection assays performed at 30 minutes and 2 hours post infection revealed that S. Enteritidis bacteria are more invasive than S. Heidelberg. Furthermore, flow cytometry results showed increased apoptotic/dead cell population in S. Enteritidis infections compared to S. Heidelberg. These results show different immunometabolic responses of HD11 macrophages to S. Enteritidis and S. Heidelberg infections.Item The immunometabolic and molecular effects of potential antibiotic alternatives as feed supplements in broilers(University of Delaware, 2023) Perry, FamattaIn this project, I aimed to expand our understanding of the immunometabolic effects and the mechanism of action of feed additives in broiler chickens’ gastrointestinal health. Poultry is the number one consumed meat in the world, thus improved production efficiency and health are important to feed the world. I determined the effects of protected biofactors and antioxidants (P(BF+AOx)), and protected biofactors and antioxidants with protected organic acids and essential oils (P(BF+AOx)+P(OA+EO)) on the immune and metabolic health of Ross 308 broiler chickens. In the jejunum, the kinome peptide array data analysis showed increased activity of cytoskeletal, cell growth and proliferation proteins, and metabolic signaling in the jejunum of P(BF+AOx)+P(OA+EO) treated chickens. There was a significant decrease in IL-6 gene expression in the jejunum of P(BF+AOx)+P(OA+EO) samples compared to control at day 15. P(BF+AOx)+P(OA+EO) treatments in the jejunum showed strong immunomodulatory effects, perhaps to control inflammation. P(BF+AOx)+P(OA+EO) improves gut health via growth and metabolic signaling in the jejunum while inducing stronger immunomodulation. While in the liver, the kinome peptide array analysis and gene expression showed stimulation of the interleukin 6 receptor (IL-6R) signal transduction for host protection via heightened immune response while maintaining immunomodulation and reduction of damaging inflammation in both P(BF+AOx) and P(BF+AOx)+P(OA+EO) treated groups at day 15. Significant changes were observed via IL-6R signaling in the metabolic profiles of both groups when compared to control with no significant differences when compared to each other. These two feed additives induced immunometabolic homeostasis predominantly via the IL-6 receptor family signaling cascade in the liver. ☐ The effects of sodium butyrate (SB) on metabolism the gastrointestinal tract and immune cells were also studied. Using the kinome peptide array, I examined the proteomic effects of 0.1% SB supplementation in the jejunum and ileum of Cobb 500 broiler chickens at days 21 and 35. Using cell viability assays, we assessed the effects of treating HD11 cells with 1 mM to 16 mM SB at different time points (6, 18, 50 hours). Trypan blue exclusion assays showed no significant differences (p>0.05) in live cell counts between control (ctrl) and treated groups at 6 hours, nor between the number of dead cells in the ctrl versus treated groups for 6, 18 and 50 hrs. At 18 hours, there were more live cells in the ctrl group than the 8mM and 16mM SB groups. At 50 hours, there were more live cells in the ctrl group than the 4- and 16-mM SB groups. Alamar blue cell viability assay showed no statistical differences between the ctrl group and cells treated with 16 mM SB at 6 hours. The kinome peptide array results showed more changes in phosphorylation for reduced diets with SB supplementation in the jejunum (401 peptides) than control with SB supplementation (261 peptides). There were also differences in the number of proteins observed in cell growth regulatory pathways like mTOR and cellular senescence. The phosphorylation statuses of cell cycle proteins were assessed, activation of CDK2, Cyclin-D1, CDK9 and CDK6, phosphorylation of Cyclin E; deactivation of mTOR, CDK1 and CDK7 indicates active G1 phase but not M or S phase in day 21 groups. These results suggest that SB does not decrease cell viability but may lead to reduction in cell growth and/or halt cell proliferation overtime by inducing changes in the cell cycle regulation. In the ileum, the kinome peptide array results showed significant changes in proteins involved in the major metabolic pathways, that is, glycolysis, tricarboxylic acid cycle and beta oxidation. Further analysis showed increased activity of beta oxidation enzymes including hydroxyacyl-coenzyme A dehydrogenase and acetyl-CoA carboxylase. SB increases beta oxidation which may induce hypoxia in segments with larger microbial populations like the ileum, indicated by increased HIF-1 and PKC activity. This increased hypoxia can trigger inflammatory responses via immune and immunometabolic pathways such as T-cell and C-type lectin signaling pathways. These immunometabolic changes by butyrate in the gut indicate more complex dual processes can only be understood in the context of metabolic state.