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Title:Immunoprotective effects of lipopolysaccharide detoxification by a novel microbial-derived alkaline phosphatase
Author(s):Zimmerman, Jalisa D.
Advisor(s):Johnson, Rodney W.
Contributor(s):Steelman, Andrew; Escobar, Jeffery
Department / Program:Animal Sciences
Discipline:Animal Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Alkaline phosphatase
Abstract:Lipopolysaccharide (LPS) is a major outer cell wall component of Gram-negative bacteria, including Escherichia coli and Salmonella enterica, which have important health and economic consequences in animal agriculture. LPS is a recognized endotoxin that stimulates toll-like receptors, which are present on cells that contribute to inflammatory responses and, via downstream signaling pathways, results in the release of pro- and anti-inflammatory mediators to assist with pathogen clearance and tissue repair. During weaning, piglets are subjected to numerous psychological and environmental stressors including mixing, lower ambient temperature and change in diet. Due to the piglets’ immature digestive and immune system, weaning is associated with growth plateaus, increased incidence of diarrhea, increased bacterial translocation and endotoxin load, and increased mortality. Increased concentrations of LPS in circulation perpetuate post-wean syndrome and drive further gastrointestinal tract (GIT) morphological and digestive perturbations. Weaning is also characterized by reductions in intestinal alkaline phosphatase (IAP), which has biological roles including detoxifying LPS in the intestinal lumen and maintaining an alkalotic pH to modulate gut microbiota. IAP is a homodimeric enzyme that catalyzes the release of an inorganic phosphate from the lipid A moiety of LPS, resulting in the considerably less immunostimulatory product, monophosphoryl lipid A (MPLA). Based on IAP’s ability to reduce the inflammatory effects of LPS in the GIT and the difficulty in creating a viable commercial product from a mammalian-derived enzyme; a microbe was engineered to produce an alkaline phosphatase (MAP). However, the ability of MAP to detoxify LPS in vitro and in vivo has not been investigated. In chapter 2, to test the hypothesis that MAP would detoxify LPS similarly to IAP, RAW264 macrophages and primary porcine alveolar macrophages were treated with vehicle, IAP, MAP, E. coli or S. enterica LPS, LPS that had been pre-incubated with MAP (MPLA), or LPS that had been pre-incubated with IAP (iMPLA). In support of the hypothesis, cells treated with MPLA had significantly lower TNFα, IL-6, IL-1β, and IL-10 gene expression as compared to cells treated with LPS alone. Furthermore, novel findings indicate an increased efficacy for MAP to detoxify LPS as compared to IAP. MPLA treated cells had significantly lower levels of pro- and anti-inflammatory cytokine production as compared to iMPLA-treated cells. We then determined the serum cytokine profiles of piglets 2 days post-wean after being challenged with saline, MPLA, or LPS for four hours. Piglets i.p. injected with MPLA were more active and ate more over the time period than LPS injected piglets. Furthermore, MPLA piglets had altered serum cytokine profiles indicating an ameliorated immune response to the challenge. In chapter 3 we investigated the effects of exogenous MAP supplementation on post-wean syndrome in piglets on a standard phase 2 diet. We found that adding 4,000 IU/kg body weight to a phase 2 diet for two weeks post-weaning resulted in a higher average daily gain and body weight. MAP-fed piglets had increased villus height and decreased crypt depth, and an increased villus height to crypt depth ratio in the duodenum, jejunum, and ileum. Furthermore, MAP-fed piglets were protected against weaning-induced downregulation of tight junction protein ZO-1 and inflammation-induced increases in claudin-1. IAP is known to be downregulated post-weaning due to increased inflammatory mediators which inhibit gene expression. Therefore, we sought to determine the effects of exogenous MAP supplementation on endogenous IAP gene expression and found that dietary MAP-supplementation significantly increased IAP gene expression in the duodenum, jejunum, and ileum. Furthermore, alkaline phosphatase activity was significantly higher in the digesta and mucosa of the duodenum, jejunum, and ileum of weaned pigs eating a MAP-supplemented diet. This is the first study, to our knowledge, to characterize the effects of exogenous alkaline phosphatase supplementation on post wean syndrome in piglets. Our data indicates that MAP may serve as a potential dietary additive to mitigate the effects of LPS- and weaning-induced inflammation on innate immune cell cytokine production, GIT permeability and morphology, tight junction protein perturbations, and downregulation of IAP gene expression and activity.  
Issue Date:2019-07-17
Rights Information:Copyright 2019 Jalisa Zimmerman
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08

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