A kinomic analysis of the immunometabolic effects of antibiotic alternatives in necrotic enteritis disease models
Date
2021
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Publisher
University of Delaware
Abstract
Since the restriction and removal of antibiotic growth promotors, resulting from both new regulations as well as public pressure, the poultry industry has seen a re-emergence of financially devastating disease, most notably necrotic enteritis. Necrotic enteritis has been estimated to cost the global poultry industry $6 billion annually. The economic consequences attributable to necrotic enteritis mostly result from the sub-clinical form of the disease, which can go unnoticed until slaughter due to a lack of overt clinical signs. Finding alternatives to antibiotic growth promotors has become a major area of research and many facets of this problem need to be considered in order to begin to find solutions. Looking at disease pathogenesis is one necessary component to finding more effective and efficient alternatives and to be able to better target problems such as necrotic enteritis more specifically. Looking at the mode of action of antibiotic alternative feed additives shown to have beneficial disease mitigating and growth promoting effects in addition to beginning to understand the host effects of antibiotic growth promotors will also be necessary to begin to replace the beneficial effects lost with the removal of antibiotic growth promotors. This work has been done to advance all of these frontiers. Necrotic enteritis and its predisposing factors were used both as disease challenge models and as a means to evaluate the disease mitigating effects and mechanism of action of antibiotic alternatives. ☐ Necrotic enteritis highlights how dysregulation of gut immune responses can have devastating consequences. The gut is the site of nutrient absorption and an important immune organ. As such, the gut has been considered the prototypical immunometabolic organ. Immunometabolism, being the study of the cross-talk between immune responses and metabolic pathways represents a rational research approach for studying an enteric disease and the effects of potentially disease mitigating feed additives. ☐ Most immune signaling and metabolic pathways contain key intermediates regulated by protein kinases. Kinomics involves the analysis of phosphorylation events, catalyzed by protein kinases. Phosphorylation is a predominant post-translational modification of proteins that plays a key role in regulating and mediating most cellular signaling. Species-specific kinome peptide arrays allow for a high-throughput method of measuring these phosphorylation events. Differences in phosphorylation target sites between species requires the use of species-specific kinome peptide arrays. The kinome peptide array protocol generates data regarding immunometabolic changes occurring between treatments and controls. This research approach enables us to explore the immunometabolic effects of our disease challenge models compared to healthy controls as well as the effects of antibiotic alternatives in the same context. ☐ The yeast cell wall components of Saccharomyces cerevisiae have been the focus of studies searching for antibiotic alternatives. Feed additives containing these components are currently in use by the broiler industry and have shown beneficial effects on growth and performance. In chapter 2, the effects of a crude yeast cell wall extract (YCW) and purified yeast cell wall components beta-glucan (BG) and mannoproteins (MPT) in the context of an experimental model of necrotic enteritis (NE) are reported on. The NE model used for this study involved the use of an infectious bursal disease virus vaccine and a Clostridium perfringens (C. perfringens) challenge. All groups other than unchallenged control were given the same NE challenge. Groups included unchallenged control, NE challenged, YCW, BG, MPT, and BG+MPT. Weight gain was recovered to control in the BG+MPT group. The weight gain in all other groups was not statistically significantly different from either the control or the NE challenged groups. Kinome peptide array and subsequent STRING analyses on jejunal tissue revealed changes in innate immune response and cell death or apoptosis between the BG+MPT and NE challenge groups. Closer analysis of peptide function revealed in the NE challenge group most phosphorylation changes indicated a decrease in functions promoting cell growth and survival and induced cell death. The BG+MPT group showed a reversal of these changes. While we observed clear differences in the phosphorylation status of key peptides between challenge and BG+MPT treated groups, often these peptides phosphorylation status was not significantly altered in the BG alone, MPT alone, and YCW treatment groups. It is possible that the differences in these peptides phosphorylation state are a key aspect of the difference in growth response we observed here, either allowing the disease to take hold and negatively affect growth in the case of the challenge group or reducing disease severity and limiting growth effects in the case of the BG+MPT group. Thus, the lack of significant change in phosphorylation of these specific peptides may be the reason we do not see a significant improvement in growth due to treatment with YWC, BG alone, or MPT alone. In other words, these peptides described previously may be critical determinants of disease severity and the growth effects due to this NE challenge model. The mRNA expression of pro- and anti-inflammatory cytokines were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results indicated an effect on cytokine gene expression in response to the NE challenge and further changes in response to the various treatment groups. These results were consistent with the kinome peptide array analysis, showing that rather than a return to control, the treatments had distinct impacts further separating the treatment groups from either the control or NE challenged groups. In summary, this paper indicates that a combination compound used as a feed additive, BG+MPT, is able to recover weight gain in NE challenged birds as well as confer unique cellular signal transduction in the guts of challenged broilers. The responses appear centered on inducing cell growth responses and reducing cell death or apoptosis and innate inflammatory responses, but rather than returning the tissue to a control-like state, the treatment appears to generate compensatory signaling to reduce disease severity. ☐ In chapter 3, the mechanism of action of a postbiotic product in the context of a C. perfringens challenge in broiler chickens is reported on. A postbiotic product contains the byproducts of bacteria fermentation including metabolites, short-chain fatty acids, and functional peptides, among others. These products are thought to have the potential for a direct effect on the host rather than relying on the microbiome to produce the same byproducts. C. perfringens is one of, if not the major, contributing factor to the development of necrotic enteritis in broilers. Administration of the postbiotic product improved weight gain, decreased C. perfringens colony count, and reduced lesion scores and mortality between the two replicate trials. Kinome peptide array analysis showed a distinct response to the postbiotic product in the jejunum of both disease challenged and unchallenged broilers. Looking at peptide phosphorylation events unique to the postbiotic, postbiotic plus C. perfringens challenge, and the C. perfringens challenge groups, STRING analysis results showed immune related signaling in the top 20 GO biological processes in the postbiotic and C. perfringens challenge groups, this signaling was absent in the postbiotic plus C. perfringens challenge group. The protein identifiers input into STRING were unique to each group, so despite immune signaling showing up in both the postbiotic and C. perfringens challenge tables, the members of the signaling pathways were unique suggesting key differences in these signaling pathways. This is further supported by a visualization comparing the phosphorylation events of these two groups that shows that many of the events on the same peptide were differentially phosphorylated in different directions when each group was compared to control, which indicates a differential effect between the two groups. The lack of immune signaling pathways in the postbiotic plus C. perfringens challenge group suggest that the combination treatment may mitigate the disease inducing effects of the C. perfringens challenge. When the complete lists of significantly differentially phosphorylated peptides for the postbiotic and C. perfringens challenge groups were input into STING, the results showed signaling changes indicating an impact on innate immune signaling in the C. perfringens challenge group not observed in the postbiotic treatment group. The postbiotic treatment group results reflect an immune-modulatory/anti-inflammatory impact by the metabolite on jejunal tissue. The phosphorylation events indicate inhibition of such signaling pathways as mTOR, NF-κB, and PI3K-Akt. These are pathways commonly seen being activated during an active/proinflammatory immune response. In summary, the metabolite product being evaluated seems to impart an immune-modulatory effect on jejunal tissue in broilers. This could be important in maintaining a healthy gut, especially considering the withdraw of in-feed antibiotics which are widely believed to impart an anti-inflammatory effect in the gut. The gut needs to maintain both a state of tolerance (to commensal microbes) as well as a readiness to respond (to pathogenic organisms). This balance must be maintained in order to allow for optimum growth efficiency and health.
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Keywords
Kinomic, Immunometabolic effects, Antibiotic alternatives, Necrotic enteritis