Enhancing Chicken Innate Immunity and Disease Resistance by Boosting Host Defense Peptide Synthesis

Routine use of antibiotics for growth promotion and disease prevention in animal agriculture has caused the concern for rapid emergence of antimicrobial resistance in public health worldwide. In this study, we attempted an antibiotic-alternative approach to disease control and prevention by enhancing host defense peptide (HDP) synthesis and animal immunity. We evaluated a diverse group of dietary supplements for their capacity to stimulate chicken HDP gene expression in vitro and in vivo by real-time RT-PCR. Chicken infection studies were further conducted to confirm an enhanced resistance to Salmonella enteritidis following oral supplementation of selected dietary factors. We also evaluated the role of histone acetylation as well as cAMP and MAP kinase signaling in the transcriptional regulation of HDP synthesis in chicken HD11 macrophage cells.Findings and Conclusions: Butyrate, a short - chain fatty acid and a well-known histone deacetylase inhibitor, enhances a large set of chicken HDPs and confers resistance to S. enteritidis . In addition, the induction of chicken HDP synthesis is largely inversely correlated with the aliphatic carbon chain length of free fatty acids, with short-chain fatty acids being the most potent, medium-chain fatty acids moderate, and long-chain fatty acids mostly ineffective. Desirably, free fatty acids enhance HDP expression with a minimum impact on proinflammatory response. Additionally, a combination of three short-chain fatty acids, namely acetate, propionate, and butyrate, induced HDP expression in a synergistic manner, leading to more significant reduction of the S. enteritidis load in the chicken than individual fatty acids. Moreover, cAMP signaling agonists stimulated chicken HDP gene expression and synergized with butyrate in HDP induction. We confirmed that p38 and JNK, but not ERK&half, MAP kinase signaling pathways are involved in butyrate-mediated chicken HDP induction. Identification of potent HDP-inducing dietary supplements and a better understanding of transcriptional regulatory mechanisms of HDP gene expression will undoubtedly facilitate development of antibiotic-free approaches to disease control and prevention with applications in both animal and public health.Department of Animal Scienc

Butyrate Enhances Disease Resistance of Chickens by Inducing Antimicrobial Host Defense Peptide Gene Expression

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Avian Antimicrobial Host Defense Peptides: From Biology to Therapeutic Applications

Host defense peptides (HDPs) are an important first line of defense with antimicrobial and immunomoduatory properties. Because they act on the microbial membranes or host immune cells, HDPs pose a low risk of triggering microbial resistance and therefore, are being actively investigated as a novel class of antimicrobials and vaccine adjuvants. Cathelicidins and β-defensins are two major families of HDPs in avian species. More than a dozen HDPs exist in birds, with the genes in each HDP family clustered in a single chromosomal segment, apparently as a result of gene duplication and diversification. In contrast to their mammalian counterparts that adopt various spatial conformations, mature avian cathelicidins are mostly α-helical. Avian β-defensins, on the other hand, adopt triple-stranded β-sheet structures similar to their mammalian relatives. Besides classical β-defensins, a group of avian-specific β-defensin-related peptides, namely ovodefensins, exist with a different six-cysteine motif. Like their mammalian counterparts, avian cathelicidins and defensins are derived from either myeloid or epithelial origin expressed in a majority of tissues with broad-spectrum antibacterial and immune regulatory activities. Structure-function relationship studies with several avian HDPs have led to identification of the peptide analogs with potential for use as antimicrobials and vaccine adjuvants. Dietary modulation of endogenous HDP synthesis has also emerged as a promising alternative approach to disease control and prevention in chickens

Modulation of antimicrobial host defense peptide gene expression by free fatty acids.

Routine use of antibiotics at subtherapeutic levels in animal feed drives the emergence of antimicrobial resistance. Development of antibiotic-alternative approaches to disease control and prevention for food animals is imperatively needed. Previously, we showed that butyrate, a major species of short-chain fatty acids (SCFAs) fermented from undigested fiber by intestinal microflora, is a potent inducer of endogenous antimicrobial host defense peptide (HDP) genes in the chicken (PLoS One 2011, 6: e27225). In the present study, we further revealed that, in chicken HD11 macrophages and primary monocytes, induction of HDPs is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids, with SCFAs being the most potent, medium-chain fatty acids moderate and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs in water resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. Taken together, oral supplementation of SCFAs is capable of boosting host immunity and disease resistance, with potential for infectious disease control and prevention in animal agriculture without relying on antibiotics

Hydrological Modeling with Respect to Impact of Land-Use and Land-Cover Change on the Runoff Dynamics in Godavari River Basin Using the HEC-HMS Model

Hydrological modeling and the hydrological response to land-use/land-cover changes induced by human activities have gained enormous research interest over the last few decades. The study presented here analyzes the spatial and qualitative changes in the rainfall–runoff that have resulted from the land-cover changes between 1985–2014 in the Godavari River Basin using the Hydrologic Engineering Centre-Hydrologic Modeling System(HEC-HMS) model and remote sensing—GIS (geographic information system) techniques. The purpose of this paper is to analyze the dynamics of land-use/land-cover (LULC) changes for the years 1985, 1995, 2005, and 2014 for the Godavari Basin. The findings reveal an increase of 0.64% of built-up land, a decrease of 0.92% in shrubland, and an increase of 0.56% in waterbodies between 1985–2014. The LULC change detection results between the years 1985–2014 indicated a drastic change in the cropland, forest, built-up land, and water bodies among all of the other classes. The urbanization and agricultural activities are the major reasons for the increase of cropland, built-up land, and water bodies, at the expense of decreases in shrubland and forest. The study had an overall classification accuracy of 92% and an overall Kappa coefficient of 0.9. The HEC-HMS model is used to simulate the hydrology of the Godavari Basin. The analyses carried out were mainly focussed on the impact of LULC changes on the streamflow pattern. The surface runoff was simulated for the year 2014 to quantify the changes that have taken place due to changes in LULC. The observed and the simulated peak streamflow was found to be the same i.e., 56,780 m3/s on 9 September 2014. In the validation part, the linear regression method was used to correlate the observed and simulated streamflow data at the prominent gauge station of the Badrachalam outlet for the Godavari River Basin and give a correlation coefficient value of 0.83. It was found that the HEC-HMS model is compatible and works better for the rainfall–runoff modeling, as it takes into account the various parameters that are influencing the process. The hydrological modeling that was carried out using the HEC-HMS model has brought out the significant impact of LULCC on rainfall–runoff at the Pranhita sub-basinscale, indicating the model’s ability to successfully accommodate all of the environmental and landscape variables. The study indicates that deforestation at the cost of urbanization and cropland expansions leads to decreases in the overall evapotranspiration (ET) and infiltration, with an increase in runoff. The results of the study show that the integration of remote sensing, GIS, and the hydrological model (HEC-HMS) can solve hydrological problems in a river basin

Differential expression of <i>AvBD9</i> in response to unsaturated fatty acids.

<p>Chicken HD11 macrophage cells (A) and primary monocytes (B) were treated in duplicate with different concentrations of sodium stearate, sodium oleate, linoleic acid, conjugated linolenic acid (CLA), and α-linolenic acid for 24 h, followed by real-time RT-PCR analysis of <i>AvBD9</i> gene expression. Data shown are means ± standard error of a representative of 2–3 independent experiments. Because of an obvious cytotoxicity, 200 and/or 400 µM could not be tested for sodium stearate and oleate. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 (in comparison with solvent controls by unpaired Student’s <i>t</i>-test).</p

A minimum impact of free fatty acids on the expression of proinflammatory cytokines.

<p>Chicken HD11 cells were stimulated with different fatty acids at optimal HDP-inducing concentrations (80 mM acetate, 32 mM propionate, 4 mM butyrate, 16 mM hexanoate, and 2 mM octanoate) or LPS (1 µg/ml) as a positive control for 3 and 24 h, followed by real-time RT-PCR analysis of the expression of <i>IL-1β</i> (A), <i>IL-12p40</i> (B), and <i>IL-8</i> (C). Data shown are means ± standard errors from 2–3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 (in comparison with solvent controls by unpaired Student’s <i>t</i>-test).</p

Modulation of cathelicidin B1 gene expression by free fatty acids.

<p>Primary chicken monocytes were treated in duplicate with or without indicated concentrations of short-chain fatty acids (SCFA), medium-chain fatty acids (MCFA) or long-chain fatty acids (LCFA) for 24 h, followed by real-time RT-PCR analysis of <i>cathelicidin B1</i> gene expression. Data was normalized with <i>GAPDH</i>, and relative fold change of each treatment versus solvent control was calculated using ΔΔCt method. Data shown are means ± standard error of a representative of 2–3 independent experiments. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 (in comparison with solvent controls by unpaired Student’s <i>t</i>-test).</p

Synergistic reduction of the <i>Salmonella enteritidis</i> load in the cecum of chickens by a combination of acetate, propionate and butyrate.

<p>Four day-old male broiler chicks were supplemented with or without 0.5% acetate, 0.2% propionate, and 0.1% butyrate alone or in combinations in water for 2 days with 5 birds per group, followed by an inoculation with <i>S. enteritidis</i> phage type 13a (1×10⁷). SCFA supplementation was continued for another 4 days before the cecal content was collected and bacterial number enumerated. Each dot indicates the bacterial titer in a bird and the solid line represents the median value of each treatment. *<i>P</i><0.05 and **<i>P</i><0.01 (by unpaired Student’s <i>t</i>-test).</p