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

Abstract

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.

Figure 1. Regulation of AvBD9 gene expression by free fatty acids.

Chicken macrophage HD11 cells (A) and primary monocytes (B) 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 AvBD9 gene expression. Data was normalized with GAPDH, 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. It is noted that all fatty acids were used at subtoxic concentrations and, because of different toxicities to HD11 cells and primary monocytes, slightly different concentrations of free fatty acids were used in the two cell types in a few cases in order to show the optimal AvBD9-inducing activity in each cell type. *P<0.05, **P<0.01, and ***P<0.001 (in comparison with solvent controls by unpaired Student’s t-test).

Figure 2. Modulation of cathelicidin B1 gene expression by free fatty acids.

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 cathelicidin B1 gene expression. Data was normalized with GAPDH, 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. *P<0.05, **P<0.01, and ***P<0.001 (in comparison with solvent controls by unpaired Student’s t-test).

Figure 3. Differential expression of AvBD9 in response to unsaturated fatty acids.

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 AvBD9 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. *P<0.05, **P<0.01, and ***P<0.001 (in comparison with solvent controls by unpaired Student’s t-test).

Figure 4. A minimum impact of free fatty acids on the expression of proinflammatory cytokines.

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 IL-1β (A), IL-12p40 (B), and IL-8 (C). Data shown are means ± standard errors from 2–3 independent experiments. *P<0.05, **P<0.01, and ***P<0.001 (in comparison with solvent controls by unpaired Student’s t-test).

Figure 5. Synergistic induction of AvBD9 with acetate, propionate, and butyrate in chicken HD11 cells (A) and primary monocytes (B).

Cells were incubated with acetate, propionate, and butyrate alone or in combinations for 24 h, followed by real-time RT-PCR analysis of AvBD9 expression. Data shown are means ± standard errors from 3 independent experiments. The bars without common superscript letters denote significance (P<0.05 by unpaired Student’s t-test).

Figure 6. Inhibition of the HDAC activity by acetate, propionate, and butyrate.

Chicken HD11 cells were incubated in duplicate with or without three SCFAs in the presence of Fluor-de-Lys®, a fluorogenic, cell-permeable HDAC substrate for 4 h. The deacetylation reaction was stopped and the fluorescent signal was generated by addition of a developer solution containing trichostatin A and NP-40. Fluorescence was monitored at 360 nm excitation and 460 nm emission. HDAC inhibition by SCFAs was calculated relative to the cells without being exposed to any HDAC inhibitor. Data shown are means ± standard errors. The bars without common superscript letters denote significance (P<0.05 by unpaired Student’s t-test).

Figure 7. Synergistic reduction of the Salmonella enteritidis load in the cecum of chickens by a combination of acetate, propionate and butyrate.

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 S. enteritidis 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. *P<0.05 and **P<0.01 (by unpaired Student’s t-test).