Induction of porcine host defense peptide gene expression by short-chain fatty acids and their analogs

Abstract

Dietary modulation of the synthesis of endogenous host defense peptides (HDPs) represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes in response to sodium butyrate treatment by real-time PCR. We revealed that butyrate is a potent inducer of multiple, but not all, HDP genes. Porcine β-defensin 2 (pBD2), pBD3, epididymis protein 2 splicing variant C (pEP2C), and protegrins were induced markedly in response to butyrate, whereas pBD1 expression remained largely unaltered in any cell type. Additionally, a comparison of the HDP-inducing efficacy among saturated free fatty acids of different aliphatic chain lengths revealed that fatty acids containing 3-8 carbons showed an obvious induction of HDP expression in IPEC-J2 cells, with butyrate being the most potent and long-chain fatty acids having only a marginal effect. We further investigated a panel of butyrate analogs for their efficacy in HDP induction, and found glyceryl tributyrate, benzyl butyrate, and 4-phenylbutyrate to be comparable with butyrate. Identification of butyrate and several analogs with a strong capacity to induce HDP gene expression in pigs provides attractive candidates for further evaluation of their potential as novel alternatives to antibiotics in augmenting innate immunity and disease resistance of pigs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Butyrate-induced expression of pBD2, pBD3, pEP2C, and PG1-5 in porcine IPEC-J2 intestinal epithelial cells.

Cells were incubated in duplicate with indicated concentrations of butyrate for 24 h (A) or 8 mM of butyrate for 6, 12, and 24 h (B). Gene expression was analyzed by real-time RT-PCR. The relative fold changes over the unstimulated control were calculated with the ΔΔCt method using porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for normalization. Data were means ± standard error of a representative of two independent experiments. *P<0.05, **P<0.01, and ***P<0.001 (in comparison with solvent controls by unpaired Student’s t-test).

Figure 2. Butyrate-induced expression of pBD2, pBD3, and PG1-5 in porcine 3D4/31 macrophage cells (A) and pBD2 in primary monocytes (B).

Cells were incubated in duplicate with indicated concentrations of butyrate for 24 h. Gene expression was analyzed by real-time RT-PCR. The relative fold changes over the unstimulated control were calculated with the ΔΔCt method using porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for normalization. Data were 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. Regulation of pBD2, pBD3, and pEP2C expression by saturated free fatty acids.

Porcine IPEC-J2 cells were incubated in duplicate with indicated concentrations of saturated free fatty acids for 24 h. Gene expression was analyzed by real-time RT-PCR. The relative fold changes over the unstimulated control were calculated with the ΔΔCt method using porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for normalization. Data were means ± standard error of a representative of 2–3 independent experiments. Abbreviations: SCFA, short-chain fatty acids; MCFA, medium-chain fatty acids; LCFA, long-chain fatty acids. *P<0.05, **P<0.01, and ***P<0.001 (in comparison with solvent controls by unpaired Student’s t-test).

Figure 4. Regulation of pBD2, pBD3, pEP2C, and PG1-5 expression by butyrate and its analogs.

Porcine IPEC-J2 cells were inbubated in duplicate with indicated concentrations of each compound (A) for 24 h. Gene expression was analyzed by real-time RT-PCR. The relative fold changes (B) over the unstimulated control were calculated with the ΔΔCt method using porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for normalization. Data were 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 5. Regulation of pBD2, pBD3, and pEP2C expression by short-chain fatty acids and their phenyl derivatives.

Porcine IPEC-J2 cells were inbubated in duplicate with indicated concentrations of each compound (A) for 24 h. HDP gene expression was analyzed by real-time PCR. The relative fold changes (B) over the unstimulated control were calculated with the ΔΔCt method using porcine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene for normalization. Data were 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).