Sodium butyrate improves porcine host defense peptide expression and relieves the inflammatory response upon Toll-like receptor 2 activation and histone deacetylase inhibition in porcine kidney cells

Abstract

Host defense peptides (HDPs) are an important component of the innate immune system and possess direct antimicrobial and immunomodulatory activities. Dietary regulation of HDPs synthesis has emerged as a novel non-antibiotic approach to combat pathogen infection. There are species- and tissue-dependent characteristics of the regulation and mechanism of HDPs. In this study, we investigated whether the histone deacetylase inhibitor (HDACi) sodium butyrate (NaB) could induce HDP expression and the mechanism underlying NaB-regulated HDP expression in PK-15 cells. Our results revealed that NaB augmented HDP expression in PK-15 cells, including porcine β-defensin 3 (pBD3), epididymis protein 2 splicing variant C (pEP2C), pBD128, pBD123, and pBD115, but no inflammatory response occurred. Inhibition of HDAC activity was not sufficient to induce the expression of pBD3 and pEP2C in comparisons of NaB and another HDACi, trichostatin A (TSA). Concomitantly, NF-κB activation was involved in the induction of HDP expression by NaB. MAPK pathway inhibition also prevented pBD3 and pEP2C induction by NaB. Furthermore, NaB could still promote pBD3 and pEP2C expression and inhibit IL-6 production in the presence of the toll-like receptor 2 (TLR2) ligand peptidoglycan. Moreover, TLR2 could be activated by both NaB and peptidoglycan, and blocking TLR2 expression suppressed HDP induction. Finally, we further showed that increased pBD3 could decrease cytokine interleukin-18 (IL-18) and increase porcine claudin 15 (pCLDN15) contents, suggesting an immunoregulatory function of pBD3. In conclusion, this work paves the way for using HDACi-NaB to induce porcine kidney defense peptides while limiting the deleterious risk of an inflammatory response.

Keywords: TLR2; host defense peptides; mechanism; porcine kidney cells; sodium butyrate.

Figure 1. Increased expression of AMP mRNA in porcine kidney cells following NaB

(A) PK-15 cells were treated with the indicated concentrations of NaB, ranging from 0-128 mM, for 24 hours. Cell viability was measured using CCK-8. All CCK-8 values were normalized to the control serial concentrations of NaB for 24 hours. (B–F) pBD3, pEP2C, pBD128, pBD123, and pBD115 gene expression were determined by qRT-PCR after treatment with 0, 0.5, 1, 2, 4, and 8 mM NaB for 24 hours. (G, H) pBD3 and pEP2C gene expression was analyzed by qRT-PCR after incubation with 8 mM NaB for 6, 12, and 24 hours. Similar results were obtained in repeated experiments (more than two) using different cell preparations. Abbreviations: NaB, sodium butyrate. Means with different letters are significantly different at P < 0.01 (B–F). *P < 0.01, using the unpaired Student's t-test (G, H, I, and J).

Figure 2. Modulation of histone acetylation activity and AMP gene expression in response to NaB or TSA

(A) HDAC activity was monitored by excitation at 490 nm and emission at 525 nm. (B) PK-15 cells were treated using various concentrations of TSA. Bars represent means with SD of three independent experiments; means with different letters are significantly different at P < 0.01.

Figure 3. NF-κB pathway plays an important role, while AMP expressionwas ameliorated by NaB in PK-15 cells

(A) Immunoblot analysis of total IκB α expression and p65 phosphorylation in cells pretreated or not with 8 mM NaB at the indicated time points. Western blots were performed using antibodies directed against IκB α and p65 phosphorylation marks. Vehicle, without NaB treatment cells for 24 h; NaB, cells challenged with NaB for 1, 3, 6, 12, and 24 hours; “P” prefix, phosphorylation. (B) qRT-PCR analysis of NF-κB1 (p50) and NF-κB3 (p65) transcription in cells treated with 8 mM NaB. Data were analyzed using the unpaired Student's t-test at P < 0.01. *P < 0.01; n.s., not significant. C. Immunoblot analysis of total IκB α expression and p65 phosphorylation in cells pretreated or not for 3 h with 2.5 μM BAY11-7082, and then treated with 8 mM NaB for 24 hours. D, E. qRT-PCR analysis of pBD3, pEP2C, pBD128, and pBD123 transcription in cells treated with or without 8 mM NaB and 2.5 μM BAY 11-7082. Bars represent means with SD of three independent experiments. Means with different letters are significantly different at P < 0.01. F. qRT-PCR analysis of NF-κB1 (p50) transcription in cells treated with 8 mM NaB or 2.5 μM BAY 11-7082. Bars represent means with SD of three independent experiments. Means with different letters are significantly different at P < 0.01.

Figure 4. NaB controls inflammatory cytokine expression in PK-15 cells

qRT-PCR analysis of inflammatory cytokines, including IL-1α, IL-6, IL-8, and IL-18 mRNA expression, in PK-15 cells treated with 8 mM NaB. *P < 0.01; n.s., not significant at P < 0.01.

Figure 5. Effect of cell density or serum concentration on NaB governs the up-regulation of AMP expression

(A, B) qRT-PCR analysis of pBD3 and pEP2C transcription at different cell densities treated with 8 mM NaB. (C, D) qRT-PCR analysis of pBD3 and pEP2C transcription in cells cultured in different serum concentrations and treated with 8 mM NaB. *P < 0.01; n.s., not significant at P < 0.01. Data were analyzed using the unpaired Student's t-test.

Figure 6. Effect of MAPK inhibitors on the up-regulation of NaB-mediated pBD3 and pEP2C expression

PK-15 cells were pretreated with a series of concentrations of the ERK1/2 inhibitor PD98059 or p38 MAPK inhibitor SB203580 and DMSO at a dose no less than the dose required to solve SB 203580 or PD98059 for 5 hours prior to incubation with 8 mM NaB. (A and C) PD98059; (B and D) SB203580. Bars represent means with SD of three independent experiments. Means with different letters are significantly different at P < 0.01.

Figure 7. Effect of NaB on porcine kidney cells in the presence of the TLR2 ligand peptidoglycan

qRT-PCR to detect changes in gene transcription of (A) pBD3, (B) pEP2C, (C) IL-6, and (D) IL-8 in cells treated with 8 mM peptidoglycan and 250 ng/ml peptidoglycan for 24 hours. n.s., not significant at P < 0.01; *P < 0.01 (A and B). Data were evaluated using the unpaired Student's t-test. “PGN” represent peptidoglycan.

Figure 8. Role of TLR2 in AMP expression control by peptidoglycan and NaB in PK-15 cells

(A) qRT-PCR analysis of TLR2 expression in cells after treatment with 8 mM NaB or 250 μg/mL peptidoglycan or co-incubation with both for 24 hours. (B) PK-15 cells were transfected with siRNA targeting TLR2, and then qRT-PCR was performed to detect TLR2 gene expression. (C–F) PK-15 cells were transfected with siRNA against TLR2 or siControl, and 6 hours post-transfection, the culture medium was replaced with medium containing 250 μg/mL peptidoglycan or 8 mM NaB for 24 hours. qRT-PCR was then performed to detect TLR2 (C), pBD3 (D), pEP2C (E), and IL-6 (F) transcript levels. Asterisks (*) represent statistical significance at P < 0.02, n.s., not significant. “PGN” represent peptidoglycan.

Figure 9. Effect of NaB (8 mM), peptidoglycan (250 μg/mL) or the combination of both on NF-κB signaling in PK-15 cells

(A, B) qRT-PCR was performed to detect p65 and p50 mRNA levels after treatment with peptidoglycan (250 μg/mL), NaB (8 mM), or the combination of both substances for 24 hours. Different letters (a, b, c, and d) indicate significant differences at P < 0.01 among the four groups. (C) Total proteins were harvested for western blot analysis to detect protein levels of IκB α and p65 phosphorylation in PK-15 cell treated as in (A and B). β-actin served as a loading control. “PGN” represent peptidoglycan.

Figure 10. Construction of eukaryotic recombinant expression plasmids pBD3-pEGFP-N1 and expression of pBD3 fusion protein, and the effect of transient overexpression of pBD3 on cytokine, chemokine and tight junction protein production in PK-15 cells

(A) Restriction enzyme digestion analysis of plasmid pBD3-pEGFP-N1. An inserted fragment of approximately 201 bp was separated by 1% agarose gel electrophoresis following digestion of pBD3-pEGFP-N1 with Xho I and EcoR I. Lane M, DL15, 000 DNA Marker; Lane 1, pBD3-pEGFP-N1 digested with EcoR I and Xho I; Lane 2-3, pBD3-pEGFP-N1 digested with Xho I and EcoR I, respectively. (B) pBD3 gene amplification in pBD3-pEGFP-N1 plasmids was analyzed by RT-PCR. Lane M, DL5, 000 DNA Marker; Lane 1, H₂O; Lane 2, pBD3-pEGFP-N1 plasmids. (C) Transient expression of the pBD3 fusion protein in PK-15 cells transfected with pBD3-pEGFP-N1 or pEGFP-N1 and analyzed by fluorescence microscopy. pBD3 protein expression observed by green fluorescence was evaluated in PK-15 cells. (D and E) pBD3 mRNA expression in PK-15 cells was analyzed by semi-quantitative-PCR or qRT-PCR after transient overexpression of the pBD3 fusion protein. F. qRT-PCR analysis of gene expression profiles for cytokine, chemokine and tight junction protein production in cells overexpressing pBD3 protein. Asterisks (*) indicate statistical significance at P < 0.01.

Figure 11. Regulatory mechanism of AMP gene expression induced by NaB in porcine kidney cells

NaB inhibited HDAC activity and improved AMP expression, even following challenge with the TLR2 ligand peptidoglycan. The mechanism: NaB leads to IκB α degradation, a reduction of p50 transcription and an increase in p65 phosphorylation, subsequently activating the NF-κB pathway and leading to enhanced AMP transcription via TLR2. The MAPK pathway also plays a role in the regulation of AMPs by NaB. NaB reversed the down-regulation of AMPs induced by the TLR2 ligand peptidoglycan by inhibiting the levels of p65 and decreasing p50 transcription and even IκB α degradation, which are key factors in the NF-κB pathway. Finally, overexpression of pBD3 protein regulated the expression of other cytokines in porcine kidney cells.