Host Defense Peptides Genes Research Articles

PSXI-14 Effects of Footpad Dermatitis on Gene Expression of Host Defense Peptides in Laying Hens

Abstract Footpad dermatitis (FPD) has a negative impact on the welfare and health production of poultry. The present study aimed to determine the effect of FPD on the expression of host defense peptides (HPDs). A total of fifty-four 55-week-old Hy-line brown hens housed individually in furnished cages were selected for investigating the mRNA expression of HPDs in foot, spleen, liver, and small intestine tissues. FPD score 0 group with normal foot, FPD score 1 with a small lesion of the foot pad epithelium and FPD score 2 with a larger lesion were divided by visual examination. The results showed that compared with group 0, hens from FPD group 1 and 2 had increased mRNA expression of HPDs of AvBD1, AvBD3, AvBD4, AvBD5, AvBD6, AvBD7, AvBD8, AvBD9, AvBD10, AvBD11, AvBD12, AvBD13, AvBD14 and LEAP2in foot and spleen tissues (P < 0.05). The relative expression of HPDs genes AvBD2, AvBD3, AvBD4, AvBD5, AvBD7, AvBD8, AvBD11, AvBD12, AvBD13, AvBD14, and LEAP2 in liver tissue increased, while the relative mRNA expression of AvBD6, AvBD9, and AvBD10 decreased (P < 0.05), and the expression levels of AvBD1, AvBD3, AvBD5, AvBD6, AvBD7, AvBD9, AvBD10, AvBD12, and LEAP2 in small intestinal tissue increased (P < 0.05), while the expression of AvBD2, AvBD8, and AvBD14 decreased (P < 0.05). Therefore, FPD can affect the expression of AVBDs in laying hens that the foot and spleen tissue had stronger responsiveness to the FPD. It is speculated that the increase in the expression of AVBDs on account of the possible infection with pathogenic bacteria. Then the increased HPDs participated in the host's defense responses and strengthened the immune regulation after FPD occurred.

Large-Scale Identification of Multiple Classes of Host Defense Peptide-Inducing Compounds for Antimicrobial Therapy.

The rapid emergence of antibiotic resistance demands new antimicrobial strategies that are less likely to develop resistance. Augmenting the synthesis of endogenous host defense peptides (HDPs) has been proven to be an effective host-directed therapeutic approach. This study aimed to identify small-molecule compounds with a strong ability to induce endogenous HDP synthesis for further development as novel antimicrobial agents. By employing a stable HDP promoter-driven luciferase reporter cell line known as HTC/AvBD9-luc, we performed high-throughput screening of 5002 natural and synthetic compounds and identified 110 hits with a minimum Z-score of 2.0. Although they were structurally and functionally diverse, half of these hits were inhibitors of class I histone deacetylases, the phosphoinositide 3-kinase pathway, ion channels, and dopamine and serotonin receptors. Further validations revealed mocetinostat, a benzamide histone deacetylase inhibitor, to be highly potent in enhancing the expression of multiple HDP genes in chicken macrophage cell lines and jejunal explants. Importantly, mocetinostat was more efficient than entinostat and tucidinostat, two structural analogs, in promoting HDP gene expression and the antibacterial activity of chicken macrophages. Taken together, mocetinostat, with its ability to enhance HDP synthesis and the antibacterial activity of host cells, could be potentially developed as a novel antimicrobial for disease control and prevention.

High-Throughput Identification of Epigenetic Compounds to Enhance Chicken Host Defense Peptide Gene Expression

Enhancing the synthesis of endogenous host defense peptides (HDPs) has emerged as a novel antibiotic-free approach to infectious disease control and prevention. A number of epigenetic compounds have been identified as HDP inducers and several have proved beneficial in antimicrobial therapy. However, species-specific regulation of HDP synthesis is evident. In attempt to identify epigenetic compounds with potent HDP-inducing activity for poultry-specific application, we developed a stable luciferase reporter cell line, known as HTC/AvBD10-luc, following our earlier construction of HTC/AvBD9-luc. HTC/AvBD10-luc was developed through permanent integration of a chicken macrophage cell line, HTC, with a lentiviral luciferase reporter vector driven by a 4-Kb AvBD10 gene promoter. Using a high throughput screening assay based on the two stable cell lines, we identified 33 hits, mostly being histone deacetylase (HDAC) inhibitors, from a library of 148 epigenetic compounds. Among them, entinostat and its structural analog, tucidinostat, were particularly effective in promoting multiple HDP gene expression in chicken macrophages and jejunal explants. Desirably, neither compounds triggered an inflammatory response. Moreover, oral gavage of entinostat significantly enhanced HDP gene expression in the chicken intestinal tract. Collectively, the high throughput assay proves to be effective in identifying HDP inducers, and both entinostat and tucidinostat could be potentially useful as alternatives to antibiotics to enhance intestinal immunity and disease resistance.

Massive gene expansion of hepcidin, a host defense peptide, in gilthead seabream (Sparus aurata)

Host defense peptides (HDP) are among the most ancient immune molecules in animals and clearly reflect an ancestral evolutionary history involving pathogen-host interactions. Hepcidins are a very widespread family of HDPs among vertebrates and are especially diverse in teleosts. We have investigated the identification of new hepcidins in gilthead seabream (Sparus aurata), a fish farmed in the Mediterranean. Targeted gene predictions supported with expressed sequence tags (ESTs) derived from Hidden Markov Models were used to find the hamp genes in the seabream genome. The results revealed a massively clustered hamp duplication on chromosome 17. In fact, the seabream genome contains the largest number of hepcidin copies described in any vertebrate. The evolutionary history of hepcidins in seabream, and vertebrates generally, clearly indicates high adaptation in teleosts and novel subgroups within hepcidin type II. Furthermore, basal hepcidin gene expression analysis indicates specific-tissue expression profiles, while the presence and distribution of transcription factor binding sites (TFBS) in hamp promoters as well as their transcription profile upon bacterial challenge indicates different immune roles depending on the type of hepcidin and tissue. This massive duplication of HDP genes in a bony fish could point to a far more specific and adaptive innate immune system than assumed in the classic concept of immunity in mammals. Hence, a new world of knowledge regarding hepcidins in fish and vertebrates is being initiated.

Epigenetic Regulation of Host Defense Peptide Synthesis: Synergy Between Histone Deacetylase Inhibitors and DNA/Histone Methyltransferase Inhibitors.

Host defense peptides (HDPs) are an integral part of the innate immune system acting as the first line of defense. Modulation of HDP synthesis has emerged as a promising host-directed approach to fight against infections. Inhibition of histone deacetylation or DNA methylation is known to enhance HDP gene expression. In this study, we explored a possible synergy in HDP gene induction between histone deacetylase inhibitors (HDACi) and DNA/histone methyltransferase inhibitors (DNMTi/HMTi). Two chicken macrophage cell lines were treated with structurally distinct HDACi, HMTi, or DNMTi individually or in combinations, followed by HDP gene expression analysis. Each epigenetic compound was found to be capable of inducing HDP expression. To our surprise, a combination of HDACi and HMTi or HDACi and DNMTi showed a strong synergy to induce the expressions of most HDP genes. The HDP-inducing synergy between butyrate, an HDACi, and BIX01294, an HMTi, were further verified in chicken peripheral blood mononuclear cells. Furthermore, tight junction proteins such as claudin 1 were also synergistically induced by HDACi and HMTi. Overall, we conclude that HDP genes are regulated by epigenetic modifications. Strategies to increase histone acetylation while reducing DNA or histone methylation exert a synergistic effect on HDP induction and, therefore, have potential for the control and prevention of infectious diseases.

Natural Cyclooxygenase-2 Inhibitors Synergize With Butyrate to Augment Chicken Host Defense Peptide Gene Expression.

Enhancing the synthesis of microbicidal and immunomodulatory host defense peptides (HDP) is a promising host-directed antimicrobial strategy to combat a growing threat of antimicrobial resistance. Here we investigated the effect of several natural cyclooxygenase-2 (COX-2) inhibitors on chicken HDP gene regulation. Our results indicated that phenolic COX-2 inhibitors such as quercetin, resveratrol, epigallocatechin gallate, anacardic acid, and garcinol enhanced HDP gene expression in chicken HTC macrophage cell line and peripheral blood mononuclear cells (PBMCs). Moreover, these natural COX-2 inhibitors showed a strong synergy with butyrate in augmenting the expressions of multiple HDP genes in HTC cells and PBMCs. Additionally, quercetin and butyrate synergistically promoted the expressions of mucin-2 and claudin-1, two major genes involved in barrier function, while suppressing lipopolysaccharide-triggered interleukin-1β expression in HTC macrophages. Mechanistically, we revealed that NF-κB, p38 mitogen-activated protein kinase, and cyclic adenosine monophosphate signaling pathways were all involved in the avian β-defensin 9 gene induction, but histone H4 was not hyperacetylated in response to a combination of butyrate and quercetin. Because of their HDP-inducing, barrier-protective, and antiinflammatory activities, these natural COX-2 inhibitors, when combined with butyrate, may be developed as novel host-directed antimicrobial therapeutics.

Synergistic Induction of Chicken Antimicrobial Host Defense Peptide Gene Expression by Butyrate and Sugars.

Antimicrobial resistance is a major concern to public health demanding effective alternative strategies to disease control and prevention. Modulation of endogenous host defense peptide (HDP) synthesis has emerged as a promising antibiotic alternative approach. This study investigated a potential synergy between sugars and butyrate in inducing HDP gene expression in chickens. Our results revealed that sugars differentially regulated HDP expression in both gene- and sugar-specific manners in chicken HD11 macrophage cells. Among eight mono- and disaccharides tested, all were potent inducers of avian β-defensin 9 (AvBD9) gene (p<0.05), but only galactose, trehalose, and lactose obviously upregulated cathelicidin-B1 (CATHB1) gene expression. The expression of AvBD14 gene, on the other hand, was minimally influenced by sugars. Moreover, all sugars exhibited a strong synergy with butyrate in enhancing AvBD9 expression, while only galactose, trehalose, and lactose were synergistic with butyrate in CATHB1 induction. No synergy in AvBD14 induction was observed between sugars and butyrate. Although lactose augmented the expression of nearly all HDP genes, its synergy with butyrate was only seen with several, but not all, HDP genes. Mucin-2 gene was also synergistically induced by a combination of lactose and butyrate. Furthermore, lactose synergized with butyrate to induce AvBD9 expression in chicken jejunal explants (p<0.05). Mechanistically, hyper-acetylation of histones was observed in response to both butyrate and lactose, relative to individual compounds. Mitogen-activated protein kinase, NF-κB, and cyclic adenosine monophosphate signaling pathways were also found to be involved in butyrate- and lactose-mediated synergy in AvBD9 induction. Collectively, a combination of butyrate and a sugar with both HDP-inducing and barrier protective activities holds the promise to be developed as an alternative to antibiotics for disease control and prevention.

Gut Microbiota Dysbiosis Aggravates Mycoplasma gallisepticum Colonization in the Chicken Lung

Mycoplasma gallisepticum (MG) is the pathogen that causes chronic respiratory diseases in chickens. Gut microbiota plays an important role in maintaining body health and resisting respiratory infection, but the correlation between gut microbiota and MG infection is poorly defined. Therefore, in this study, the correlation between gut microbiota and MG infection was explored by disturbing gut microbiota in chickens with antibiotic cocktail. The results showed that the gut microbiota dysbiosis impairs pulmonary immune response against MG infection. It has been noted that MG colonization in the lung was significantly increased following gut microbiota dysbiosis, and this could be reversed by intranasally administrated toll-like receptor 2 (TLR2) ligand, recombinant chicken IL-17 protein or recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) protein. In addition, the levels of short-chain fatty acids (SCFAs) and vitamin A were significantly reduced in gut microbiota dysbiosis group, however, butyric acid or vitamin A as feed additives promoted MG clearance in the lung of gut microbiota dysbiosis group via increasing TLR2/IL17/GM-CSF and host defense peptides genes expression. The present study revealed an important role of gut microbiota in the defense against MG colonization in the lung of chicken.

Butyrate and Forskolin Augment Host Defense, Barrier Function, and Disease Resistance Without Eliciting Inflammation.

Host defense peptides (HDPs) are an integral part of the innate immune system with both antimicrobial and immunomodulatory activities. Induction of endogenous HDP synthesis is being actively explored as an antibiotic-alternative approach to disease control and prevention. Butyrate, a short-chain fatty acid, and forskolin, a phytochemical, have been shown separately to induce HDP gene expression in human cells. Here, we investigated the ability of butyrate and forskolin to induce the expressions of chicken HDP genes and the genes involved in barrier function such as mucin 2 and claudin 1 both in vitro and in vivo. We further evaluated their efficacy in protecting chickens from Clostridium perfringens-induced necrotic enteritis. Additionally, we profiled the transcriptome and global phosphorylation of chicken HD11 macrophage cells in response to butyrate and forskolin using RNA sequencing and a kinome peptide array, respectively. Our results showed a strong synergy between butyrate and forskolin in inducing the expressions of several, but not all, HDP genes. Importantly, dietary supplementation of butyrate and a forskolin-containing plant extract resulted in significant alleviation of intestinal lesions and the C. perfringens colonization in a synergistic manner in a chicken model of necrotic enteritis. RNA sequencing revealed a preferential increase in HDP and barrier function genes with no induction of proinflammatory cytokines in response to butyrate and forskolin. The antiinflammatory and barrier protective properties of butyrate and forskolin were further confirmed by the kinome peptide array. Moreover, we demonstrated an involvement of inducible cAMP early repressor (ICER)-mediated negative feedback in HDP induction by butyrate and forskolin. Overall, these results highlight a potential for developing butyrate and forskolin, two natural products, as novel antibiotic alternatives to enhance intestinal health and disease resistance in poultry and other animals.

Butyrate, Forskolin, and Lactose Synergistically Enhance Disease Resistance by Inducing the Expression of the Genes Involved in Innate Host Defense and Barrier Function.

The rising concern of antimicrobial resistance highlights a need for effective alternatives to antibiotics for livestock production. Butyrate, forskolin, and lactose are three natural products known to induce the synthesis of host defense peptides (HDP), which are a critical component of innate immunity. In this study, the synergy among butyrate, forskolin, and lactose in enhancing innate host defense, barrier function, and resistance to necrotic enteritis and coccidiosis was investigated. Our results indicated that the three compounds synergistically augmented the expressions of multiple HDP and barrier function genes in chicken HD11 macrophages. The compounds also showed an obvious synergy in promoting HDP gene expressions in chicken jejunal explants. Dietary supplementation of a combination of 1 g/kg sodium butyrate, 10 mg/kg forskolin-containing plant extract, and 10 g/kg lactose dramatically improved the survival of chickens from 39% to 94% (p < 0.001) in a co-infection model of necrotic enteritis. Furthermore, the three compounds largely reversed growth suppression, significantly alleviated intestinal lesions, and reduced colonization of Clostridium perfringens or Eimeria maxima in chickens with necrotic enteritis and coccidiosis (p < 0.01). Collectively, dietary supplementation of butyrate, forskolin, and lactose is a promising antibiotic alternative approach to disease control and prevention for poultry and possibly other livestock species.

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening

BackgroundIn-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides (HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.MethodsTo identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3D4/31 macrophages against a Gram-negative bacterium (enterotoxigenic E. coli) and a Gram-positive bacterium (Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds.ResultsA total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening, with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment. Xanthohumol and deoxyshikonin were further found to be the most potent in inducing pBD3 mRNA expression, showing a minimum 10-fold increase in IPEC-J2, 3D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced pBD3 mRNA expression by at least 10-fold in both IPEC-J2 cells and jejunal explants, but not 3D4/31 cells. In addition to pBD3, other porcine HDP genes such as pBD2, PG1-5, and pEP2C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene- and cell type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3D4/31 macrophages (P < 0.05) against both Gram-negative and Gram-positive bacteria.ConclusionsOur results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.

Profiles of expression pattern and tissue distribution of host defense peptides genes in different chicken (Gallus gallus) breeds related to body weight.

Host defense peptides (HDPs) are an important first line of defense with antimicrobial and immunomodulatory properties. Selection for increased body weight is hypothesized to be related to reduced immune response. We studied the relationships among body weight, age, and the HDP expression patterns in intestine and immune organs. We used chickens with marked differences of body sizes. The non-selected Daweishan mini chickens showed the highest indexes of immune organs and the lowest concentrations of the plasma immune parameters C3, C4, IgA, and IgY, while the commercial Avian broiler showed the opposite results. The Daweishan mini chickens showed the highest mRNA expressions of HDP genes in small intestine followed by the semi-selected Wuding chickens. Compared with local breeds, broiler chickens showed higher mRNA expression of HDP genes in spleen, thymus, and bursa. Body weight and HDP expression levels were negatively correlated in the intestine and positively in the immune organs. Our results indicated that the HDP immune regulatory roles in small intestine acted as first line of defense in innate immunity in local breeds, and as an adaptive immunity in broiler chickens. Selection was associated with different expression expressions of HDP genes in breed-, age-, and organ-specific manners.

Development of a Cell-Based High-Throughput Screening Assay to Identify Porcine Host Defense Peptide-Inducing Compounds.

Novel alternatives to antibiotics are needed for the swine industry, given increasing restrictions on subtherapeutic use of antibiotics. Augmenting the synthesis of endogenous host defense peptides (HDPs) has emerged as a promising antibiotic-alternative approach to disease control and prevention. To facilitate the identification of HDP inducers for swine use, we developed a stable luciferase reporter cell line, IPEC-J2/PBD3-luc, through permanent integration of a luciferase reporter gene driven by a 1.1 kb porcine β-defensin 3 (PBD3) gene promoter in porcine IPEC-J2 intestinal epithelial cells. Such a stable reporter cell line was employed in a high-throughput screening of 148 epigenetic compounds and 584 natural products, resulting in the identification of 41 unique hits with a minimum strictly standardized mean difference (SSMD) value of 3.0. Among them, 13 compounds were further confirmed to give at least a 5-fold increase in the luciferase activity in the stable reporter cell line, with 12 being histone deacetylase (HDAC) inhibitors. Eight compounds were subsequently observed to be comparable to sodium butyrate in inducing PBD3 mRNA expression in parental IPEC-J2 cells in the low micromolar range. Six HDAC inhibitors including suberoylanilide hydroxamine (SAHA), HC toxin, apicidin, panobinostat, SB939, and LAQ824 were additionally found to be highly effective HDP inducers in a porcine 3D4/31 macrophage cell line. Besides PBD3, other HDP genes such as PBD2 and cathelicidins (PG1–5) were concentration-dependently induced by those compounds in both IPEC-J2 and 3D4/31 cells. Furthermore, the antibacterial activities of 3D4/31 cells were augmented following 24 h exposure to HDAC inhibitors. In conclusion, a cell-based high-throughput screening assay was developed for the discovery of porcine HDP inducers, and newly identified HDP-inducing compounds may have potential to be developed as alternatives to antibiotics for applications in swine and possibly other animal species.

Molecular characterization of a novel ovodefensin gene in chickens

Host defense peptides (HDPs) represent a large group of diverse small peptides that play important roles in host defense and disease resistance. In vertebrates, one of the main types of HDPs belong to defensins, which are less than 100 amino acid residues and characterized by a highly conserved motif of cysteine residues. Recently, a subfamily of defensins, namely ovodefensins (OvoDs), has been identified in birds and reptiles. However, both their family members and evolutionary relationships remain unclear. In the present study, we cloned and characterized a novel gene namely OvoDBβ in chickens. Our results showed that the full length of chicken OvoDBβ mRNA contains 344 bp nucleotides and encodes a 61-amino acid protein. We further revealed that the mRNA of OvoDBβ is abundant in the oviduct of laying hens but absent in many other tissues. Additionally, sequences comparison and analyses suggested that OvoDBβ is orthologous to the gene previously known as zebra finch OvoDB1, albeit it might exhibit specific structures. Furthermore, both OvoDBα and OvoDBβ were existent in the genome of each bird, implying that two types of OvoDBs sharing same cysteine motif have already emerged before the species divergence. More importantly, recombinant OvoDBβ mature peptide exerted antibacterial activity against Escherischia coli (CICC23657 strain) in vitro. These results collectively indicated that the putative sequence, namely chicken OvoDBβ, is a function gene with potential antimicrobial property. Discovery and function characterization of novel HDP genes may help us develop novel antimicrobial agents in the future.

High Throughput Screening for Natural Host Defense Peptide-Inducing Compounds as Novel Alternatives to Antibiotics.

A rise in antimicrobial resistance demands novel alternatives to antimicrobials for disease control and prevention. As an important component of innate immunity, host defense peptides (HDPs) are capable of killing a broad spectrum of pathogens and modulating a range of host immune responses. Enhancing the synthesis of endogenous HDPs has emerged as a novel host-directed antimicrobial therapeutic strategy. To facilitate the identification of natural products with a strong capacity to induce HDP synthesis, a stable macrophage cell line expressing a luciferase reporter gene driven by a 2-Kb avian β-defensin 9 (AvBD9) gene promoter was constructed through lentiviral transduction and puromycin selection. A high throughput screening assay was subsequently developed using the stable reporter cell line to screen a library of 584 natural products. A total of 21 compounds with a minimum Z-score of 2.0 were identified. Secondary screening in chicken HTC macrophages and jejunal explants further validated most compounds with a potent HDP-inducing activity in a dose-dependent manner. A follow-up oral administration of a lead natural compound, wortmannin, confirmed its capacity to enhance the AvBD9 gene expression in the duodenum of chickens. Besides AvBD9, most other chicken HDP genes were also induced by wortmannin. Additionally, butyrate was also found to synergize with wortmannin and several other newly-identified compounds in AvBD9 induction in HTC cells. Furthermore, wortmannin acted synergistically with butyrate in augmenting the antibacterial activity of chicken monocytes. Therefore, these natural HDP-inducing products may have the potential to be developed individually or in combinations as novel antibiotic alternatives for disease control and prevention in poultry and possibly other animal species including humans.

Host Defense Peptide Expression in Human Cervical Cells and Regulation by 1,25-Dihydroxyvitamin D3 in the Presence of Cytokines and Bacterial Endotoxin.

Host defense peptides (HDPs) in the pregnant female reproductive tract provide protection against infection. The relationship between HDPs and infection/inflammation is poorly understood. Therefore, we investigated the regulation of HDPs by 1α, 25-dihydroxyvitamin D3 (1,25-(OH)2) in the presence/absence of infectious/inflammatory agents. Endocervical epithelial cells (END1/E6E7, n = 6) were exposed to 1,25-(OH)2, calcipotriol, interleukin 1β (IL-1β), granulate-macrophage colony-stimulating factor (GM-GSF), and lipopolysaccharide (LPS). Elafin, human beta defensin (hBD2), cathelicidin, secretory leucocyte protease inhibitor, interleukin 8, 1,25-(OH)2 receptor, and toll-like receptor 4 (TLR4) expression was determined using quantitative polymerase chain reaction and/or enzyme-linked immunosorbent assay. Host defense peptide gene and protein expression was assessed in cervicovaginal cells/fluid, respectively, from first trimester pregnant women (n = 8-12). Interleukin 1β induced elafin and hBD2. The 1,25-(OH)2 induced cathelicidin expression in the presence of IL-1β and LPS. The 1,25-(OH)2 also attenuated IL-1β-induced IL-8 expression and LPS enhancement of TLR4. Host defense peptides and TLR4 profiles in cervicovaginal cells and fluid samples from pregnant women were similar to END1/E6E7 cells. In conclusion, HDPs are differentially regulated in END1/E6E7 cells. The 1,25-(OH)2 induction of cathelicidin and suppression of IL-8 highlights a mechanism by which 1,25-(OH)2 supplementation could enhance the pregnant innate immune defenses.

Induction of Porcine Host Defense Peptide Gene Expression by Short-Chain Fatty Acids and Their Analogs

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.

Role of histone acetylation, cAMP signaling, and mitogen-activated protein kinase pathways in butyrate-induced host defense peptide gene expression in chicken HD11 macrophage cells. (P1283)

Abstract Host defense peptides (HDPs), critical components of innate immunity of animals, act as direct antimicrobials and immunomodulators. We found that, butyrate, a short-chain fatty acid and histone deacetylase inhibitor, enhances HDP gene expression and reduce Salmonella enteritidis colonization in chickens. However, the molecular mechanisms of butyrate-mediated HDP gene induction remain elusive. Here, we dissected the role of histone acetylation, cAMP signaling, and mitogen-activated protein kinases (MAPKs) in HDP expression in butyrate treated HD11cells. Our real-time PCR results revealed that, in addition to butyrate, several other histone deacetylase inhibitors also enhanced the expression of AvBD9, a chicken beta-defensin gene. In contrast, histone acetyltransferase inhibitors suppressed butyrate-mediated AvBD9 gene induction. Moreover, inhibition of p38 MAP kinase or c-Jun N-terminal kinase, but not extracellular signal regulated kinase, diminished AvBD9 gene expression with butyrate. Interestingly, agonists of the cAMP signaling pathway increased AvBD9 expression either alone or synergistically with butyrate. Collectively, our results showed that histone acetylation, and MAPKs and cAMP pathways play an important role in butyrate-regulated AvBD9 gene expression. A detailed understanding of the transcriptional regulatory mechanisms of HDP expression may facilitate the development of antibiotic-free strategies to disease control and prevention in both animals and humans.

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.

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.