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.


Introduction
Subtherapeutic antibiotics are commonly used in the swine industry for growth promotion and disease control and prevention. However, due to a rise in antimicrobial resistance, such a practice has been largely phased out in the US. Novel approaches to disease intervention are needed. As a major component of the innate immune system, host defense peptides (HDPs) are broadly active against Grampositive and Gram-negative bacteria, viruses, and fungi [1][2][3]. HDPs are also capable of controlling and limiting infections by recruiting different types of immune cells to the site of infection and further neutralizing endotoxininduced inflammation [1][2][3]. A few HDPs were recently found to have barrier protective properties [4]. HDPs and their mimetics are, therefore, being actively explored as vaccine adjuvants or novel antimicrobials to treat resistant infections [1][2][3].
Cathelicidins and defensins constitute two major families of HDPs in mammals. The human genome harbors one cathelicidin (LL-37), six α-defensins, and more than 30 βdefensins [5]. Approximately a dozen cathelicidins such as protegrins (PG1-5) and 29 porcine β-defensins (PBDs) have been reported in pigs [6,7]. As an important first line of host defense, HDPs are mainly produced in phagocytic leukocytes as well as in mucosal epithelial cells on the surface of gastrointestinal, respiratory, and urogenital tracts. Besides infection and inflammation, a number of small-molecule compounds such as butyrate, vitamin D 3 , isoleucine, and bile acids were recently found to induce HDP genes in humans and other animals [8][9][10]. In fact, augmenting the synthesis of endogenous HDPs has been found to enhance bacterial clearance and could be explored as a novel approach to host-directed antimicrobial therapy [8][9][10].
Butyrate together with other short-and medium-chain fatty acids is capable of inducing HDP synthesis not only in humans but also in rabbits, cattle, chickens, and pigs [11][12][13][14][15][16][17][18][19][20]. For example, multiple porcine HDP genes such as PBD2, PBD3, and PG1-5 have been shown to be induced by a number of fatty acids including butyrate in porcine IPEC-J2 intestinal epithelial cells, 3D4/31 lung alveolar macrophage cells, and primary monocyte cells [20,21]. However, a few other HDP inducers such as vitamin D 3 are strong HDP inducers in humans [22,23] but are weaker in chickens and cattle [24][25][26]. Vitamin D 3 fails completely to induce cathelicidin gene expression in mice due to a lack of the vitamin D receptor in the mouse cathelicidin gene promoter, suggesting the existence of species-specific regulation of HDP genes [27]. To identify potent HDP inducers specific for swine applications, we developed a cell-based highthroughput screening (HTS) assay in this study and identified multiple small-molecule compounds that could be potentially developed as alternatives to antibiotics for swine disease control and prevention.

2.2.
Cloning of a Porcine PBD3 Gene Promoter-Driven Luciferase Reporter Vector. Porcine genomic DNA was isolated from a segment of the jejunum using the Quick-gDNA MiniPrep Kit (Zymo Research, Irvine, CA) and subsequently used as the template for PCR amplification of a 1121 bp promoter fragment using the Advantage 2 PCR Kit (Clontech, Mountain View, CA). The forward and reverse primers are 5 ′ -TGG CCT AAC TGG CCG GTA CCT GAA CTG CCC CTC TTT GCA TCT-3 ′ and 5 ′ -CCG GAT TGC CAA GCT TTA AAG ATT CCA GGT CCA CAG CCA-3 ′ , respectively, where gene-specific sequences are located at the 3 ′ -regions and underlined sequences are included for In-Fusion PCR Cloning (Takara Bio USA, Mountain View, CA) as recommended by the manufacturer. It is noted that the 5 ′ -end of the gene-specific reverse primer was designed immediately upstream of the porcine PBD3 mRNA reference sequence (GenBank accession number NM_214444), and the entire PBD3 gene promoter fragment is located at chr15: 38,063,074-38,064,194 of the UCSC Genome Browser assembly ID susScr11. The PCR product was then cloned into a pGL4.21[luc2P/Puro] luciferase reporter vector (Promega, Madison, WI) predigested with KpnI and HindIII (Promega) using the In-Fusion HD Cloning Kit (Takara Bio USA) following the manufacturer's instructions. Recombinant plasmids were confirmed for the presence of a genespecific insert by Sanger sequencing.

Construction of a Stable Luciferase Reporter Cell Line.
Recombinant reporter plasmid was linearized with KpnI and transfected into IPEC-J2 cells using FuGene HD (Promega) by following the manufacturer's recommendations. In brief, 2 × 10 5 IPEC-J2 cells/well were seeded in a 6-well plate in 2 ml complete DMEM/F12 medium and incubated overnight, followed by transfection of 750 ng of the linearized plasmid with 2.25 μl FuGene HD reagent. After 48 h incubation, cells were replenished every 2-3 days with fresh complete DMEM/F12 medium containing 1 μg/ml puromycin. After a week of puromycin selection, cells in each well were expanded to a 10 cm cell culture plate for another week. A stable puromycin-resistant luciferase reporter cell line, named IPEC-J2/PBD3-luc, was maintained in complete DMEM/F12 medium containing 1 μg/ml puromycin for another week and subcultured every 3-4 days, before being used in the HTS assay development.

Development and Optimization of a High-Throughput
Screening (HTS) Assay. The responsiveness of the stable IPEC-J2/PBD3-luc reporter cell line to butyrate was first evaluated by seeding 2 × 10 4 cells/well in 50 μl of complete DMEM/F12 medium in a 96-well tissue culture plate overnight prior to stimulation with or without different concentrations (4,8,16,32, and 64 mM) of sodium butyrate (Sigma-Aldrich, St. Louis, MO) for 24 h. The luciferase activity was measured with Modulus Luminometer (Turner BioSystems, Sunnyvale, CA) using the Steady-Glo Luciferase Assay System (Promega, Madison, WI) as instructed by the manufacturer. To further assess the robustness of the HTS assay, the Z ′ -factor [30] was calculated by treating the stable reporter cells in a 96-well plate with or without 16 mM butyrate for 24 h, followed by the luciferase assay.

HTS Assay for HDP-Inducing
Compounds. An Epigenetics Screening Library was purchased from Cayman Chemical (Ann Arbor, MI) and consists of 148 smallmolecule compounds that are involved in different epigenetic modifications such as DNA/histone methylation and demethylation as well as histone acetylation and deacetylation. A natural product library containing 502 compounds and a rare natural product library containing 82 compounds were previously procured from BIOMOL International (Plymouth Meeting, PA) [31]. The compounds in the Epigenetics Screening Library were provided as 10 mM stocks in DMSO, while those in natural product libraries were provided at 1 mg/well and reconstituted in DMSO to 10 mg/ml stocks. The HTS assays were conducted in 96-well plates by seeding the stable IPEC-J2/PBD3-luc cells at 2 × 10 4 /well. After overnight culture, the cells were stimulated for 24 h with individual compounds at the final concentration of 20 μM for the Epigenetics Screening Library or 20 μg/ml for the natural product libraries. The luciferase activity was measured with an L-Max II Luminescence Microplate Reader (Molecular Devices, Sunnyvale, CA). To assess relative toxicity of each compound, 1/50 volume of alamarBlue Reagent (Invitrogen, Carlsbad, CA) was added to each well 4 h prior to the luciferase assay. The fluorescence was read in live cells at 545 nm excitation and 590 nm emission using an FLx80 Microplate Fluorescence Reader (BioTek Instruments, Winooski, VT). The relative luciferase activity of each compound was normalized to the cell viability to calculate the strictly standardized mean difference (SSMD) for hit selection as described [32].
2.6. Validation of Hit Compounds. The compounds with a normalized SSMD value of no less than 3.0 were further assayed for their luciferase activity in the stable IPEC-J2/ PBD3-luc cell line at three different concentrations in 96well plates. After normalization to the cell viability, the fold change in the luciferase activity of each compound relative to the nonstimulation control was calculated. Compounds showing a minimum 5-fold increase at any of the three concentrations examined were further evaluated in the parental IPEC-J2 cell line and a porcine lung alveolar macrophage cell line 3D4/31 for their ability to induce the mRNA expression of porcine HDP genes. All individual compounds were purchased from Cayman Chemical (Ann Arbor, MI), except for (−)-depudecin, which was procured from BioVision (Milpitas, CA) and MyBioSource (San Diego, CA). Three different concentrations of each compound were applied to the cells in 12-well plates for 24 h, followed by total RNA isolation, reverse transcription, and real-time PCR as described below. All treatments were performed in duplicate and repeated 2-4 times.
2.7. RNA Isolation, Reverse Transcription, and Real-Time PCR. After stimulation, cells were directly lysed in RNAzol® RT Reagent (Molecular Research Center, Cincinnati, OH), followed by total RNA isolation as recommended by the manufacturer. The first-strand cDNA was synthesized with 0.3 μg of total RNA in 4 μl reactions using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). Real-time PCR was conducted in 10 μl reactions on the iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories) using the Maxima SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instruction. The expression levels of porcine HDP genes including PBD2, PBD3, and PG1-5 as well as a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were evaluated using gene-specific primers as described [20], and relative fold changes in gene expression were calculated using the ΔΔCt method [17,20].

Minimum Inhibitory Concentrations of HDAC Inhibitors.
The minimum inhibitory concentrations of the selected HDAC inhibitors were determined by a standard broth microdilution assay as recommended by the Clinical and Laboratory Standards Institute [33] as we previously described [34][35][36]. Briefly, Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 43300) were cultured in trypticase soy broth (TSB) with shaking at 250 rpm at 37°C for 3 h to reach the midlog phase of growth. Bacteria were then washed twice in 10 mM sodium phosphate buffer (pH 7.4) and diluted to 5 × 10 5 CFU/ml in Mueller Hinton Broth (MHB, Fisher Scientific). After dispensing 90 μl/well in a 96-well tissue culture plate, 10 μl of SAHA, apicidin, and HC toxin was added in duplicate to final concentrations of 5, 10, 20, 40, 80, 160, and 320 μM. The MIC value of each compound was determined as the lowest concentration of the compound that gave no visible bacterial growth after overnight incubation at 37°C.
2.10. Data Analysis. One-way or two-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, was performed for statistical analyses using GraphPad Prism (San Diego, CA). P ≤ 0 05 was considered statistically significant.

Construction and Optimization of a Cell-Based HTS
Luciferase Assay. Because PBD3 is among the most responsive porcine HDP genes to butyrate and many other fatty acids and their analogs [20], the PBD3 gene promoter was selected for the development of a HTS luciferase assay. A 1121 bp PBD3 gene promoter fragment immediately upstream of the transcriptional start site was cloned into the pGL4.21[luc2P/Puro] luciferase reporter vector. The recombinant plasmid was then transfected into porcine IPEC-J2 cells, followed by 2-week puromycin selection to achieve stable integration of the luciferase reporter gene. The newly established luciferase reporter cell line, IPEC-J2/ PBD3-luc, showed a clear concentration-dependent increase in the luciferase activity to sodium butyrate, with an approximately 5.5-fold increase in response to 64 mM butyrate (Figure 1(a)). To further evaluate the quality of the cell-based HTS assay, we treated the cells with or without 16 mM butyrate in a 96-well plate, assayed for the luciferase activity, and calculated the Z ′ -factor [30] to be 0.56 (Figure 1(b)). The Z ′ -factor in subsequent HTS assays in different plates was calculated to be in the range of 0.56-0.74, which is considered rather robust in a typical HTS assay [30].

High-Throughput Identification of HDP-Inducing
Compounds. After screening 148 epigenetic and 584 natural compounds, a total of 41 nonredundant compounds showed a SSMD value of no less than 3.0, which are considered to be strong hits [32] (Figure 2). Among these, 33 compounds were from the Epigenetics Screening Library (Table 1), and Library (compound #1-148) and two natural product libraries (compound #149-732). The IPEC-J2/PBD3-luc luciferase reporter cell line was stimulated with 20 μM of each compound in the Epigenetics Screening Library or 20 μg/ml of each compound in the natural product libraries for 24 h, followed by the luciferase assay. The alamarBlue Dye was added 4 h before the luciferase assay to measure cell viability. The luciferase activity of each compound was normalized to cell viability before the SSMD value was calculated. another 10 compounds were from two natural product libraries (Table 2), with apicidin and Helminthosporium carbonum (HC) toxin being identified from both sources. It was apparent that most compounds identified are histone deacetylase (HDAC) inhibitors, whose action leads to hyperacetylation of histones [39] and often increased HDP gene transcription in humans [40]. Additionally, a few histone or DNA methylation inhibitors such as UNC0642 [41], UNC0224 [42], UNC1215 [43], and Sadenosylhomocysteine [44] were also found to have potential HDP-inducing activities (Table 1). Several natural products such as ikarugamycin [45,46], cynaroside [47], and sanguinarine [48] that are known to have anti-inflammatory, antioxidative, and/or antimicrobial activities but with no confirmed epigenetic modification properties were also identified ( Table 2). It is surprising to observe that a number of natural products appeared to inhibit HDP gene expression with a SSMD value of less than −3.0 ( Figure 2). The putative inhibitory role of these compounds in HDP gene expression is being verified, but characterizations of the HDP-inducing compounds were the focus of this study.
Follow-up concentration-response experiments in the stable IPEC-J2/PBD3-luc reporter cell line with 41 hits revealed that most compounds increased the luciferase activity in an obvious concentration-dependent manner  i.e., (−)-depudecin, sanguinarine, apicidin, and HC toxin, from two natural product libraries (Figure 3). HC toxin was again identified from both sources.

Validation of HDP-Inducing Compounds in Porcine Cell
Lines. Among the 13 compounds identified, 12 happened to be HDAC inhibitors. Sanguinarine is the only exception; however, it was found to be obviously toxic to IPEC-J2 cells at HDP-inducing concentrations. Another compound, (−)-depudecin, showed no HDP-inducing activity at various concentrations when purchased from BioVision (Milpitas, CA) and MyBioSource (San Diego, CA), which are the only two vendors that we found selling the chemical. Both sanguinarine and (−)-depudecin were, therefore, excluded from further evaluations. We also excluded TSA, a well-known  toxic HDAC inhibitor, as well as two SAHA analogs (4iodo-SAHA and coumarin-SAHA) to minimize redundancy with SAHA. Each of the eight remaining compounds was then assessed at three different concentrations using realtime PCR in the parental IPEC-J2 cell line for their ability to induce the mRNA expression of PBD3 and other representative porcine HDP genes including PBD2 and five highly homologous cathelicidins (PG1-5), which could not be differentiated by the primers that were used [20,49]. All eight compounds induced PBD2 mRNA expression by 10-to 30-fold at a minimum of one concentration examined (Figure 4). They also increased PBD3 mRNA expression by 15-to 40-fold, while enhancing PG1-5 expression by 5-to 15-fold (Figure 4). A majority of the eight compounds achieved a similar HDP-inducing efficacy to butyrate. It is also clear that three HDP genes were differentially regulated by these compounds. For example, SAHA concentration dependently increased PBD3 and PG1-5 gene expression, but the same three concentrations caused a graduated decrease in PBD2 gene induction. ITF-2357 was among the most potent compounds in inducing PBD2 expression but became the least effective in PBD3 and PG1-5 gene induction (Figure 4).
To further compare relative HDP-inducing activities of these compounds in a different cell type, we selected six compounds including SAHA, panobinostat, HC toxin, apicidin, LAQ824, and SB939 that were among the most effective HDP inducers in IPEC-J2 cells and evaluated them in a porcine lung alveolar macrophage cell line, 3D4/31. As expected, all six compounds approached or exceeded the efficacy of butyrate and induced the expression of PBD2 mRNA by up to 15-fold, PBD3 by as much as 150-fold, and PG1-5 by up to 20-fold ( Figure 5). Again, we observed differential regulation among HDP genes. For example, LAQ824 and SB939 showed a concentration-dependent decrease in inducing PBD2 and PBD3 gene expression; however, such a concentration-response pattern was not obvious with PG1-5 ( Figure 5). Panobinostat at 80 μM appeared to be the least effective in inducing PBD2 and PBD3 but was the most effective in inducing PG1-5. Nevertheless, it can be concluded that the HTS assay that we developed in this study is highly effective in identifying HDP inducers and that most compounds identified are capable of inducing the expression of multiple porcine HDP genes in different cell types. Enhancing the expression of HDP genes is expected to improve bacterial clearance and disease outcomes [8][9][10]. The results are means ± SEM of three independent experiments. One-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, was performed. * P < 0 05, * * P < 0 01, * * * P < 0 001, and * * * * P < 0 0001 (relative to the unstimulated control).

No Direct Antibacterial Activity of HDP-Inducing HDAC
Inhibitors. To examine whether the HDAC inhibitors identified above have direct antibacterial activities, a representative Gram-negative bacterium (E. coli, ATCC 25922) and a Gram-positive bacterium (S. aureus, ATCC 43300) were used to determine the minimum inhibitory concentrations of SAHA, apicidin, and HC toxin using a standard broth microdilution assay [33]. However, none of the compounds showed any obvious antibacterial activity, and their minimum inhibitory concentrations were all beyond 320 μM, the highest concentration that we tested (data not shown), implying that these compounds could potentially enhance HDP synthesis and bacterial clearance without exerting selective pressure on bacteria.
3.5. Augmentation of the Antibacterial Activity of Porcine 3D4/31 Cells by HDAC Inhibitors. To further confirm whether HDAC inhibitor-mediated HDP mRNA induction can lead to an increase in the antibacterial activity of host cells, porcine 3D4/31 macrophages were left untreated or treated with 16 mM sodium butyrate, 20 μM SAHA, 20 μM apicidin, or 5 μM HC toxin for 24 h, followed by incubation of the cell lysate with E. coli (ATCC 25922) or S. aureus (ATCC 43300) and measurement of the bacterial turbidity [17,37,38]. Consistent with our earlier observation [17], butyrate enhanced the antibacterial activity of 3D4/31 cells ( Figure 6). SAHA, apicidin, and HC toxin also similarly improved the ability of porcine macrophages to suppress the growth of both Gram-negative and Gram-positive bacteria, albeit with varying antibacterial efficacies ( Figure 6). Further characterization of the efficacy of these compounds in HDP induction and pathogen clearance in live animals could potentially lead to the identification of antibiotic alternatives for use in pigs without triggering bacterial resistance to these HDP-inducing compounds.

Discussion
A variety of small-molecule compounds have been found to augment HDP synthesis in different animal species [8][9][10]; however, not all compounds can regulate HDP gene expression with similar efficacy across species [27], which necessitates the identification of species-specific HDP-inducing compounds. A HTS assay was previously developed and led to the discovery of a number of compounds with the ability to induce human cathelicidin LL-37 [50]. In this study, we developed a cell-based HTS assay to identify HDP inducers for use in pigs. Porcine IPEC-J2 intestinal epithelial cells were permanently integrated with a luciferase reporter vector driven by a PBD3 gene promoter construct One-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, was performed. * P < 0 05, * * P < 0 01, * * * P < 0 001, and * * * * P < 0 0001 (relative to the unstimulated control).
through puromycin selection. After optimization, such a HTS assay had a Z′-factor in the range of 0.57-0.74, which suggested that it is a rather robust system and equivalent to a human HTS assay [50]. However, instead of fusing a 4 kb LL-37 gene promoter plus its entire four-exon open reading frame with the luciferase reporter gene as in the human HTS system [50], our system only needed a 1.1 kb PBD3 gene promoter ( Figure 2) and has resulted in the identification of a number of compounds that are capable of inducing multiple porcine HDPs in both an intestinal epithelial cell line and a macrophage cell line. Nevertheless, it is also possible to employ a longer PBD3 gene promoter to achieve a better outcome. The PBD3 gene promoter was selected to drive the luciferase gene expression in this HTS assay due to its strong responsiveness to a panel of fatty acids and their structural analogs in both porcine IPEC-J2 and 3D4/31 cells [20]. Because several other porcine HDP genes such as PBD2, epididymis protein 2 splicing variant C (EP2C), and protegrins are also readily induced by fatty acids [20], cloning of those HDP gene promoters into the pGL4.21 luciferase reporter vector is expected to yield a similar outcome to the current HTS assay. Indeed, the compounds identified here using the PBD3 gene promoter-based system are also capable of triggering the induction of PBD2 and protegrin genes.
Epigenetic modifications of chromatin such as histone acetylation, histone methylation, and DNA methylation play an important role in regulating gene expression [51]. Histone acetylation is regulated by opposing effects of histone acetyltransferases (HATs) and HDACs [49], whereas histone methylation is controlled by histone methyltransferases (HMTs) and histone demethylases [52]. On the other hand, DNA methylation occurs through the action of DNA methyltransferases (DNMTs), while the enzymes and mechanisms for DNA demethylation are only beginning to be elucidated [53]. HDAC inhibitors suppress the removal of the acetyl groups from acetylated histones resulting in hyperacetylation of histones and increased gene transcription [39]. Consistently, a number of HDAC inhibitors have been identified to be capable of inducing human HDP gene expression [40]. Similarly, HMT and DNMT inhibitors reduce methylation of histones and DNA and often lead to enhanced gene expression [51,52]. Hypermethylation of DNA and histones has been revealed to suppress human HDP gene expression [54][55][56][57].
In this study, a screening of a total of 732 epigenetic and natural product compounds has led to identification of 41 nonredundant compounds with a SSMD value of no less than 3.0. Among them, a great majority are known inhibitors of HDACs, HMTs, and/or DNMTs, which is in contrast with the outcome of an earlier human HTS, where a number of non-HDAC inhibitors were identified [50], which may be due to a difference in the selection of compound libraries. Subsequent validation has resulted in discovery of the 13 most potent HDP inducers that gave a minimum 5-fold increase in the luciferase activity in the stable IPEC-J2/ PBD3-luc reporter cell line. Out of the 13 compounds, 12 are HDAC inhibitors, with sanguinarine being the only exception. Sanguinarine is a benzophenanthridine alkaloid extracted from the bloodroot plant Sanguinaria canadensis known to have anti-inflammatory, antioxidative, antiproliferative, and proapoptotic properties [58]. Although its mechanisms of action remain unclear [48], sanguinarine was recently identified as a putative inhibitor of heat shock protein 90 (HSP90) [31], whose activity has been demonstrated to be regulated by reversible acetylation [59].
HDACs are classified into four classes (I-IV) based on their sequence homology and domain organization, while HDAC inhibitors are often grouped according to their structures [49]. Both broad-spectrum and class-specific HDAC inhibitors exist [49]. Among the 12 HDAC inhibitors identified, most are known pan-HDAC inhibitors that are capable of suppressing the activity of multiple classes of HDACs and the majority are hydroxamates (Table 3). For example, SAHA is a well-known pan-HDAC inhibitor that has been approved by the FDA in 2006 for treatment of advanced primary cutaneous T-cell lymphoma [60]. In this study, we found that SAHA is highly active in inducing multiple porcine HDP gene expression in both IPEC-J2 and  Two-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test, was performed. * P < 0 05, * * P < 0 01, and * * * P < 0 001 (relative to the control at each time point).  [61], is also highly efficient in enhancing HDP expression in both porcine intestinal epithelial and alveolar macrophages. Similarly, SB939, also known as pracinostat, is a synthetic pan-HDAC inhibitor [62,63] and effective in HDP induction in both cell types as well.
Interestingly, MS-275, also known as entinostat, was identified in humans to be among the most effective HDAC inhibitors in LL-37 and β-defensin 1 (HBD1) gene expression [64,65]; however, it failed to give more than 5-fold increase in the luciferase activity in follow-up evaluations in the stable IPEC-J2/PBD3-luc luciferase reporter cell line in our study, albeit with a SSMD value of 7.39 at 20 μM in the initial screening (Table 1), suggesting a difference in the potency of various HDP-inducing compounds among animal species. In fact, species-specific HDP induction by HDP inducers has been observed. Vitamin D 3 , which is a potent human cathelicidin LL-37 inducer, completely loses its ability to induce the cathelicidin gene expression in mice [27]. Mediumchain fatty acids such as hexanoate (C6) and heptanoate (C7) are more potent than short-chain fatty acids such as butyrate (C4) and valeric acid (C5) in HDP induction in human cells [19], while the opposite is true in chickens and pigs [18,20].
Besides showing a species-specific regulation pattern, many HDP genes are regulated in cell-and gene-specific manners as well. First, a HDP gene is often modulated differently by the same inducer in different cell types. For example, human LL-37 is strongly induced by butyrate in intestinal epithelial cells but with a minimum change in mRNA expression in response to butyrate in monocytes or skin keratinocytes [37]. In this study, PBD2, PBD3, and PG1-5 are induced to different magnitudes by many of the same compounds in porcine IPEC-J2 epithelial and 3D4/31 macrophages. Secondly, different HDP genes are often regulated differently in a cell type by the same inducer. Human LL-37 is readily induced by vitamin D 3 in monocytes and keratinocytes, where HBD1 and HBD2 are barely altered by vitamin D 3 [37]. Here, we observed that 20 μM SAHA increased PBD3 expression by nearly 150-fold in 3D4/31 cells but only enhanced PBD2 or PG1-5 expression by 10-to 15-fold ( Figure 5). It is, therefore, desirable to identify HDP inducers with the capacity to induce the synthesis of multiple HDPs in multiple cell types across animal species. To date, most HDAC inhibitors appear to have this capacity by simultaneously inducing multiple HDP synthesis in different cell types and different animal species, albeit with varying potency [40]. Besides their use in swine, the HDAC inhibitors identified in this study could thus see potential application in other animals.

Conclusions
We have successfully developed a cell-based HTS assay and identified several highly active HDP inducers with the potential for further development as antibiotic alternatives for swine use. Because most HDAC inhibitors work across species, these small-molecule compounds are likely to be able to induce HDP synthesis and enhance disease resistance in other animals and even humans. Therapeutic and prophylactic applications of those HDP-inducing compounds with no direct antimicrobial activities constitute a novel host-directed approach to infectious disease control and prevention with virtually no risk of triggering microbial resistance.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.