On a global basis, it is estimated that 5.9 million children under the age of five years died in 2015, most of which are caused by infectious diseases associated with bacteria that are resistant to antibiotics [1
]. As key components of the innate immune system, host defense peptides (HDPs) play critical roles in fighting against infections for their ability of possessing antimicrobials and a low propensity for the development of bacterial resistance in younger children with immature neonatal immune systems [2
]. HDPs have been commonly studied for their antimicrobial properties and have been shown to kill bacteria, viruses, fungi, protozoa, and even cancer cells [3
]. Due to their potential therapeutic activities, HDPs are attractive candidates as alternatives for antibiotics [4
]. Swine and humans share high similarity in physiologic and anatomic characteristics, which makes the former the ideal model for human health and disease [5
]. In vertebrate animals, HDPs are generally grouped into two major families; defensins and cathelicidins [7
As an important first line of defense, HDPs are produced mainly by intestinal epithelial cells and phagocytes in the gastrointestinal tract. In addition to infection or inflammation, HDPs can also be induced by dietary compounds, including saccharides, essential amino acids, butyrate, vitamin D3, and zinc [4
]. Moreover, probiotic lactobacilli could stimulate HDP expression in human cells and piglets without provoking inflammatory responses like pathogenic strains [13
]. However, different lactobacilli strains show a varying magnitude of HDP-inducing activity [13
is considered to be an indigenous species in the gastrointestinal tract of humans and animals [15
]. Numerous studies have demonstrated that L. reuteri
has excellent probiotic properties and has been widely used as a probiotic in humans and animals [16
]. L. reuteri
I5007, initially known as L. fermentum
I5007, was isolated from the colonic mucosa of healthy weaning piglets [17
]. Compelling evidence shows that L. reuteri
I5007 has several important probiotic properties including: (1) resistance to gastric acid and bile [18
]; (2) strong adhesion [17
]; (3) competitive exclusion against pathogens [19
]; (4) alleviation of weaning stress in piglets [20
]; (5) improvement of piglet performance [21
]; (6) and positive regulation of redox status and immune function in piglets [23
]. Notably, oral administration of L. reuteri
I5007 increased the concentration of butyrate and branched chain fatty acids in the colonic digesta of suckling piglets [22
]. It has been shown that butyrate, produced by butyrate-producing bacterial strains, has strong capacity to induce HDP expression in vitro. However, whether L. reuteri
I5007 could modulate intestinal HDP expression through modifying gut microbiota and its metabolite butyrate in neonatal piglets is still unknown.
The aim of the current study was to investigate the effects of L. reuteri I5007 on the gut microbiota and HDP expression. We initially studied the in vitro effect of L. reuteri I5007 by inducing HDP expression in a porcine intestinal epithelial cell line. We subsequently determined the effects of L. reuteri I5007 supplementation on the colonic bacterial community and HDP expression in formula-fed neonatal piglets.
2. Materials and Methods
2.1. Ethics Statement
The procedures used in this experiment were approved by the China Agricultural University Institutional Animal Care and Use Committee (CAU20144-2, Beijing, China).
2.2. Bacterial Strain, Growth and Storage Conditions
I5007 was grown in De Man Rogosa Sharpe media under anaerobic conditions at 37 °C for 20 h. For cell culture assays, after incubation, bacterial cells were obtained by centrifugation (8000× g
for 10 min at 4 °C). Then the bacterial cells were washed with phosphate-buffered saline (PBS, a balanced salt solution used for a variety of cell culture applications), reconstituted in DMEM/F12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12, 1:1 mixture of DMEM and Ham’s F-12) medium supplemented with 10% fetal bovine serum (FBS) and adjusted to the required cell concentration. After centrifugation, the culture supernatant of L. reuteri
I5007 was passed through a 0.2-μm-pore-size filter (Corning Inc., Corning, NY, USA), and it was preserved for subsequent treatment with a 10% (v
) concentration. For heat killed bacteria, heat inactivation was carried out in a water bath at 65 °C for 1 h. The bacterial cells were centrifugated, and the pellet was washed with PBS and adjusted to a density of 1 × 108
colony forming units (CFU)/mL with DMEM/F12 medium supplemented with 10% FBS. The freeze-dried powder, containing 5 × 1010
CFU/g, was produced according to Liu et al. [22
2.3. Cell Culture and Treatment
The porcine small intestinal epithelial cell line (IPEC-J2) was kindly provided by Dr. Wu at Texas A & M University (College Station, TX, USA). IPEC-J2 cells were cultured in DMEM/F12 medium supplemented with 10% FBS at 37 °C in a 5% CO2 and 95% air atmosphere with 90% humidity.
For stimulation experiments, undifferentiated cells were seeded at a density of 1 × 106 cells per well in 6-well plates (Costar, Corning Inc., Corning, NY, USA). After overnight growth (cells were grown to ~80% confluence in the culture wells), the cells were treated in duplicate with L. reuteri I5007. To prevent any influence of antibiotics on the immune response, the medium did not contain antibiotics. The FBS showed no effect on expression.
For dose-dependent L. reuteri I5007 stimulation experiments, IPEC-J2 cells were incubated with a control or 105, 106, 107, 108, or 109 CFU/mL L. reuteri I5007 for 6 h. For time-dependent L. reuteri I5007 stimulation experiments, IPEC-J2 cells were incubated with 108 CFU/mL L. reuteri I5007 for 3, 6, or 12 h.
IPEC-J2 cells were also treated for 6 h with 108 CFU/mL L. reuteri I5007 exposed to different processing conditions. The processing conditions included a solvent control without L. reuteri I5007 (Control, DMEM/F12 medium supplemented with 10% FBS), 108 CFU/mL live L. reuteri I5007 (Live I5007), 108 CFU/mL heat-killed L. reuteri I5007 (Dead I5007, incubated in a water bath at 65 °C for 1 h), adhered L. reuteri I5007 (Adhered I5007, treated with 108 CFU/mL L. reuteri I5007 for 1 h, rinsed three times in PBS with fresh medium added, followed by continued incubation for 5 h), and 200 μL of L. reuteri I5007-free culture supernatant of L. reuteri I5007 (Supernatant, diluted 1:10 in basal medium). In addition, a Transwell Insert System (Costar, Corning Inc., Corning, NY, USA) was used to avoid direct contact between the IPEC-J2 cells and L. reuteri I5007 (Separate I5007). Herein, L. reuteri I5007 cells in an upper chamber and IPEC-J2 cells in a lower chamber were separated by a 0.2-μm-pore-size filter membrane support (Corning Inc., Corning, NY, USA), thereby minimizing any direct contact between the L. reuteri I5007 cells and IPEC-J2.
2.4. Animals and Treatments
The in vivo experiment was conducted in the Metabolism Laboratory of the Ministry of Agriculture Feed Industry Centre (Beijing, China). Twenty-two, full-term, crossbred (Duroc × Large White × Landrace) male piglets, obtained from six litters, were used in this study. The piglets were delivered vaginally and allowed colostrum for 48 h after birth. The piglets were individually housed in stainless steel cages (1.4 m × 0.45 m × 0.6 m) in a temperature (32 ± 1 °C) and relative humidity (65–70%) controlled room programmed to deliver a light:dark cycle of 16:8 h.
On the third day after parturition, the piglets were trained to suckle from bottles filled with milk replacer (Jiaduonai H001, DaChan Tianyao, Tianjin, China, Table 1
), which was dissolved in warm previously boiled water (45 °C, w
1:9). The fresh liquid milk replacer was fed to piglets individually from a feeder five times daily (6:00, 10:00, 14:00, 18:00, and 22:00 h) for 20 days. After feeding, the remaining milk was measured and the feeders were cleaned before adding new fresh milk replacer. The formula did not contain any antibiotics or other medicine.
On day 4, the neonatal piglets were allocated to one of two treatments balanced for litter of origin and body weight (initial body weight of 1.81 ± 0.31 kg) with 11 piglets assigned to each treatment (n = 11). The treatments were comprised of a control treatment (the piglets were given a placebo of 4 mL of 0.1% peptone) and a L. reuteri I5007 treatment, which involved oral administration of 1.0 × 1010 CFU L. reuteri I5007 dissolved in 4 mL of 0.1% peptone water daily for 20 days.
The health status for each piglet was recorded, and the occurrence of diarrhea was assessed two times a day (monitoring time: 10 a.m. and 4 p.m.) according to the method of Marquardt et al. [25
] and Ou et al. [26
]. Scores were 0 = normal, solid feces; 1 = slight diarrhea, soft and loose feces; 2 = moderate diarrhea, semi-liquid feces; or 3 = severe diarrhea, liquid and unformed feces. The occurrence of diarrhea was defined as maintaining a score of two or three for one day. The incidence of diarrhea (%) was calculated as ((number of piglets with diarrhea × number of days of diarrhea)/(total number of experiment piglets × number of days of the whole experiment)) × 100%.
On days 4, 14, and 24, the piglets were weighed. On day 24, all piglets were euthanized with Zoletil 50® (Virbac, Carros, France), and all the intestinal tissues from the jejunum, ileum, and proximal distal colon were collected, frozen in liquid nitrogen, and then stored at –80 °C until total RNA was extracted. The colonic digesta were gently squeezed into sterile Eppendorf tubes, frozen in liquid nitrogen, and subsequently stored at −80 °C until processing.
2.5. Analysis of Porcine Gene Expression by Real Time PCR
The cells and tissues (about 0.04 mg) were lysed directly in TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA was extracted according to the manufacturer’s instructions. RNA concentrations were measured using a NanoDrop Spectrophotometer (P330, Implen, Germany). The purity was determined by the ratio of A260:A280 and A260:A230 by NanoDrop, and then the quality was checked with 1% Agarose Gel Electrophoresis following the procedures outlined by Aranda et al. [27
The first-strand cDNA was synthesized by reverse transcription of 1 μg of total RNA using a PrimeScript 1st Strand cDNA Synthesis Kit (Takara, Dalian, China) according to the manufacturer’s protocol and stored at –80 °C. The primers used are listed in Supplemental Table S1
. Porcine β-Defensin (PBD) 1, pBD2, pBD3, pBD114, pBD129, Protegrins (PG) 1-5, Epididymis Protein 2 Splicing Variant C (PEP2C), toll-like Receptors (TLR) 2, TLR4, TLR6, TLR9, Nucleotide-Binding Oligomerization Domain (NOD) 1, Mucin 1 (MUC1), Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) ,G Protein-Coupled Receptor (GPR) 41, and GPR43 were determined [4
Real-time PCR was performed on an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Singapore) using SYBR Green PCR Master Mix (Takara, Dalian, China). All reactions were run in triplicate. Relative gene expression was calculated according to the ΔΔCt method ((Ct gene of interest − Ct internal control) treatment − (Ct gene of interest − Ct internal control) control) using porcine β-actin as the reference gene.
2.6. Colonic Short Chain Fatty Acid Concentrations
The concentrations of SCFA (short-chain fatty acid) were determined with a Dionex ICS-3000 Ion Chromatography System (Dionex Corporation, Sunnyvale, CA, USA) following the procedures of Qiu and Jin [32
] with modification. Samples of colonic digesta (0.5 g) were weighed, diluted in a ratio of 1:5 with ultrapure water, homogenated with 8 mL ultrapure water, and then centrifuged at 10,000× g
for 20 min at 4 °C. The supernatant was kept in a 2 mL screw-capped vial. The concentrations of formic, acetic, propionic, butyric, and lactic acid were measured with the Dionex ICS-3000 Ion Chromatography System (Dionex, Sunnyvale, CA, USA).
2.7. Fecal Microbiota Analysis
Microbial genomic DNA was extracted and purified from colon digesta samples using a QIAmp DNA stool mini kit (Qiagen, GmbH Hilden, Germany) modified to contain a bead-beating step. Successful DNA isolation was confirmed by agarose gel electrophoresis. PCR primers flanking the V3-V4 hyper variable region of bacterial 16S rDNA were designed. The barcoded fusion forward primer was 341F(5′-CCTAYGGGRBGCASCAG-3′), and the reverse primer was 806R(5′-GGACTACNNGGGTATCTAAT-3′). The optimized conditions for amplification were as follows: one pre-denaturation cycle at 95 °C for 5 min, 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s, and a final extension at 72 °C for 10 min. The resulting amplicons were gel purified, quantified, pooled, and sequenced on the Illumina HiSeq 2500 platform. Microbiota sequences were processed through QIIME 1.8 (QIIME Team). After quality filtering, the sequences were denoised using denoise_wrapper.py. The denoised sequences were clustered into operational taxonomic units (OTUs) at a 97% sequence similarity against the GreenGenes OTU database (gg_13_8_otus). The chimeric OTUs were removed using UCHIME v4.2. Representative sequences for each OTU were picked and aligned using QIIME 1.8. Taxon-dependent analysis was conducted using the Ribosomal Database Project (RDP) classifier. The OTUs were counted for each sample to express the richness of bacterial species with an identity cutoff of 97%. Alpha and beta diversity calculations and taxonomic community assessments were performed using QIIME 1.8 scripts.
2.8. Statistical Analysis
Statistical analyses were performed using SPSS 17.0 Software (SPSS Inc., Chicago, IL, USA). All pairwise comparisons for the in vivo and in vitro data were examined using an unpaired Student’s two-tailed t-test. Chi square was used to test differences in diarrhea incidence between the two groups. The level of significance was set at p < 0.05. The results were expressed as mean ± standard error of the mean (SEM). Principle component analysis (PCA) plots were used to visualize differences in bacterial community composition among samples. The PCA plots were produced based on a euclidean metric. Linear discriminant analysis effect size (LEfSe) analysis was used to identify the OTUs or taxa, which were responsible for the differences between the groups. An effect size threshold of two was used for the biomarkers discussed in this study. The metastats program from R-script was used to identify statistically different phylotypes among groups. Only taxa with average abundances greater than 10−3, p < 0.05 and low Q values (low risk of false discovery) were considered significant.
In the present study, we observed that the probiotic L. reuteri I5007 induced the expression of HDP in a porcine small intestinal epithelial cell line (IPEC-J2). In addition, we observed that oral administration of L. reuteri I5007 stimulated colonic HDP expression in neonatal piglets. Finally, we found that L. reuteri I5007 increased concentrations of butyric acid in neonatal piglets but did not affect the colonic bacterial community structure.
Probiotics have been shown to induce β-Defensin (hBD-2) in human cells, and different probiotic strains show different HDP-inducing activity [13
]. In pigs, L. salivarius
can induce pBD2 production in the digestive tract [14
]. L. reuteri
is one of dominant species in the gastrointestinal tracts of humans and animals and is currently used as a probiotic in pigs [16
]. Oral administration of L. reuteri
modulates ileum microbial composition, intestinal development, and immune status in pigs [22
]. However, there are no reports of the effects of L. reuteri
on the stimulation of HDP gene expression in IPEC-J2 cells and pigs.
Previous studies have indicated that pBD1, pBD2, pBD3, pBD114, pBD129, PG1-5, and pEP2C are expressed in IPEC-J2 cells [4
]. In this study, we found that L. reuteri
I5007 administration increased pBD2, pBD3, pBD114, pBD129, and PG1-5 gene expression in these same cells. The time-dependent experiments showed a similar pattern, as previously described by Wehkamp et al. [33
] and Schlee et al. [13
], with the maximum level of HDP being induced after 6 h of incubation. The dose-dependent experiment showed that L. reuteri
I5007 induced HDP production when the concentration of L. reuteri
I5007 reached 106
Previously, Schlee et al. [13
] used heat-killed bacteria, but we decided to research the effects of live strains. Since L. reuteri
I5007 is used as a probiotic, the results for live strains may be helpful for future in vivo studies. Wehkamp et al. [33
] also found a living form of E. coli
Nissle 1917, which showed a strong induction of hBD-2 after incubation with Caco-2 cells for 4.5 h. A previous study demonstrated that heat-killed bacteria induced hBD-2 [13
], but we found that only pBD3 expression was significantly increased with heat killed L. reuteri
in the present study. Compared with the live strain, the capacity of the heat-killed L. reuteri
I5007 to induce HDP was visibly diminished.
The suspension without bacteria did not induce HDP, which is consistent with the findings described by Wehkamp et al. [33
] for E. coli
Nissle 1917. In order to determine whether L. reuteri
I5007 induced HDP by cell-to-cell contact, a Transwell Insert System was used. Our results indicate that L. reuteri
I5007 without contact with IPEC-J2 cells also induced HDP, which suggests that a metabolite produced by L. reuteri
I507 may be playing a role.
Neonatal piglets have an immature immune system, are susceptible to infections, and often suffer from diarrhea and growth retardation if infected [4
]. Our previous studies showed that the optimum dosage (about 1010
CFU/day of L. reuteri
I5007) could improve performance and reduce diarrhea incidence in neonatal piglets [22
]. In this study, we obtained a similar result. We also found that the administration of L. reuteri
I5007 induced pBD2 in the jejunum and pBD2, pBD3, pBD114, and pBD129 in the colon. Similar results have been obtained for L. salivarius
induced expression of pBD2 in the pig jejunum [14
]. The spatial heterogeneity patterns of this induction effect were probably due to the production of butyric acid, mainly triggered in the hindgut after L. reuteri
I5007 administration [34
]. It has been reported that most of the porcine HDP (e.g., pBD1, pBD2, pBD114, pBD129, PG1-5, pEP2C) show various activity against Gram-negative and Gram-positive bacteria, including Salmonella typhimurium
, Escherichia coli
, and Clostridium perfringen
, the blooms of which are involved in the occurrence of diarrhea [35
]. In addition to its antimicrobial properties, pBD3 could also regulate the expression of IL-8 and intestinal tight junction protein and exhibits a strong immunoregulatory ability [37
]. In addition, β-defensins (hBD2) have been observed to have the capacity to recruit leukocytes. These actions can directly modify the inflammatory response [38
]. The induction of the expression of HDP genes allows the immature intestinal epithelial surfaces of neonatal piglets to cope with these continuously complex microbial challenges [38
]. Furthermore, previous studies showed that the supplementation of synthetic HDP could improve nutrient digestibility, intestinal morphology, and growth performance in weanling pigs [39
] and broiler chickens [40
]. These results suggest that the induction of HDP gene expression by L. reuteri
I5007 may be responsible for the body weight increase and decrease in diarrhea incidence, which was mostly caused by the bloom of pathogens that can be eliminated by the HDPs [35
Short chain fatty acids are the major metabolites of microbial digestion in the colon and have been considered as contributing an important role in normal colonic morphology and function [34
]. Recent published data have proven that HDP could be induced by SCFA, especially by butyrate [4
]. Our previous work and that of others indicated that administration of probiotics increased butyrate levels in the colonic digesta and fecal samples [22
]. In this experiment, after the administration of L. reuteri
I5007, the quantity of butyric acid in the colonic digesta was increased, which is consistent with the finding of Liu et al. [22
] and may be the mechanism through which L. reuteri
I5007 induces the expression of HDP.
Exposure to SCFA, such as butyrate, triggers profound changes in epithelial gene expression in vitro [43
], which are mediated at least in part through the SCFA sensor PPAR-γ [45
]. GPR41 (Free Fatty Acid Receptor 3, FFA3) and GPR43 (FFA2) are related G Protein-Coupled Receptors that are activated by short chain carboxylic acids [46
]. In order to further confirm the changes of SCFA, we determined the changes of short-chain fatty acid receptors in the colon. PPAR-γ and GPR41 were observed to be higher in mRNA expression after L. reuteri
I5007 administration compared with piglets in the control treatment.
Changes in the structure of gut microbiota alter the gut-microbial metabolism and eventually influence intestinal mucosal immunity and host metabolism [48
]. Previous studies have reported increases of lactic acid bacteria after the intake of individual lactobacilli strains [50
]. The administration of Lactobacillus rhamnosus
GG increases the fecal butyrate level through expanding butyrate-producing bacterial strains [54
]. To examine whether the significant difference in butyrate concentration of colonic digesta was induced by the change of microbiota structure after L. reuteri
I5007 administration, the colonic microbial community was determined. Unexpectedly, the results of the next generation high throughput sequencing showed no significant changes in the colonic microbiota composition or stability, which was consistent with our previous finding using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) profiling [22
]. Similar observations have been reported in a recent review [55
], suggesting that probiotics do not significantly modify the gut microbiota composition of healthy subjects. Additionally, microbiomes were compared at different taxonomic levels, and no differences were detected except the specific increase of genera Sharpea
, which is a member of Clostridium XVII
and exhibits a close phylogenetic association with Lactobacillus catenaformis
and Lactobacillus vitulinus
]. However, no significant correlation was found between the butyrate level and genera Sharpea
, which indicated that the increase of butyrate levels was not due to the alteration of genera Sharpea
. It has recently been reported that the introduction of probiotics significantly changes the microbiome’s transcriptional profile but has no significant impact on the structure, leading us to speculate that L. reuteri
I5007 may increase the butyrate concentration through modulating the microbial metabolic activities. Taken together, these results suggest that L. reuteri
I5007 might increase butyrate levels through modulating the microbiota at the transcript level, rather than modifying the bacterial community structure in the colonic digesta [57
]. PRRs could recognize conserved molecular motifs present on a wide range of different microbes, which have been termed Microbe-Associated Molecular Patterns (MAMPs). TLRs and NOD proteins are two classes of PRRs involved in innate immune detection [59
]. Agreeing with the result of colonic bacterial community analysis, the mRNA levels for PRRs were unaffected by L. reuteri
I5007 treatment, which indicated that L. reuteri
I5007 induction of HDP expression was not induced through regulating the structure of the colonic bacterial community or expression of PRRs [57