The establishment of drug-free feeding systems has been required for secure and healthy livestock production. Although functional feed materials containing microorganisms as alternatives to enhance intestinal immunity are expected to be beneficial for reducing diarrhoea caused by pathogens in weaned piglets, the effects of such materials on porcine intestinal cells have not been investigated in detail. Therefore, this work evaluated the immunoregulatory functions of microbial feed materials in porcine intestinal immune and epithelial cells. Porcine immune cells isolated from Peyer’s patches and mesenteric lymph nodes were stimulated with six different feed materials containing microorganisms, and evaluated for lymphocyte mitogenicity and cytokine inductions. In addition, porcine intestinal epithelial cells were stimulated with the materials before treatment with heat-killed enterotoxigenic Escherichia coli (ETEC), and analyzed for the proinflammatory cytokine expressions. The material containing Bifidobacterium thermophilum significantly augmented lymphocytes’ mitogenicity and also induced a high expression of IL-2, IL-6 and IFN-γ in immune cells, and inhibited ETEC-induced overexpression of IL-6 and IL-8 via regulation of Toll-like receptor signaling. These results suggest that this feed material stimulates intestinal epithelial and immune cells to exert immunoregulation, suggesting that this feed is expected to contribute to promoting the health of piglets without using antimicrobial feed materials.
The establishment of drug-free feeding systems has been required for secure and healthy livestock production. Although functional feed materials containing microorganisms as alternatives to enhance intestinal immunity are expected to be beneficial for reducing diarrhoea caused by pathogens in weaned piglets, the effects of such materials on porcine intestinal cells have not been investigated in detail. Therefore, this work evaluated the immunoregulatory functions of microbial feed materials in porcine intestinal immune and epithelial cells. Porcine immune cells isolated from Peyer’s patches and mesenteric lymph nodes were stimulated with six different feed materials containing microorganisms, and evaluated for lymphocyte mitogenicity and cytokine inductions. In addition, porcine intestinal epithelial cells were stimulated with the materials before treatment with heat-killed enterotoxigenic Escherichia coli (ETEC), and analyzed for the proinflammatory cytokine expressions. The material containing Bifidobacterium thermophilum significantly augmented lymphocytes’ mitogenicity and also induced a high expression of IL-2, IL-6 and IFN-γ in immune cells, and inhibited ETEC-induced overexpression of IL-6 and IL-8 via regulation of Toll-like receptor signaling. These results suggest that this feed material stimulates intestinal epithelial and immune cells to exert immunoregulation, suggesting that this feed is expected to contribute to promoting the health of piglets without using antimicrobial feed materials.
Keywords:Feed Microbial Material; Immunoregulatory Effect; Porcine Intestinal Epitheliocytes; Porcine Immune Cells
Weaning-associated intestinal inflammation occurs in various animal species including the pig. Following the withdrawal of sow’s milk, young piglets are highly susceptible to enteric diseases partly as a result of the altered balance between developing beneficial microbiota and the establishment of intestinal bacterial pathogens. In addition to the changes in microbiota composition, the intestinal immune system of the newborn piglet undergoes a rapid period of maturation, expansion, and specialization that is not achieved before commercial weaning [
Various nutritional approaches for optimizing the weaning transition and minimizing gut inflammation and enteric diseases have been tested in the past decade. Among the novel dietary strategies investigated that are focused on improving gut health in pigs, probiotics and prebiotics are clear nutritional options. A growing body of evidence supports the therapeutic and preventive application of probiotics for several gastrointestinal disorders in pigs. Qiao et al. [
In addition, some recent studies have specifically evaluated the capacity of probiotics to improve the resistance of piglets against enterotoxigenic Escherichia coli (ETEC). It was shown that the probiotic strain L. plantarum CJLP243 may serve as a potential alternative to antibiotic supplementation to improve the growth and health performance of weaning pigs because of its capacity to reduce the severity of ETEC-induced diarrhoea [
Although these studies demonstrated that it is possible to modulate piglets’ gut microbiota and immunity resulting in improvement of growth performance by using appropriate probiotics strains, the true efficacy of probiotics in livestock animals remains unclear because of incomplete studies of mechanisms. In this regard, some feed microbial materials (FMAs) containing Aspergilli, Lactobacilli and Bifidobacteria have been used to successfully improve growth performance in livestock in Japan [
Our laboratory has shown that probiotic microorganism with immunoregulatory functions (immunobiotics) can beneficially modulate the immune response in the gut by modulating the functions of intestinal epithelial cells (IECs) and immune cells [
The following feed microbial materials (FMAs) used as livestock feed in Japan were evaluated in this study. FMA1: Grinded freeze dry soymeal fermented with Enterococcus faecium and Aspergillus oryzae. FMA2: Freeze dry of Candida utilis cultures. FMA3: Freeze dry of Saccharomyces cerevisiae cultures. FMA4: Freeze dry of Enterococcus faecalis cultures. FMA5: Freeze dry of Bifidobacterium thermophilum cultures. FMA6: Freeze dry of Leuconostoc mesenteroides cultures.
Enterotoxigenic Echerichia coli (ETEC) strain 987P was kindly provided by the National Institute of Animal Health (Tsukuba, Japan). ETEC cells were plated into tryptic soy agar (TSA, Becton, Dickinson and Company) supplemented with 5% sheep blood (Nippon Biotest Laboratories Inc., Tokyo, Japan). After overnight incubation at 37˚C, ETEC was transferred to tryptic soy broth (TSB, Becton) and grown for 5 days without shaking to form a pellicle containing the piliated phase. Then, ETEC cells were collected from the pellicle and transferred to TSB and cultured 20 hours at 37˚C with shaking (200 rpm). Finally, the subculture of the ETEC strain was centrifuged at 1900 × g for 10 min at 4˚C, washed with phosphate buffered saline (PBS) and heat-killed at 65˚C for 30 min, and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen corporation, Carlsbad, CA) as described previously [
Porcine intestinal epitheliocyte (PIE) cells were obtained as described before [
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Guidelines for Animal Experimentation of Tohoku University, Sendai, Japan. The present study was approved by the Institution Animal Care and Use Committee of Tohoku University with a permitted No. 2011-noudou-5. All efforts were made to minimize suffering. Immunocompetent cells from Swine peyer’s patches (PPs) and mesenteric lymph nodes (MLNs) were prepared from adult swine intestine as described previously [
The mitogenicity assay was performed as described previously [
[
Evaluation of the immunoregulatory activity of FMAs was performed using mononuclear cells from PPs and MLNs or PIE cells. PP and MLM cells ware plated at a density of 4.0 × 106 cells/well in 12-well type I collagencoated plates (Iwaki, Tokyo, Japan). PIE cells were plated at a density of 3.0 × 104 cells/well in 12-well type I collagen-coated plates (Iwaki) and cultured for 3 days before stimulation of FAMs. FMAs were added into each well and cells were incubated for 48 h. Expression of IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13 and MCP-1 was evaluated with quantitative real time RT-PCR as described below. In addition, expression levels of six TLR negative regulators, A20, Bcl-3, IRAK-M, MKP-1, SIGIRR, and Tollip, were evaluated in PIE cells as described previously [
PIE cells were seeded at 3 × 104 cells/12-well plate on type I collagen-coated plates (Iwaki) and cultured for 3 days. After changing medium, FMAs were added; 48 h later, each well was washed vigorously with medium at least three times to eliminate all stimulants, and then cells were stimulated with ETEC (equivalent to 5 × 107 cells/ml) for 12 h. Expression of cytokines was evaluated with quantitative real time RT-PCR as described below. In blocking experiments, unlabeled anti-porcine TLR2- or TLR4-rabbit IgG (Biolegend, San Diego, CA) were used. Cultured porcine cells were incubated with unlabeled anti-TLR2 of TLR4 antibodies for 12 h before stimulation with FMAs.
We performed two-step real-time quantitative PCR to characterize the expression of mRNAs in PIE cells and immune cells. Total RNA was isolated from each PIE or immune cell sample using TRIzol reagent (Invitrogen). All cDNAs were synthesized using a Quantitect reverse transcription (RT) kit (Qiagen, Tokyo, Japan) according to the manufacturer’s recommendations. Real-time quantitative PCR was carried out using a 7300 real-time PCR system (Applied Biosystems, Warrington, UK) and the Platinum SYBR green qPCR SuperMix uracil-DNA glycosylase with 6-carboxyl-X-rhodamine (Invitrogen). The primers for IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10, IL- 13, MCP-1, A20, SIGIRR, Tollip, Bcl-3, MKP-1, and IRAK-M used in this study were previously described [
PIE cells were resuspended in 200 μl CelLytic M cell lysis reagent (Sigma) including protease and phosphate inhibitors (Complete Mini, PhosSTOP; Roche, Mannheim, Germany). Cells were transferred into Eppendorf tubes and boiled for 5 min at 95˚C. Protein concentration was measured using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Total protein samples (2 μg/sample) were loaded onto 10% SDS-polyacrylamide gels. Separated proteins were electrophoretically transferred to a nitrocellulose membrane. Phosphorylation of p38, and IκBα degradation were evaluated using anti-phosphorylated p38, and anti-IκB antibodies, respectively (Cell Signaling Technology, Beverly, MA). After detecting the phosphorylation, the membranes were stripped with Ten Minute Western Blot Re-Probe Kit (Jacksun Easy Biotech, Inc., New York, USA) for the detection of each total protein using p38 antibody (Cell Signaling Technology) and -actin antibody (Cell Signaling Technology). Anti-rabbit IgG, AP-linked antibody (Cell Signaling Technology) was used as secondary antibody. The optical protein bands were detected by ECF substrate (GE Healthcare Japan Co., Tokyo, Japan) and estimated from the peak area of densitogram by using Image J software (National Institute of Health, Bethesda, MD, USA).
Statistical analyses were performed using the GLM and REG procedures available in the SAS computer program (SAS, 1994). Comparisons between mean values were carried out using one-way analysis of variance and Fisher’s least-significant-difference test. For these analyses, P values of 0.05 were considered significant.
We first evaluated the mitogenic capacity of the feed microbial materials (FMAs) in porcine intestinal immune isolated from Peyer’s patches (PPs) or MLN (
All the FMAs were able to significantly increase the mitogenic activity of PPs and MLN cells compared with the control group, in a dose dependent manner. However, FMA5 was the most effective treatment to increase this parameter in MLN cells, being the doses of 100 μg/ml and 1000 μg/ml able to increase mitogenic activity more than 10 folds (
We also evaluated the changes in cytokine expression in immune isolated from PPs or MLN after the treatments with the FMAs. As shown in
The immunoregulatory capacities of FMAs were then evaluated in PIE cells. First, PIE cell were stimulated with the different FMAs and the expression of IL-6, IL-8 and MCP-1 was evaluated (
FMA5 significantly increased the expression of IL-6 in PIE cells while FMA6 upregulated the expression of MCP-1 (
In addition, the effect of FMAs in the response of PIE cell to heat-killed ETEC challenge was evaluated. As shown in
We next evaluated whether FMA5 attenuated the ETEC-mediated proinflammatory response by modulating the NF-κB pathway (
tion and the regulation of proinflammatory cytokines in PIE cells by FMA5 (
We also studied regulators that inhibit the TLR signaling pathway. The expression of SIGIRR, Tollip, A20, Bcl-
3, MKP-1, and IRAK-M mRNAs in PIE cells was determined after the challenge with heat-killed ETEC (
To study the role of TLR2 and TLR4 in the immunomodulatory effect of FMA5, we next performed comparative studies with FMA5 and FMA1 and used anti-TLR2 and anti-TLR4 blocking antibodies, in PIE cells challenged with heat-killed ETEC (
The weaning transition is a complex period during which the piglets have to face abrupt changes. They are separated from their mother, mixed with other litters in a new environment, and switched from milk to a solid feed which involves a change from a highly digestible to a less-digestible and more-complex feed. In consequence, several physiological changes occur in the intestine of pigs during weaning which increase the susceptibility to uncontrolled inflammation and infections [reviewed in 17]. Probiotic microorganisms have been proposed to functionally modulate the microbiota and the immune system during the weaning transition in the pig, in order to avoid gut alterations and increase pig productivity. In Japan, several FMAs are used as expected alternatives to antibiotics to improve pig health. However, the exact cellular and molecular mechanisms involved in the beneficial effects obtained in pigs have not been studied in detail. In this work we evaluated the immunoregulatory capacities of several FMAs using porcine in vitro systems, and we found that the freeze dry of B. thermo-
philum cultures (called FMA5 in this work) was notably able to modulate porcine immune cells as well as PIE cells.
Mitogenic activities are generally considered to be one of the most important properties of immune modulators, and have been used to evaluate the immunomodulating activities of potential probiotic microorganisms. Here, we used this methodology to evaluate the immunoregulatory effect of different FMAs in porcine immune cells. Results showed that FMA5 significantly increased mitogenic activity of both PPs and MLN cells. Moreover, FMA5 was able to increase the expression of IL-2, IL-6, IL-10 and IFN-γ in porcine immune cells indicating a clear immunostimulatory. In fact, early studies from Sasaki et al. [
improve both resistance against infection and protection from inflammatory tissue damage [
In addition, we showed that FMA5 is able to functionally modulate PIE cells. Previously, we demonstrated that the stimulation of PIE cells with ETEC significantly increases the levels of IL-6, IL-8, and MCP-1 [
To dissect the mechanism(s) involved in the immunoregulatory effect of FMA5 we evaluated the expression of the negative TLR regulators in PIE cells. The expression of SIGIRR, Tollip, A20, Bcl-3, MKP-1, and IRAKM was studied, and it was found that SIGIRR, Tollip, A20 and IRAK-M expression was upregulated in PIE cells stimulated with FMA5.
It was shown in vitro that overexpression of SIGIRR inhibits TLR-induced NF-κB activation and attenuates the production of inflammatory cytokines [
innate immunity through a negative feedback loop [
We also demonstrated previously that L. jensenii TL2937, B. longum BB536 and B. breve M-16V significantly upregulated A20 in PIE cells in a TLR2-dependent manner [
In addition, TLR4 seems to have an important role in the immunoregulatory effect of FMA5 since blocking experiments with anti-TLR4 antibodies inhibit the reduction of IL-6 and IL-8 induced by FMA5. It was demonstrated that IECs shortly after birth exhibit a transient transcriptional activation induced by exposure to environmental endotoxin since it is almost completely abolished in TLR4-deficient mice [
The scientific research into probiotic mode of actions has come to age and has shown how immunobiotics are able to induce beneficial changes in the host. The study of immunobiotics—intestinal cell interactions has unraveled several molecular mechanisms and cellular pathways, showing that these interactions play a crucial role in the regulation of several immunological functions in the gut. Our present research work allows us to give a view of the cellular and molecular mechanisms involved in the immunoregulatory effects of Bifidobacterium thermophilum. These results improve the scientific basis to use this feed material to modulate intestinal epithelial and immune cells and exert beneficial effects in pigs. This feed is expected to contribute to the healthy growth of young pigs without using antimicrobial feed materials.
This study was supported by a Grant-in-Aid for Scientific Research (B) (2) (No.21380164, 24380146) and Challenging Exploratory Research (No. 23658216) from the Japan Society for the Promotion of Science (JSPS) to Dr. H. Kitazawa. Dr. Paulraj Kanmani was supported by JSPS (Postdoctoral Fellowship for Foreign Researchers, Program No. 25-03397).