Dietary Bacillus subtilis-based direct-fed microbials alleviate LPS-induced intestinal immunological stress and improve intestinal barrier gene expression in commercial broiler chickens

Accepted Manuscript Dietary Bacillus subtilis-based direct-fed microbials alleviate LPS-induced intestinal immunological stress and improve intestinal barrier gene expression in commercial broiler chickens

Ujvala Deepthi Gadde, Sungtaek Oh, Youngsub Lee, Ellen Davis, Noah Zimmerman, Tom Rehberger, Hyun Soon Lillehoj PII: DOI: Reference:

S0034-5288(17)30360-0 doi: 10.1016/j.rvsc.2017.05.004 YRVSC 3320

To appear in:

Research in Veterinary Science

Received date: Revised date: Accepted date:

29 March 2017 1 May 2017 5 May 2017

Please cite this article as: Ujvala Deepthi Gadde, Sungtaek Oh, Youngsub Lee, Ellen Davis, Noah Zimmerman, Tom Rehberger, Hyun Soon Lillehoj , Dietary Bacillus subtilisbased direct-fed microbials alleviate LPS-induced intestinal immunological stress and improve intestinal barrier gene expression in commercial broiler chickens, Research in Veterinary Science (2017), doi: 10.1016/j.rvsc.2017.05.004

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ACCEPTED MANUSCRIPT Dietary Bacillus subtilis-based direct-fed microbials alleviate LPS-induced intestinal immunological stress and improve intestinal barrier gene expression in commercial broiler chickens

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Ujvala Deepthi Gadde 1,*, Sungtaek Oh1,*, Youngsub Lee1, Ellen Davis2, Noah Zimmerman2,

Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center,

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Tom Rehberger2 and Hyun Soon Lillehoj1,†

Agro Biosciences Inc., 10437 Innovation Drive, Wauwatosa, WI 53226, USA

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Agricultural Research Service, USDA, Beltsville, MD 20705, USA

Corresponding Author: Hyun S. Lillehoj

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Mailing Address: Animal Biosciences and Biotechnology Laboratory

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Beltsville Agricultural Research Center 10300 Baltimore Ave, Bldg. 1043, Beltsville, MD 20705

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Phone: 301-504-8771 Fax: 301-504-5103

Email: [email protected] *

Oak Ridge Institute for Science and Education (ORISE) Research Fellow at the Animal

Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, Beltsville, MD 20705

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ACCEPTED MANUSCRIPT Abstract

This study investigated the effects of Bacillus subtilis-based probiotics on the performance, modulation of host inflammatory responses and intestinal barrier gene expression of broilers

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subjected to LPS challenge. Chickens were randomly allocated to one of the 3 dietary treatment groups - control, antibiotic, or probiotic. At 14 days, half of the chickens in each treatment were

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injected with LPS (1 mg/kg body weight), and the other half injected with sterile PBS. Chickens

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fed probiotics weighed significantly more than controls at 15 days of age, irrespective of immune challenge. LPS challenge significantly reduced weight gain at 24 hr post-injection, and the

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probiotics did not alleviate the LPS-induced reduction of weight gain. Serum α-1-AGP levels

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were significantly higher in LPS-injected chickens, and probiotic supplementation significantly reduced their levels. The percentages of CD4+ lymphocytes were significantly increased in

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probiotic groups in the absence of immunological challenge but were reduced during LPS

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challenge compared to controls. CD8+ lymphocytes were significantly reduced in probiotic-fed birds. The LPS-induced increase in the expression of cytokines IL8 and TNFSF15 was reduced

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by probiotic supplementation, and IL17F, iNOS expression was found to be significantly

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elevated in probiotic-fed birds subjected to LPS challenge. The reduced gene expression of tight junction proteins (JAM2, occludin and ZO1) and MUC2 induced by LPS challenge was reversed by probiotic supplementation. The results indicate that B. subtilis-based probiotics differentially regulate intestinal immune and tight junction protein mRNA expression during states of LPSmediated immunological challenge.

Keywords: chicken, probiotic, B. subtilis, cytokine, tight junction, LPS 2

ACCEPTED MANUSCRIPT 1. Introduction

With an increase in concerns regarding the development of antibiotic resistance and efforts to promote the judicious use of antimicrobials in food-producing animals, there is a timely need for

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the development of viable alternatives that can ensure and maintain optimal animal health and performance. Direct-fed microbials (DFMs), often referred to as probiotics, are one such

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potential non-antibiotic replacement that has been studied extensively and used in commercial

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applications. DFM comprise either mono or mixed cultures of beneficial organisms known to exert positive effects on health when administered in appropriate quantities (Huyghebaert et al.,

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2011; Shivaramaiah et al., 2011). A variety of bacterial species (Bacillus, Bifidobacterium,

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Enterococcus, Lactobacillus, Streptococcus, Lactococcus, Pediococcus), yeast (Saccharomyces), and in some cases undefined cultures have been tested as probiotics in poultry (Griggs and Jacob,

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2005; Patterson and Burkholder, 2003). In particular, Bacillus sp. have been shown to improve

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performance, positively modulate intestinal microflora, inhibit pathogen colonization, improve nutrient digestibility, and enhance immune activities in the gut of broiler chickens (La Ragione

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2011).

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and Woodward, 2003; Lee et al., 2010; Park and Kim, 2014; Sen et al., 2012; Wolfenden et al.,

Bacillus-based probiotics modulate various parameters of inflammation, humoral and cellular immunity and augment macrophage and heterophil function in chickens (Farnell et al., 2006; Lee et al., 2010, 2011, 2013, 2014; Rajput et al., 2014). Several studies in mice and pigs have shown that probiotics alter systemic and intestinal immunological pathways during the acute phase response and stabilize the intestinal epithelium by increasing the expression of tight junction (TJ) 3

ACCEPTED MANUSCRIPT proteins (Bai et al., 2004; Thomas and Versalovic, 2010; Yang et al., 2015). Very few studies have been conducted in chickens to investigate the role of probiotics in regulating immune responses during the acute phase response and the maintenance of intestinal integrity. Thus, the present study was conducted with the objective of investigating the modulation of the host

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inflammatory response and changes in intestinal TJ gene expression induced by LPS challenge

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upon the supplementation of diets with probiotics in comparison to non-supplemented controls or

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those supplemented with antibiotics. Specific parameters monitored to judge the effect of probiotics included performance, specific T-cell subsets in blood, and the expression of various

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cytokines, mucin (MUC) and TJ genes in the intestines of broilers exposed to LPS challenge.

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2. Materials and methods

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Probiotic strains

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DFM strains were identified by genetic isolation and 16S ribosomal RNA sequence identification. DFM 16S rRNA sequences were compared to existing bacterial type strain 16S

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rRNA sequences using Bionumerics version 7.1. The relevant strains were shown to be members of the B. subtilis group according to full-length 16s rDNA sequence comparisons. DFM strains

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were screened for diarrheal toxin using the Tecra™ Bacillus cereus Diarrhoeal Enterotoxin Visual Immunoassay kit (3M, Maplewood, MN) and demonstrated no toxin production. Antibiotic resistance was assessed by analysis of full length genome sequencing which demonstrated that there are no transferrable antibiotic resistance genes present in the genomes of any of the Bacillus DFM used in this study.

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ACCEPTED MANUSCRIPT Birds and husbandry

Day (d)-old commercial broiler chickens (Ross/Ross, Longenecker’s Hatchery, Elizabethtown, PA) were obtained, weighed and housed in electrically heated battery brooders (Petersime

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Incubator Company, Gettysburg, OH). Chickens were raised in starter cages until 14 days of age

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and then transferred to finisher cages where they were kept until the end of the experimental

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period. Feed and water were provided ad libitum. Husbandry followed guidelines for the care and use of animals in agricultural research (NRC, 2010). All experimental protocols were

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Experimental design and LPS challenge

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approved by the Small Animal Care Committee of the Beltsville Agricultural Research Center.

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Birds at d 0 of age (n=90) were randomly allocated to one of the three dietary treatments (30 birds/treatment) - non-supplemented (control), antibiotic-supplemented (antibiotic), and

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probiotic-supplemented (probiotic)]. The basal diets were corn and soybean meal-based and were

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formulated to meet or exceed the National Research Council’s nutrient requirements for broiler chickens (NRC, 1994). The basal diets (without any antibiotics or anticoccidial drugs) were

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supplemented with either Bacitracin Methylene Disalicylate (Zoetis, Inc.; 50 g/ton inclusion rate), probiotics (Bacillus subtilis strain 1781 @ 1.5x105 CFU/g of feed) or none (controls). Birds were fed with the experimental diets throughout the course of the study. At 14 d of age, chickens in each dietary treatment were randomly assigned to finisher cages (6 cages; 5 chickens/cage) and subjected to LPS challenge. One-half of the chickens in each dietary treatment (3 cages; total 15 chickens) were injected intraperitoneally with Escherichia coli O55:B5 LPS (Sigma, St.

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ACCEPTED MANUSCRIPT Louis, MO) at 1 mg/kg body weight, and the other half were injected with sterile PBS. Body weight was measured on d14 and 15 (0, 24 hr post-LPS injection, respectively), and the weight gain was calculated. Body weight was also measured on d21 and d28 of age.

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Sample collection

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At 24 hr post-LPS injection, three chickens were randomly selected from each of the 6 treatment groups and used for the collection of blood and small intestine samples. Blood samples (3/trt)

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with or without anticoagulants were collected by cardiac puncture immediately following

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euthanasia and used for the isolation of peripheral blood mononuclear cells (PBMC) and serum separation, respectively. Intestinal (ileum) sections were collected and stored in RNA

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preservation buffer (RNAlater®, Applied Biosystems, Foster City, CA) at −20°C until further use

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for the isolation of RNA and gene expression analysis.

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Serum alpha-1 acid glycoprotein ELISA

Chicken α-1-Acid Glycoprotein (α-1-AGP) levels in the serum were measured by ELISA (Life

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Diagnostics Inc., West Chester, PA) according to the manufacturer’s instructions. Briefly, diluted serum samples were added to microtiter wells with immobilized anti-chicken α-1-AGP antibodies and incubated for 45 minutes. Following incubation, the plates were washed and treated with HRP-conjugated anti-chicken α-1-AGP antibodies and incubated for 45 minutes, which was followed by color development with substrate. The OD450 values were determined with an automated microplate reader (Bio-Rad Laboratories Inc., Hercules, CA). OD values were

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ACCEPTED MANUSCRIPT converted to concentrations (ng/mL) based on a standard curve generated using known quantities of recombinant α-1-AGP standard protein.

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PBMC subpopulations-Flow Cytometry

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PBMC were isolated from whole blood samples as described previously (Gadde et al., 2009).

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Briefly, the blood samples were diluted with HBSS (Sigma Chemical Company, St. Louis, MO) at a 1:1 ratio. The diluted blood was then layered on Histopaque®-1077 (Sigma) and centrifuged

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at 400 × g for 30 min at room temperature. The PBMC interface between the Histopaque and the

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plasma layers was collected and washed 2 times with HBSS at 250 × g for 10 min at 4°C. The CD4+ and CD8+ defined lymphocyte subsets in the PBMC suspensions were identified using

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direct fluorescence staining followed by flow cytometric cell population analysis. The antibodies

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employed were mouse anti-chicken CD45-fluorescein isothiocyanate (FITC) (clone LT-40), mouse anti-chicken CD4- phycoerythrin (PE) (clone CT-4) and mouse anti-chicken CD8-FITC

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(clone CT-8) (Southern Biotechnology Associates Inc., Birmingham, AL). Staining controls

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included the incubation of cells with FITC- and PE-labeled isotype control (mouse IgG1 with irrelevant specificity; Sigma) to test for the nonspecific binding of the specific antibodies (mouse

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IgG1) and to set the cut-off between fluorescence-positive versus fluorescence-negative PBMC for FL-1 (FITC) and FL-2 (PE), respectively. The percentages of live CD4+ and CD8+ cells in the PBMC populations were determined by flow cytometry using FACSCalibur instrument (BD Biosciences, San Jose, CA, USA). Data were obtained from a total of 1.0 x 104 viable cells (n=3) and cell population analysis was conducted using Cell-Quest software (BD Biosciences). The percentages of CD4+ and CD8+ lymphocytes in the live, small mononuclear cell population,

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ACCEPTED MANUSCRIPT determined by flow cytometry, were expressed as the percentage of CD45 (pan leukocyte marker)-positive cells for each sample.

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Isolation of RNA, reverse transcription, and Real-Time PCR

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Total RNA was extracted from ileal samples (3/trt) using TRIzol (Invitrogen, Carlsbad, CA).

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One microgram of RNA was reverse transcribed to cDNA using the QuantiTect® reverse transcription kit (Qiagen Inc., Valencia, CA) according to the instructions of the manufacturer.

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The cDNA samples were divided into aliquots and stored at −20°C. Quantitative RT-PCR

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oligonucleotide primers for chicken pro-inflammatory cytokines, including interleukin (IL)-8, tumor necrosis factor (TNF) superfamily 15 (TNFSF), lipopolysaccharide-induced TNFα-factor

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(LITAF) and inducible nitric oxide synthase (iNOS), and intestinal TJ proteins, including

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junctional adhesion molecule (JAM)2, occludin, zona occludens (ZO)1, intestinal MUC2 protein, and glyceraldehyde 3 phosphate dehydrogenase (GAPDH) as internal controls, are listed in

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Table 1. The primer sequences of TJ proteins and MUC2 were adapted from Chen et al., 2015.

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The differential expression of the mRNA transcripts of various cytokines (IL8, IL17F, TNFSF15, LITAF), iNOS, intestinal barrier proteins (JAM2, occludin, ZO1), and MUC2 in the

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ileal cDNA samples was assessed by real-time PCR. Amplification and detection were carried out using the Stratagene Mx3000P system (Agilent Technologies Inc., Santa Clara, CA) and RT2 SYBR Green qPCR master mix (Qiagen). All the samples were analyzed in triplicate, and negative controls were included to check for the nonspecific amplification of primers. Standard curves were generated using log10 diluted RNA, and the levels of individual transcripts were normalized to those of GAPDH using the Q-gene program (Muller et al., 2002). To normalize

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ACCEPTED MANUSCRIPT RNA levels between samples within an experiment, the logarithmic-scaled threshold cycle (Ct) values were transformed to linear units of normalized expression prior to calculating the means and SEM for the references and individual targets, followed by the determination of mean

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normalized expression (MNE) using the Q-gene program.

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Statistical analysis

Statistical analysis was carried out using SAS software (SAS, 2014). The data were analyzed by

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two-way ANOVA to examine the interaction of main effects (diet and immune challenge). When

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there was a significant interaction (P < 0.05), the main effects were ignored, and the group means within each diet or immune challenge treatment were compared by Duncan’s multiple

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range test. In the absence of interaction, significant main effects (P < 0.05) were separated by

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Duncan’s multiple range test. All the data were expressed as the mean ± SEM for each treatment.

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Body weight

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3. Results

At 24 hr post-LPS injection (d15 of age), there was a significant effect of diet on body weight and the results are shown in Figure 1. The chickens fed diets supplemented with probiotic or antibiotic weighed more than those that were fed non-supplemented diets. There was no significant effect of LPS challenge on body weight at 24 hr post-challenge, and no significant diet x immune challenge interactions were observed. However, LPS challenge showed a 9

ACCEPTED MANUSCRIPT significant effect (P < 0.0001) on body weight gain at 24 hr post-LPS injection and the results are presented in Figure 2. The chickens challenged with LPS, irrespective of the diet, had reduced body weight gain compared to the chickens injected with PBS. The effects of diet and diet x immune challenge interaction on body weight gain were not significant. No significant

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differences were observed in the body weight or weight gain at d21 and d28 of age (data not

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shown).

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Serum α-1-AGP levels

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There was a significant diet x immune challenge interaction effect on the serum concentrations of α-1-AGP at 24 hr post-LPS injection. Due to this interaction, the levels were compared

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between various dietary treatments within each immunological challenge group. In the chickens

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that were given PBS injection, there were no significant differences in the serum levels of α-1AGP among the birds fed different diets. LPS injection significantly increased serum α-1-AGP

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concentration (significant main effect of LPS, P < 0.0001), and within the LPS-injected groups,

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chickens fed diets with probiotic had reduced serum α-1-AGP levels compared to control or

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antibiotic-fed chickens (Figure 3).

CD4+ and CD8+ lymphocyte percentage changes in PBMC

At 24 hr post-LPS challenge, the analysis of CD4+ lymphocyte percentages in PBMC showed significant diet x immune challenge interaction. In chickens that were not subjected to immune challenge, the CD4+ lymphocyte % in PBMC was higher in the probiotic group than in the

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ACCEPTED MANUSCRIPT antibiotic group or controls (Figure 4). LPS challenge (main effect) reduced CD4+ lymphocyte percentage irrespective of diet, and within the LPS-challenged chickens, probiotic-supplemented chickens had reduced percentages compared to chickens fed control diets. For CD8+ lymphocyte percentages in PBMC, the main effects of diet (P = 0.0014) and immune challenge (P < 0.0001)

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were significant, and there was no significant interaction. As analyzed by diet, the chickens fed

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diets with probiotics had a significantly lower % of CD8+ lymphocytes compared to controls or

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antibiotic fed chickens (Figure 5). Irrespective of the diet, CD8+ lymphocyte percentages were significantly increased in LPS-injected chickens than PBS-injected chickens (P < 0.0001) (data

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not shown).

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Cytokine gene expression-Ileum

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For all the immune target genes tested for expression analysis, there was a significant diet x immunological challenge interaction except for LITAF expression, for which only the main

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effects were significant (Figure 6). Among the LPS-injected groups, IL8, TNFSF15 expression

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was significantly reduced in chickens fed probiotic or antibiotic compared with those that were given control diets. In the absence of immunological challenge, the expression of TNFSF15 was

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elevated in probiotic-fed chickens compared to chickens given antibiotic or control diets, whereas no differences were seen in IL8 expression among various dietary treatments. In the LPS-challenged chickens, the expression of iNOS and IL17F was significantly elevated in probiotic-fed groups compared to other treatments. In chickens not challenged with LPS and raised on different dietary treatments, no differences were seen in iNOS expression, but IL17F expression was significantly elevated in the antibiotic group compared to the control or probiotic

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ACCEPTED MANUSCRIPT group. For LITAF expression, because no interaction was observed, the main effect of diet and LPS challenge were used to separate the means. LITAF expression was increased in chickens raised on probiotic diets compared to those raised on antibiotic or non-supplemented diets.

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Intestinal TJ and mucin gene expression in the Ileum

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The results of gene expression analysis for TJ proteins and mucin in the ileum are shown in

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Figure 7. LPS reduced the expression of tight junction proteins in the intestine (significant main effect of LPS challenge). There was a significant diet x immunological challenge interaction for

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all the target genes tested. Among the chickens challenged with LPS, there was a significant increase in the expression of all TJ genes (JAM2, occludin and ZO1) and MUC2 in those fed

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diets with probiotic compared to those fed control diets. In the chickens injected with PBS,

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occludin expression was significantly elevated in the probiotic group compared to the other

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groups and no differences in JAM2, ZO1 expression were observed in probiotic groups compared to control chickens; however, their expression was downregulated compared to the

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antibiotic-fed group.

4. Discussion

This study was undertaken to investigate whether dietary B. subtilis-based DFM would alter the performance and inflammatory immune activities in the peripheral blood and intestine and influence intestinal barrier function during immunological stress induced by LPS injection. Our results demonstrated that chickens fed diets with B. subtilis-based probiotics show significantly 12

ACCEPTED MANUSCRIPT higher body weight at d15 of age compared to chickens fed non-supplemented diets. The beneficial effects of supplementing B. subtilis probiotics on body weight of chickens, seen in this study, are in agreement with previous reports. Shivaramaiah et al. (2011) showed that broiler chickens fed diets with two strains of B. subtilis showed increased body weight gain at 11 days

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of age. Aliakbarpour et al. (2012) demonstrated a similar increase in the body weight of chickens

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fed B. subtilis-based probiotics at 42 days of age compared to controls. An increase in average

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daily weight gain was also shown upon supplementation of diets with Bacillus sp. in broiler chickens (Bai et al., 2017; Nguyen et al., 2015; Sen et al., 2012). However, several reports also

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showed that no differences in body weight were seen when birds were given diets supplemented

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with various strains of B. subtilis (Lee et al., 2010, 2014; Park and Kim, 2014; Waititu et al., 2014). In our study, LPS challenge resulted in a significant reduction in body weight gain, as

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observed at 24 hr post-injection, and probiotic supplementation did not alleviate the LPS-induced

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weight reduction. In contrast, Li et al. (2015) reported that the supplementation of diets with Bacillus amyloliquefaciens alleviated the LPS induced reduction in the average daily gain of

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broiler chickens. This difference could be due to the difference in Bacillus sp. or functional

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differences between individual Bacillus probiotic strains.

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An acute phase protein, α-1-AGP, produced by the liver in response to inflammatory cytokines, has been used as a marker for systemic non-specific inflammation in chicken (Adler et al., 2001). In this study, serum α-1-AGP levels were found to be significantly elevated following LPS injection compared to PBS-injected chickens, indicating that the LPS challenge triggered an acute phase response. Within the LPS-injected groups, B. subtilis DFM-fed chickens showed significantly reduced α-1-AGP concentration in the serum compared to controls or antibiotic-fed

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ACCEPTED MANUSCRIPT chickens, indicating that B. subtilis DFM was successful in reducing the effects of the systemic inflammation. A similar reduction in serum α-1-AGP levels was reported in DFM-fed birds at 14 and 21 days of age compared to controls (Lee et al., 2010).

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In this study, we characterized the protective effects of B. subtilis in broiler chickens subjected to

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LPS-induced immune stress. We chose LPS challenge to induce immunological stress, as its

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effects are well-characterized in chickens and are defined by compromised growth performance and the enhanced production of pro-inflammatory cytokines(Hong et al., 2006b; Lee et al., 2010;

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Xie et al., 2000). LPS is the primary component of the outer membranes of Gram-negative

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bacteria and serves as a potent activator of the innate immune response (Ulevitch and Tobias, 1995). The binding of LPS to toll-like receptor (TLR)-4 initiates a signaling cascade that results

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in the activation of nuclear factor-kB (NF-kB), which in turn increases the transcription and

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subsequent release of various cytokines and inflammatory mediators (Li and Verma, 2002). Specifically, LPS stimulates the increased production of proinflammatory cytokines such as

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TNFα, which in turn induces the intestinal epithelial cells to secrete IL8 and express TLR4

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excessively. Increased TLR4 expression makes the epithelia hyper-reactive in response to LPS and IL8 attracts and stimulates more leucocytes to the lamina propria, resulting in the

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amplification of the inflammatory reaction (Bai et al., 2004; Hausmann et al., 2002). Though inflammation is an essential physiological response to infection and tissue protection, nonregulated inflammatory processes will result in tissue injury and compromised growth (Hakansson and Molin, 2011). We hypothesized that dietary probiotics would modulate the inflammatory activities that occur in response to LPS challenge and play a role in the restoration of cytokine balance to minimize inflammation-induced damage.

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Following LPS challenge, alterations in the T-lymphocyte subpopulations in the blood of chickens fed diets with probiotics or antibiotics or none were characterized by flow cytometric analysis. The results show that dietary B. subtilis significantly increased CD4+ T-lymphocytes in

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the peripheral blood in the absence of immunological stress, but significantly reduced CD4+ T-

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cells during LPS challenge compared to control chickens. Similar results were reported by

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Asgari et al. (2016), who showed that Lactobacillus acidophilus supplementation increased the percentage of CD4+ cells in the blood of chickens at 21 days of age. CD8+ T-cell populations in

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the blood were found to be significantly reduced in probiotic-fed birds, irrespective of the

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immune challenge. Though the exact cause of the differential regulation of CD4+ and CD8+ Tcell levels in the blood observed in this study is not known, it could be attributed to the tissue

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redistribution of lymphocytes in response to LPS challenge.

Several studies have also demonstrated the inhibitory effect of probiotics on LPS-induced pro-

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inflammatory cytokine production in vitro. Bai et al. (2004) reported that Bifidobacterium

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longum and Lactobacillus bulgaricus can suppress IL8 secretion in human intestinal epithelia when stimulated by proinflammatory cytokines such as TNFα. Carey and Kostrzynska (2012)

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showed a similar reduction in Salmonella typhimurium-induced IL8 secretion by intestinal epithelial cells upon pre-incubation with several Bifidobacterium and Lactobacillus strains. Pretreatment of porcine epithelial cells with Lactobacillus reuteri I5007 was also shown to have reduced the expression of TNFα and IL6 following LPS challenge (Yang et al., 2015). In this experiment, B. subtilis-based probiotics showed similar effects and reduced the expression of IL8 and TNFSF15 in the ileum following LPS challenge in broiler chickens. IL8 (CXCL8), a CXC

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ACCEPTED MANUSCRIPT chemokine produced by macrophages, epithelial cells and endothelial cells, is an important mediator of inflammation and is responsible for the recruitment of leukocytes, primarily neutrophils, into the mucosa. The differential expression of IL8 seen in probiotic-supplemented groups seen in this study can be attributed to the probiotic interactions with intestinal enterocytes

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and lamina propria immune cells. It was shown that probiotics can reduce TNF-induced IL8

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secretion in intestinal epithelial cells by inhibiting one or more components of the NF-kB

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pathway, thus preventing its activation or nuclear translocation or inhibiting other signaling pathways, such as MAPKs (Thomas and Versalovic, 2010). Nevertheless, the pathways leading

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to the altered expression in this study remain unclear and needs to be explored. TNFSF15 is

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abundantly expressed by immune cells and is critically involved in the differentiation, proliferation, and apoptosis of immune cells (Collette et al., 2003). In addition to the altered

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signaling pathways, the decrease in the IL8 expression seen in probiotic-fed and LPS-challenged

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chickens may also be attributed to reduced levels of TNFSF15. IL17F is a proinflammatory cytokine that plays an important role in gut homeostasis. It is produced by CD4+ T cells, CD8+

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T cells, NK cells, γδ T cells, and neutrophils and induces the production of other

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proinflammatory cytokines such as IL1 and IL6, chemokines such as CXCL1, CXCL5, and IL8, and antimicrobial peptides and matrix metallo-proteinases by a large variety of cells (Weaver et

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al., 2007). Inducible nitric oxide synthase (iNOS) is a key enzyme involved in the generation of nitric oxide (NO), which in turn plays an important role in various physiological and pathophysiological conditions (Lirk et al., 2002). The expression of iNOS and IL17F was found to be elevated in the ileum of chickens fed probiotics and subjected to immune challenge compared to other groups. The increase in IL7F production seen in chickens fed with probiotics and subjected to LPS challenge suggests that the decrease in IL8 and TNFSF15 production seen

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ACCEPTED MANUSCRIPT in these birds is mediated by pathways other than through IL17F levels. LITAF, predominantly expressed in lymphoid cells, including peripheral blood leukocytes, lymph nodes leukocytes, and intestinal IELs, is a transcription factor that upregulates TNF-α gene expression. The expression of LITAF was shown to be upregulated following the in vitro stimulation of macrophages with

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E. coli or Salmonella endotoxin (Hong et al., 2006a). In this trial, the expression of

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proinflammatory cytokine LITAF in the ileum was significantly upregulated in probiotic-fed

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chickens irrespective of the immune challenge.

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Intestinal epithelium forms the gut mucosal barrier and is an integral part of the innate immune

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responses in the intestine. The intestinal epithelial cells are the first line of defense against various pathogenic organisms and communicate extensively with commensal microbes and

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probiotics (Thomas and Versalovic, 2010). Many probiotics were shown to increase barrier

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function, enhance mucin and heat shock protein production, and modulate signaling pathways and cell survival of the intestinal epithelial cells (Otte and Podolsky, 2004; Thomas and

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Versalovic, 2010). Tight junctions, the multiprotein complexes located at the apical end of the

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lateral membrane of epithelial cells, play an important role in the regulation of intestinal permeability by sealing the paracellular space between the adjacent epithelial cells. The major

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structural and functional components of TJs include occludin, claudin, JAM2, JAM3, and ZO1 (Anderson, 2001; Tsukita et al., 2001). In this trial, LPS challenge significantly reduced the expression of occludin, JAM2, and ZO1 in the ileum 24 hr post-injection. This is in agreement with the results from studies conducted previously in pigs and mice, in which it was shown that LPS treatment reduced the expression of occludin and ZO1 in porcine intestinal epithelial cells and pulmonary cells in mice (Xie et al., 2013; Yang et al., 2015). B. subtilis-based probiotic

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ACCEPTED MANUSCRIPT increased TJ gene expression in LPS-challenged chickens compared to controls. This increased expression of TJ gene mRNA transcripts may translate to improved barrier function in the intestine, especially during times of barrier disruption, as in LPS challenge. MUC2, the major mucin produced by goblet cells, is an important constituent of the mucus layer covering the

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intestinal epithelium. It has been shown that supplementation of probiotics increased the

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expression of the MUC2 gene in chickens (Aliakbarpour et al., 2012; Smirnov et al., 2005). In

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this study, chickens challenged with LPS showed significantly reduced MUC2 expression compared to chickens injected with PBS. The supplementation of diets with B. subtilis

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counteracted the LPS-induced MUC2 down-regulation and increased its expression.

In this trial, we demonstrated that B. subtilis-based probiotics, in addition to improving the

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performance, beneficially modulate the intestinal immune activities and has the potential to

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stabilize gut integrity by increasing the expression of TJ and mucin mRNA transcripts during states of inflammation. Though evidence exists that probiotics communicate with intestinal

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epithelial cells, macrophages, dendritic cells and lymphocytes in the gut, the mechanisms behind

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these interactions have yet to be defined. Further studies involving the characterization of biological signaling pathways involved in the intestinal modulation of immune activities by

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probiotics should be performed.

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ACCEPTED MANUSCRIPT Table 1. List of primers used for qRT-PCR

PCR product Target gene

Primer sequence* (5´-3´)

F-GGTGGTGCTAAGCGTGTTAT R-ACCTCTGCCATCTCTCCACA F-GGCTTGCTAGGGGAAATGA

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IL8

264

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GAPDH

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size (Kb)

200

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R-AGCTGACTCTGACTAGGAAACTGT IL17F

F-TGAAGACTGCCTGAACCA

117

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R-AGAGACCGATTCCTGATGT TNFSF15

F-CCTGAGTATTCCAGCAACGCA

292

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R-ATCCACCAGCTTGATGTCACTAAC LITAF

F-TGTGTATGTGCAGCAACCCGTAGT

229

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R-GGCATTGCAATTTGGACAGAAGT iNOS

F-TGGGTGGAAGCCGAAATA

241

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Occludin

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R-GTACCAGCCGTTGAAAGGAC

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ZO1

JAM2

F-GAGCCCAGACTACCAAAGCAA

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R-GCTTGATGTGGAAGAGCTTGTTG F-CCGCAGTCGTTCACGATCT

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R-GGAGAATGTCTGGAATGGTCTGA F-AGCCTCAAATGGGATTGGATT

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R-CATCAACTTGCATTCGCTTCA MUC2

F-GCCTGCCCAGGAAATCAAG

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R-CGACAAGTTTGCTGGCACAT

*F = Forward primer; R = Reverse primer

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ACCEPTED MANUSCRIPT

Body weight-24hr post-LPS

*

*

Antibioitc

Probiotic

500 450 400

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350 300 250

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Average body weight (g)

550

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Control

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200

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Figure 1. Average body weight of broiler chickens (24 hr after LPS challenge) fed diets nonsupplemented or supplemented with antibiotics or probiotics and challenged with LPS or PBS at

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14 days of age. The data were analyzed by two-way ANOVA, a significant main effect of diet

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was seen, and there were no interactions. The asterisk (*) denotes significantly increased body

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weight compared to controls (P < 0.05).

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ACCEPTED MANUSCRIPT Body weight gain-24 hr post LPS

*

40

30 #

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20

10

0 PBS

LPS Antibiotic

Probiotic

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Control

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Average body weight gain (g)

50

AN

Figure 2. Average body weight gain of broilers (24 hr after LPS challenge) fed diets non-

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supplemented or supplemented with antibiotics or probiotics and challenged with LPS or PBS at 14 days of age. The data were analyzed by two-way ANOVA, and a significant main effect of

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immune challenge was seen; there were no interactions. * and # represent significant main

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effects of immunological challenge (P < 0.05).

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ACCEPTED MANUSCRIPT Serum α-1-AGP levels a

160

a

140 120 100 80

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b

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60 40 20 0 PBS

LPS Antibiotic

Probiotic

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Control

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Average A-1-AGP conc. (ng/mL)

180

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Figure 3. α-1-AGP concentration in the serum of chickens (n=3) fed diets non-supplemented or

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supplemented with antibiotics or probiotics and challenged with LPS or PBS at 14 days of age. Serum samples were collected 24 hr post-LPS injection. Each bar represents the mean ± SEM of

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triplicate samples. The data were analyzed by two-way ANOVA and significant main effects

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(diet, LPS), and diet x immune challenge interactions were seen. Owing to these interactions, means within each immunological challenge group were compared. Bars with different letters

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within each immunological challenge group differ significantly (P < 0.05).

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ACCEPTED MANUSCRIPT CD4+ cell percentage 80

a b

60 50

a

c

b c

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40

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30 20 10 0 PBS

LPS Antibiotic

Probiotic

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Control

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Mean % CD4 cells

70

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Figure 4. CD4+ cells expressed as a percentage of CD45 cells in the peripheral blood of

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chickens fed diets non-supplemented or supplemented with antibiotics or probiotics and challenged with LPS or PBS at 14 days of age. Each bar represents the mean ± SEM of triplicate

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samples. The data were analyzed by two-way ANOVA, and significant main effects (diet, LPS)

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and diet x immune challenge interactions were seen. Owing to these interactions, means within each immunological challenge group were compared to each other. Bars with different letters

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within each immunological challenge group differ significantly (P < 0.05).

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ACCEPTED MANUSCRIPT CD8+ cell percentage 30

a

b

c

20

T

15 10

IP

Mean % CD8 cells

25

CR

5 0 Antibiotic

Probiotic

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Control

AN

Figure 5. CD8+ cells expressed as a percentage of CD45 cells in the peripheral blood of

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chickens fed diets non-supplemented or supplemented with antibiotics or probiotics and challenged with LPS or PBS at 14 days of age. Each bar represents the mean ± SEM. The data

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were analyzed by two-way ANOVA, and significant main effects (diet, LPS) and no interactions

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were seen. Bars with no common letter among different dietary treatments differ significantly (P

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< 0.05).

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ACCEPTED MANUSCRIPT B)

a

6.0E-03 5.0E-03 4.0E-03 3.0E-03

b

2.0E-03

b

1.0E-03

2.0E-03 b

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a

1.0E-03 b

0.0E+00

LPS Probiotic

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PBS Control Antibiotic

ED

c

b

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E)

AC

5.0E+00

b

b

LPS Probiotic

6.0E-02

iNOS a

AN

a

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TNFSF15

1.5E-03

b

b

PBS Control Antibiotic

LPS

Mean normalized mRNA

Mean normalized mRNA

b

1.0E-04

D)

2.5E-03

Mean normalized mRNA

a

2.0E-04

Antibiotic

C)

3.0E+00

3.0E-04

CR

PBS

Control

4.0E+00

a

4.0E-04

0.0E+00

0.0E+00

5.0E-04

IL17F

5.0E-04

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Mean normalized mRNA

Mean normalized mRNA

IL8 7.0E-03

IP

A)

5.0E-02 4.0E-02 3.0E-02 2.0E-02

b

1.0E-02

b

0.0E+00 Control

PBS Antibiotic

LPS Probiotic

a

b

2.0E+00 1.0E+00 0.0E+00 Control

Antibiotic

Probiotic

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ACCEPTED MANUSCRIPT Figure 6. Effects of dietary probiotics or antibiotics on the levels of transcripts of pro-inflammatory cytokines. Chickens were fed diets non-supplemented or supplemented with antibiotics or probiotics and challenged with LPS or PBS at 14 days of age. Transcript levels of IL8 (A), IL17F (B), TNFSF15 (C), iNOS (D) and LITAF (E) in ileum were measured by quantitative RT-PCR and

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normalized to GAPDH transcript levels. Each bar represents the mean ± SEM (n = 3). The data were

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analyzed by two-way ANOVA, and significant main effects (diet, LPS) and diet x immune challenge

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interaction were seen for all genes except LITAF (significant main effects only, no interactions). Bars with no common letter within each immunological challenge treatment differ significantly (P <

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0.05). For LITAF, bars with no common letter among different dietary treatments differ

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CE

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ED

M

AN

significantly (P < 0.05)

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ACCEPTED MANUSCRIPT

JAM2

4.0E-03

a

3.0E-03 a

b

b c

T

1.0E-03

IP

2.0E-03

ab

0.0E+00 PBS Control Antibiotic

LPS Probiotic

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Mean normalized mRNA

A)

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Occludin

1.5E-02

AN

a

a

1.0E-02

b

5.0E-03

M

b c

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c

0.0E+00

PT

PBS Antibiotic

LPS Probiotic

CE

Control

AC

Mean normalized mRNA

B)

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ACCEPTED MANUSCRIPT

ZO1

3.5E-02 3.0E-02 2.5E-02

a

a

b

b

a

2.0E-02 1.5E-02

b

T

1.0E-02

IP

5.0E-03 0.0E+00 PBS Control Antibiotic

LPS Probiotic

CR

Mean normalized mRNA

C)

1.5E-01

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a

a b

a

M

2.0E-01

b

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MUC2

2.5E-01

1.0E-01

c

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5.0E-02 0.0E+00 Control

PBS Antibiotic

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Mean normalized mRNA

D)

LPS Probiotic

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Figure 7. Effects of dietary probiotics or antibiotics on the levels of transcripts of intestinal tight junction proteins and mucin. Chickens were fed diets non-supplemented or supplemented with

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antibiotics or probiotics and challenged with LPS or PBS at 14 days of age. Transcript levels of JAM2 (A), occludin (B), ZO1 (C), and MUC2 (D) in the ileum were measured by quantitative RTPCR and normalized to GAPDH transcript levels. Each bar represents the mean ± SEM (n = 3). The data were analyzed by two-way ANOVA, and significant main effects (diet, LPS) and diet x immune challenge interactions were seen. Bars with no common letter within each immunological challenge treatment differ significantly (P < 0.05).

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ACCEPTED MANUSCRIPT References Adler, K.L., Peng, P.H., Peng, R.K., Klasing, K.C., 2001. The kinetics of hemopexin and α1-acid glycoprotein levels induced by injection of inflammatory agents in chickens. Avian Dis, 289296. Aliakbarpour, H., Chamani, M., Rahimi, G., Sadeghi, A., Qujeq, D., 2012. The Bacillus subtilis

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Anderson, J.M., 2001. Molecular structure of tight junctions and their role in epithelial transport. Physiology 16, 126-130.

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Asgari, F., Madjd, Z., Falak, R., Bahar, M., Nasrabadi, M.H., Raiani, M., Shekarabi, M., 2016. Probiotic feeding affects T cell populations in blood and lymphoid organs in chickens.

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Carey, C.M., Kostrzynska, M., 2012. Lactic acid bacteria and bifidobacteria attenuate the proinflammatory response in intestinal epithelial cells induced by Salmonella enterica serovar

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feed supplement to promote weight gain in broilers. Lett Appl Microbiol 60, 580-588. NRC, 1994. Nutrient requirements of poultry. National Academy Press Washington, DC.

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NRC, 2010. Guide for the care and use of laboratory animals. National Academies Press. Otte, J.-M., Podolsky, D.K., 2004. Functional modulation of enterocytes by gram-positive and

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gram-negative microorganisms. American Journal of Physiology-Gastrointestinal and Liver Physiology 286, G613-G626.

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Saccharomyces boulardii and Bacillus subtilis B10 modulate TLRs mediated signaling to induce immunity by chicken BMDCs. J Cell Biochem 115, 189-198. SAS, 2014. Base SAS 9. 4 Procedures Guide: Statistical Procedures. SAS Institute. Sen, S., Ingale, S., Kim, Y., Kim, J., Kim, K., Lohakare, J., Kim, E., Kim, H., Ryu, M., Kwon, I., 2012. Effect of supplementation of Bacillus subtilis LS 1-2 to broiler diets on growth performance, nutrient retention, caecal microbiology and small intestinal morphology. Res Vet Sci 93, 264-268.

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nutrient digestibilities, and immune responses. Poultry Sci 93, 625-635. Weaver, C.T., Hatton, R.D., Mangan, P.R., Harrington, L.E., 2007. IL-17 family cytokines and

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lipopolysaccharide on broiler chickens. Poultry Sci 79, 33-40. Xie, W., Wang, H., Wang, L., Yao, C., Yuan, R., Wu, Q., 2013. Resolvin D1 reduces deterioration of tight junction proteins by upregulating HO-1 in LPS-induced mice. Laboratory Investigation 93, 991-1000. Yang, F., Wang, A., Zeng, X., Hou, C., Liu, H., Qiao, S., 2015. Lactobacillus reuteri I5007 modulates tight junction protein expression in IPEC-J2 cells with LPS stimulation and in newborn piglets under normal conditions. BMC microbiology 15, 32.

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ACCEPTED MANUSCRIPT Highlights  Dietary probiotics effects LPS-induced host inflammatory responses and intestinal barrier gene expression in broiler chickens.  Peripheral blood CD4+ T-lymphocytes were increased in the LPSchallenged chickens.

fed chickens irrespective of the immune challenge

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 LITAF expression in the ileum was significantly upregulated in probiotic-

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 LPS challenge significantly reduced the expression of occludin, JAM2, and

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ZO1 in the ileum 24 hr post-injection

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 B. subtilis counteracted the LPS-induced MUC2 down-regulation.

33