Influence of orally fed a select mixture of Bacillus probiotics on intestinal T-cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge

Yang et al. Vet Res (2016) 47:71 DOI 10.1186/s13567-016-0355-8 Open Access RESEARCH ARTICLE Influence of orally fed a select mixture of Bacillus probiotics on intestinal T‑cell migration in weaned MUC4 resistant pigs following Escherichia coli challenge Gui‑Yan Yang†, Yao‑Hong Zhu†, Wei Zhang, Dong Zhou, Cong‑Cong Zhai and Jiu‑Feng Wang* Abstract Efficient strategies for treating enteritis caused by F4+ enterotoxigenic Escherichia coli (ETEC)/verocytotoxigenic Escherichia coli (VTEC)/enteropathogenic E. coli (EPEC) in mucin 4 resistant (MUC4 RR; supposed to be F4ab/ac recep‑ tor–negative [F4ab/acR−]) pigs remain elusive. A low (3.9 × 108 CFU/day) or high (7.8 × 108 CFU/day) dose of Bacillus licheniformis and Bacillus subtilis spore mixture (BLS-mix) was orally administered to MUC4 RR piglets for 1 week before F4+ ETEC/VTEC/EPEC challenge. Orally fed BLS-mix upregulated the expression of TLR4, NOD2, iNOS, IL-8, and IL-22 mRNAs in the small intestine of pigs challenged with E. coli. Expression of chemokine CCL28 and its receptor CCR10 mRNAs was upregulated in the jejunum of pigs pretreated with high-dose BLS-mix. Low-dose BLS-mix pretreatment induced an increase in the proportion of peripheral blood CD4−CD8− T-cell subpopulations and high-dose BLS-mix induced the expansion of CD4−CD8− T cells in the inflamed intestine. Immunostaining revealed that considerable IL-7Rα–expressing cells accumulated at the lamina propria of the inflamed intestines after E. coli challenge, even in pigs pretreated with either low- or high-dose BLS-mix, although Western blot analysis of IL-7Rα expression in the intestinal mucosa did not show any change. Our data indicate that oral administration of the probiotic BLS-mix partially ameliorates E. coli-induced enteritis through facilitating upregulation of intestinal IL-22 and IκBα expression, and preventing loss of intestinal epithelial barrier integrity via elevating ZO-1 expression. However, IL-22 also elicits an inflammatory response in inflamed intestines as a result of infection with enteropathogenic bacteria. Introduction Enterotoxigenic Escherichia coli bearing F4 fimbriae (F4+ ETEC) is the most prevalent ETEC strain in causing postweaning diarrhea in pigs [1]. The fimbriae-mediated recognition of specific receptors on host enterocytes is the prerequisite for infection. Breeding programs with F4 receptor–negative pigs is preferable for prevention of F4+ ETEC infection, and a polymorphism in the mucin 4 (MUC4) gene has been developed to allow genotyping for determining F4ab/ac ETEC resistance/susceptibility [2, 3]. According to this DNA marker-based test, pigs were *Correspondence: † Gui-Yan Yang and Yao-Hong Zhu contributed equally to this work College of Veterinary Medicine, China Agricultural University, Beijing 100193, China genotyped as resistant (RR), susceptible heterozygote (SR) and susceptible homozygote (SS). However, MUC4 RR pigs are now identified not absolutely F4ab/ac receptor–negative (F4ab/acR−) pigs, since there are more than 30% showing positive adhesion with F4ab/ac ETEC and more receptors for F4 fimbriae have been discovered [3– 5]. We recently found that an F4+ enterotoxigenic E. coli (ETEC)/verocytotoxigenic E. coli (VTEC)/enteropathogenic E. coli (EPEC) hybrid can cause enteritis and/or fever in MUC4 RR pigs. This is possibly due to the ability of this strain to adhere to the intestinal mucosa, and subsequently secrete toxins (e.g. heat-liable, heat-stable enterotoxins, Shiga-like toxin Stx2e) and release LPS [1, 6]. The probiotics Bacillus licheniformis and Bacillus subtilis are widely used in both humans and animals with a © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yang et al. Vet Res (2016) 47:71 broad spectrum of inhibitory activity against pathogenic bacteria [7, 8]. Our recent study showed that excessive generation of CD4+ interleukin (IL)-10–positive T cells following consumption of a B. licheniformis and B. subtilis mixture (BLS-mix) during episodes of intestinal inflammation caused by F4+ ETEC/VTEC/EPEC can inhibit clearance of the pathogen in newly weaned MUC4 RR pigs [6]. Effective defense against F4+ ETEC/VTEC/ EPEC achieved through coordination of complex signaling networks linking the innate and adaptive immune systems thus remains elusive. IL-22 is essential for epithelial defense against extracellular bacteria and critical for mediating mucosal host defenses against attaching and effacing bacteria in the gastrointestinal tract [9]. The central roles of IL-22 in the gut include maintaining normal barrier homeostasis, inducing the secretion of antibacterial proteins, and triggering the expression of chemokines for controlling the spread of invading pathogens [10]. However, IL-22 has both protective and pathologic roles, and the effect of BLS-mix on IL-22 secretion and its role in pigs infected with E. coli is poorly understood. The induction of IL-10–producing Foxp3− T cells by BLS-mix cannot account for the protection of newly weaned MUC4 RR pigs from F4+ ETEC/VTEC/EPEC infection [6]. CD4+CD25+CD127low cells were used as an alternative marker for regulatory T (Treg) cells, in addition to the conventional CD4+CD25+Foxp3+ population [11]. IL-7 receptor α-chain (IL-7Rα, also known as CD127) contributes to the development of IL-22–producing cells and Treg cells, IL-7/IL-7R–dependent signaling plays a crucial role in regulating the immune response in the intestinal mucosa [12, 13]. In swine, CD127 has been detected in the intestine, lymphoid tissues, and various nonlymphoid tissues [14]. Chemokines can attract specific populations of immune cells to sites of infection or inflammation [15]. Specifically, in humans and mice, the CC chemokine receptor CCR9, expressed by IgA antibody-secreting cells (ASCs) and T cells, responds to its ligand, CCL25, which is selectively expressed in the small intestine and thymus. In contrast, chemokine CCL28, a ligand for CCR10 that is expressed primarily by IgA ASCs and some T lymphocytes, is expressed in mucosa of intestine and elsewhere [16]. In pigs, CCL25 recruits T cells and IgA ASCs that express CCR9 in the gut-associated lymphoid tissues and small intestine, whereas CCL28 can be detected in both intestinal and other mucosal tissues [17]. It remains to be elucidated that the effect of BLS-mix on these two chemokines with their respective receptors in pigs. Probiotic bacteria increase tight-junction function to modul (...truncated)