A Lactobacillus rhamnosus Strain Induces a Heme Oxygenase Dependent Increase in Foxp3+ Regulatory T Cells Khalil Karimi1,3*, Nalaayini Kandiah1, Jessie Chau1,3, John Bienenstock1,4, Paul Forsythe1,2,3* 1 The Brain Body institute, McMaster University, Hamilton, Ontario, Canada, 2 Firestone Institute for Respiratory Health, McMaster University, Hamilton, Ontario, Canada, 3 Department of Medicine, McMaster University, Hamilton, Ontario, Canada, 4 Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Abstract We investigated the consequences of feeding with a Lactobacillus species on the immune environment in GALT, and the role of dendritic cells and heme oxygenase-1 in mediating these responses. Feeding with a specific strain of Lactobacillus rhamnosus induced a significant increase in CD4+CD25+Foxp3+ functional regulatory T cells in GALT. This increase was greatest in the mesenteric lymph nodes and associated with a marked decrease in TNF and IFNc production. Dendritic cell regulatory function and HO-1 expression was also increased. The increase in Foxp3+ T cells could be prevented by treatment with a heme oxygenase inhibitor. However, neither inhibition of heme oxygenase nor blockade of IL-10 and TGFb prevented the inhibition of inflammatory cytokine production. In conclusion Lactobacillus feeding induced a tolerogenic environment in GALT. HO-1 was critical to the enhancement of Foxp3+ regulatory T cells while additional, as yet unknown, pathways were involved in the down-regulation of inflammatory cytokine production by T cells. Citation: Karimi K, Kandiah N, Chau J, Bienenstock J, Forsythe P (2012) A Lactobacillus rhamnosus Strain Induces a Heme Oxygenase Dependent Increase in Foxp3+ Regulatory T Cells. PLoS ONE 7(10): e47556. doi:10.1371/journal.pone.0047556 Editor: Ciriaco A. Piccirillo, McGill University Health Center, Canada Received March 21, 2012; Accepted September 18, 2012; Published October 15, 2012 Copyright: 2012 Karimi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funded in part by a grant from AllerGen NCE (http://www.allergen-nce.ca/index.html) Grant 09-B6. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: forsytp@mcmaster.ca (PF); karimik@mcmaster.ca (KK) Introduction The gut microbiota impacts various aspects of host physiology including modulation of the immune system [1–5]. Overall balance in composition of the microbiota, together with the influence of pivotal species that induce specific responses are important determinants of immunity in the intestine and beyond. Non-pathogenic bacteria that promote beneficial health effects when ingested have been termed probiotics [6]. These ‘‘beneficial microbes’’ are most frequently Lactobacillus or Bifidobacterium species, however a number of lactic acid bacteria and non- pathogenic E.coli have also been identified as probiotics [7]. While initial attention was focused on the role of probiotics in gastrointestinal development, immune adaptation and attenuation of GI inflammatory diseases there is increasing interest in the ability of orally delivered probiotics to regulate immune responses outside the GI tract. There is evidence linking the immunomodulatory function of certain commensal bacteria, and components thereof, to induction of regulatory T cells (Treg) and their associated cytokines. In vitro, selective bacteria have been shown to induce IL-10 producing Treg [8]. While, in vivo, Mazmanian et al. demonstrated that oral ingestion of polysaccharide A (PSA) derived from Bacteroides fragilis protects animals from experimental colitis through induction of IL-10-producing CD4+ T cells [9,10] and dendritic cells co- cultured with PSA and then incubated with na¨ıve T cells promoted the generation of an IL-10 producing Treg population. Bifidobacterium infantis induces expression of Foxp3, a key tran- scription factor in programming Treg, in T cells that protect mice against Salmonella typhimurium infection [11]. We have previously demonstrated that attenuation of the allergic airway response following oral treatment with Lactobacillus rhamnosus JB-1 in mice corresponded with a significant increase in the proportion of functional CD4+CD25+Foxp3+ Treg in the spleen and mediastinal lymph nodes [12]. Furthermore, adoptive transfer of these Treg mimicked the protection from airway inflammation observed following feeding of the bacteria. In another study of three candidate probiotic strains, the ability to promote Foxp3 expression in T cells, in mesenteric lymph nodes and spleen, was associated with protection against airway inflammation in OVA- sensitized animals and inhibition of IgE induction to orally administered OVA [13]. However, while Treg may play an important role in the anti-inflammatory response to certain commensal bacteria much remains unknown about the mechan- isms underlying the induction of these cells. Dendritic cells (DC) are among the first immune cells to encounter bacteria in the intestine and can directly present antigens from commensals to the mesenteric lymph node (MLN) [14,15]. DC interact with T and B cells to maintain non- inflammatory immune responses through mechanisms that include direct suppression and deletion of T cells and the induction of a range of regulatory T cell subtypes [16,17]. Thus while Treg may be major effectors of immune regulation mediated by some probiotics, the phenotypic changes in DC following interaction PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47556