A Novel Probiotic Mixture Exerts a Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Mediated by IL-10 Producing Regulatory T Cells

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODOLOGY/PRINCIPAL FINDINGS: The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-beta1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4(+)CD25(+) Tregs are involved in the suppressive effect induced by the lactobacilli mixture. CONCLUSIONS/SIGNIFICANCE: Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases

Gut Manifestations in an Experimental Model of Multiple Sclerosis

Multiple sclerosis (MS) is an immune-mediated demyelinating and neurodegenerative disease of the central nervous system (CNS). Neuroinflammatory processes in MS are believed to lead to damage and leakage in the blood-brain barrier that protects the CNS and regulates the trafficking of soluble mediators and leukocytes. MS is thought to be triggered in genetically susceptible individuals following exposure to environmental factors, which may be responsible for loss of tolerance and activation of the myelin-reactive T cells. Based on findings in animal models the influence of gut microbiota on disease development has been suggested. In this thesis we aimed to investigate if intestinal barrier and immune function are affected during the progression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and whether probiotic bacteria could have an impact on the disease. EAE was induced in C57BL/6 mice either by active immunization using myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide, or by adoptive transfer of MOG35-55 reactive T cells. A major part of the work focuses on how, in addition to CNS, the bowel is influenced during the development of EAE. The results show increased intestinal permeability and inflammation in the intestinal mucosa, concomitant with activation of the acquired (Th1 and Th17) as well as innate immunity. An increased expression of inflammatory cytokines in antigen presenting cells and also increased activated neutrophils was noted. As a consequence of this activation increased levels of the inflammatory molecule calprotectin was also shown in EAE animals. Interestingly, we found an increased number of mucus-producing goblet cells throughout the intestinal tract. Another part of the thesis is on oral treatment of EAE diseased mice with probiotic bacteria. A screening experiment revealed immunostimulatory properties of some probiotics and a preventive effect on the development of disease. This part focuses on immunoregulatory abnormalities and a synergistic effect of different probiotic lactobacilli. It was shown that a combination of strains could suppress and reverse an ongoing inflammatory condition of diseased animals. Our analysis showed that the therapeutic effect was exerted by activated regulatory T cells in an IL-10 dependent manner. We showed an association between the intestinal mucosa and immune system during development of the disease, however, how local anti-inflammatory activities induced by probiotics can have an influence on the CNS is unclear and remains to be investigated. In addition, our findings show that disruption of intestinal homeostasis is an early and immune-mediated event, and it is proposed that this disruption will support disease progression in MS and thus represent a potential therapeutic target

Estrogen receptor genes variations and breast cancer risk in Iran

Evidence suggests that alterations in estrogen signaling pathways, including estrogen receptor alpha (ER-alpha) and estrogen receptor beta (ER-beta) occur during breast cancer development. ER-alpha and ER-beta genes polymorphisms have been found to be associated with breast cancer and clinical features of the disease in the western countries. In the current study, we evaluated the hypothesis that certain sequence variants of the ER-alpha and ER-beta genes are associated with an additively increased risk for breast cancer in Iranian women breast cancer patients. The genes were scanned in 150 Iranian patients with newly diagnosed invasive breast tumors and in healthy control individuals by PCR single-strand conformation polymorphism (SSCP) method. Three single nucleotide polymorphisms (SNPs) in codon10 (TCT -> TCC), codon 352 (CCG -> CCC) and codon 594 (ACG -> ACA) in ER-alpha gene and one SNP codon 392 (CTC -> CTG) in ER-beta were revealed have additive effects in developing breast cancer and LN metastases. Also, SNP in codon 392 of estrogen receptor-beta gene is more effective (threefold) than those SNPs in codons 10, 325, 594 of estrogen receptor-alpha gene in developing LN metastases in breast cancer patients. SNPs in estrogen receptor alpha and beta have additive effects in increasing risk for developing breast cancer with LN metastases among Iranian women breast cancer patients

Intestinal barrier dysfunction develops at the onset of experimental autoimmune encephalomyelitis, and can be induced by adoptive transfer of auto-reactive T cells.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE), the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers). These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms) and at 14 days (i.e., at the stage of paralysis) after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer's patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies

Association of estrogen receptor-alpha A908G (K303R) mutation with breast cancer risk

Genetic mutations in premalignant breast lesions may have a role in malignancy progression or influence the behavior of subsequent disease. A point mutation in estrogen receptor-alpha (ER-&ALPHA) as A908G (Lys303 -> Arg) was originally involved to hypersensitive to estrogen breast hyperplasia. We detected this mutation among Iranian women with invasive breast cancer. A population-based case-control study was conducted in 150 newly diagnosed invasive breast cancer and 147 healthy control individuals controls to screen for presence of the ER-alpha A908G mutation by using single-strand conformation polymorphism (SSCP) analysis and 33Pcycle DNA sequencing. We detected the 10.7% ER-alpha A908G mutation in the form of heterozygote genotype only among cancer patients (chi(2)=22.752, P=0.00). The allelic frequency of mutant allele AGG in codon 303 was significantly (chi(2)=29.709, P=0.001) higher in patients with the family history of breast cancer (28.9%) than those without the family history of breast cancer (1.9%). Our data suggest that ER-alpha codon 303 mutation is correlated with various aspects of breast cancer in Iran. ER-alpha genotype might represent a surrogate marker for predicting breast cancer developing later in life

Cross-reactivity of antibody responses to Borrelia afzelii OspC : Asymmetry and host heterogeneity

The tick-transmitted bacterium Borrelia afzelii consists of a number of antigenically different strains — often defined by outer surface protein C (OspC) genotype — that coexist at stable frequencies in host populations. To investigate how host antibody responses affect strain coexistence, we measured antibody cross-reactivity to three different OspC types (OspC 2, 3 and 9) in three different strains of laboratory mice (BALB/c, C3H and C57BL/6). The extent of cross-reactivity differed between mouse strains, being higher in C3H than BALB/c and C57BL/6. In one of three pairwise comparisons of OspC types (OspC2 vs OspC9), there was evidence for asymmetry of cross-reactivity, with antibodies to OspC2 cross-reacting more strongly with OspC9 than vice versa. These results indicate that the extent of antibody-mediated competition between OspC types may depend on the composition of the host population, and that such competition may be asymmetric. We discuss the implications of these results for understanding the coexistence of OspC types

Increased IL-6 and TNF-α expression in intestinal macrophages and dendritic cells.

<p>Intestinal lamina propria (LP), Peyer's patches (PP) and mesenteric lymph nodes (MLN) isolated from unimmunized controls, and EAE7 and EAE14 mice were analyzed by flow cytometry for presence of F4/80⁺ macrophages and CD11c⁺ DC expressing IL-6 (A) and TNF-α (B). Data are representative of one of three independent experiments.</p

Increased intestinal permeability and altered intestinal morphology after adoptive transfer of encephalitogenic T cells.

<p>Na-F (A) , FITC-BSA (B) in plasma from mice receiving un-stimulated lymph node cells (Control), MOG-reactive T cells (adoptively transferred EAE) and OVA-reactive T cells, (n = 3–5). Mice were gavaged with a marker molecules as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106335#pone-0106335-g001" target="_blank">Figure 1</a>. H&E-sections from duodenum, jejunum and ileum isolated from animals receiving MOG-reactive T cells (EAETransfer) (C). The sections were examined for crypt depth (D), villus length (E), submucosa (F) and muscularis thickness (G). Arrows demonstrate approximate measurements for villus length (1) crypt depth (2) submucosa thickness (3), muscularis thickness (4) and highlight the differences between the groups (C, original magnification ×40, insets ×100). Each bar represents mean ±SD of 7–9 analyzed sections per animal, (n = 5). Immunohistochemical analysis of zonulin expression from mice receiving MOG-reactive and OVA-reactive T cells (H). Arrows show zonulin both in enterocytes and lamina propria on top of the villi. Semi-quantitative analysis of zonulin staining (I). Staining intensity was expressed as positive pixels/mm² and converted as ratio to the mean values from relevant sections in the control animals. Data shown are mean ±SD from 3–5 animals for each group. ** represents a p-value≤0.01 and *** a p-value≤0.001.</p

Increased peripheral and intestinal inflammation.

<p>Intestinal lamina propria (LP), Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleen were isolated from unimmunized controls and EAE7 and EAE14 mice. Single cell suspensions were prepared and analyzed by flow cytometry for presence of T cells expressing IFN-γ, IL-17, or Foxp3. The lymphocytes were gated and cells were analyzed for expression of the indicated cytokines. The dot plots (A, C, and F) show the percentage of double positive cells (upper right quadrants) out of total T cells and the data are representative of one of three independent experiments. The histograms (B, D, and G) show results from all performed experiments, mean ±SD (n = 5). The frequency of CD4⁺IFN-γ⁺T cells (A) and relative percentage (B). The frequency of CD3⁺IL-17⁺ T cells (C; upper right), CD3⁻IL-17⁺ cells (C; upper left) and relative percentage of CD3⁺IL-17⁺ T cells (D). A histogram shows IL-17 expression by gated γδ T cells (E). The frequency of CD4⁺ Foxp3⁺ Tregs, dot plots from one representative experiment (F) and total results (G). * represents a p-value≤0.05, ** a p-value≤0.01 and *** a p-value≤0.001 in comparison with the controls.</p

Increased intestinal permeability in primary Sjögren's syndrome and multiple sclerosis

There is increasing evidence suggesting a role of intestinal dysfunction in a number of autoimmune diseases. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease with a documented increased level of intestinal inflammation, whereas multiple sclerosis (MS) is an organ-specific autoimmune disease known to exhibit increased intestinal permeability. In this study we determine to what extent intestinal inflammation, analysed by a faecal calprotectin ELISA, is accompanied by altered intestinal wall permeability, as measured by a lactulose and mannitol intestinal absorption assay. Intestinal permeability was increased in both pSS and MS patients, while faecal calprotectin was elevated in pSS but normal in MS. Our findings suggest different mechanisms mediating a leaky gut in these two diseases: in pSS there is autoimmune attack directly on the intestinal wall; in MS, with autoimmunity being limited to the CNS, it may be due to a disturbed CNS regulation of enteric nerve function