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

Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP

Background: Antimicrobial peptides (AMPs) are receiving increasing attention due to resistance development against conventional antibiotics. Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in an array of infections such as ocular infections, cystic fibrosis, wound and post-surgery infections, and sepsis. The goal of the study was to design novel AMPs against these pathogens. Methodology and Principal Findings: Antibacterial activity was determined by radial diffusion, viable count, and minimal inhibitory concentration assays, while toxicity was evaluated by hemolysis and effects on human epithelial cells. Liposome and fluorescence studies provided mechanistic information. Protease sensitivity was evaluated after subjection to human leukocyte elastase, staphylococcal aureolysin and V8 proteinase, as well as P. aeruginosa elastase. Highly active peptides were evaluated in ex vivo skin infection models. C-terminal end-tagging by W and F amino acid residues increased antimicrobial potency of the peptide sequences GRRPRPRPRP and RRPRPRPRP, derived from proline arginine-rich and leucine-rich repeat protein (PRELP). The optimized peptides were antimicrobial against a range of Gram-positive S. aureus and Gram-negative P. aeruginosa clinical isolates, also in the presence of human plasma and blood. Simultaneously, they showed low toxicity against mammalian cells. Particularly W-tagged peptides displayed stability against P. aeruginosa elastase, and S. aureus V8 proteinase and aureolysin, and the peptide RRPRPRPRPWWWW-NH2 was effective against various "superbugs'' including vancomycin-resistant enterococci, multi-drug resistant P. aeruginosa, and methicillin-resistant S. aureus, as well as demonstrated efficiency in an ex vivo skin wound model of S. aureus and P. aeruginosa infection. Conclusions/Significance: Hydrophobic C-terminal end-tagging of the cationic sequence RRPRPRPRP generates highly selective AMPs with potent activity against multiresistant bacteria and efficiency in ex vivo wound infection models. A precise "tuning'' of toxicity and proteolytic stability may be achieved by changing tag-length and adding W-or F-amino acid tags

A Novel Microparticle Based Formulation for Topical Delivery of FOL-005, a Small Peptide

Although many therapeutically active peptides and proteins have been developed there is a lack of topical pharmaceutical products on the market containing these sensitive molecules. The main reasons may be lack of stability and a limitation of larger molecules to penetrate into the skin. In this study we investigated the possibility to develop a peptide formulation which enables follicular permeation of peptides and passes the following criteria: 1) The formulation should be chemically and physically stable, 2) The formulation should have appealing cosmetical properties and 3) The formulation should be compatible with skin as well as sebum. The hypothesis was that increased stability of the peptide could be obtained by keeping the peptide in solid form and in a water-free environment, and that permeation into skin could be facilitated by reducing the particle size to < 10 µm and by formulating the peptide in sebum compatible excipients. By this method a safe and a cosmetically attractive formulation, facilitating the local distribution of the model peptide FOL-005 into the skin and at the same time securing chemical and physical stability, was successfully developed

Administration of three potential probiotic strains suppresses inflammatory autoimmune disorders in mice with established EAE.

<p>C57BL/6 mice were immunized and scored for clinical signs of EAE. The animals were orally treated, starting 14 days after the onset of EAE, with <i>L. paracasei</i> DSM 13434 (n = 18), a mixture of lactobacilli (Lacto-mix) containing <i>L. paracasei</i> DSM 13434, <i>L. plantarum</i> DSM 15312 and DSM 15313 (n = 18) or saline (vehicle) as control (n = 18). (A) The mean clinical score for each group of mice is shown. Data represent pooled values from two independent experiments. The presence of infiltrating CD4⁺ T cells into the spinal cord parenchyma and perivascular cuffs was assessed by immunohistochemical staining in sections from (B) control EAE and (C) Lacto-mix-treated animals after 20 days of therapeutic treatment. (D) In the histogram, each bar represents the mean percentage of stained area (± SEM) from 5 mice per treatment group. Levels of pro-inflammatory cytokines (E) TNF-α, (F) IFN-γ, (G) IL-17, and (H) anti-inflammatory IL-10 were measured in supernatants of MOG_35–55 peptide restimulated spleen cell cultures from the Lacto-mix-treated (n = 3) or control mice (n = 3), after 20 days of therapeutic treatment. Data are representative of two experiments. Each bar represents mean ± SEM of three samples per group. * represents a p-value ≤0,05 and ** a p-value ≤0,01.</p

Probiotic-induced Treg cells suppress EAE in an IL-10-dependent fashion.

<p>Naïve C57BL/6 wild type (WT) or IL-10-KO mice were treated with either the probiotic Lacto-mix or saline (control) during 14 days. MLN cells were isolated from these animals and administered intravenously into recipient mice which were immunized for EAE 24 h later. (A) EAE development in recipient mice after adoptive cell transfer of total MLN cells (isolated from mice treated with either saline or Lacto-mix), MLNs (isolated from WT mice treated with Lacto-mix) depleted for CD4⁺CD25⁺ cells, isolated CD4⁺CD25⁺ cells (from MLNs of Lacto-mix treated WT mice), isolated CD4⁺CD25⁺ cells (from MLNs of Lacto-mix treated IL-10 KO mice) (n = 3−5). (B) Serum levels of IL-10 were measured by ELISA in plasma obtained from recipient animals at 28 days after cell transfer. Data are representative of two experiments. Each bar represents mean ± SEM of three samples per group. * represents a p-value ≤0,05 and ** a p-value ≤0,01.</p

Distinct probiotic strains prevents EAE and suppresses MOG-reactive T cells.

<p>C57BL/6 mice received either <i>L. paracasei</i> DSM 13434, <i>L. plantarum</i> DSM 15312, <i>L. plantarum</i> DSM 15313 <i>L. paracasei</i> PCC 101, <i>L. delbrueckii</i> DSM 20081 (10⁹ cfu, daily) or vehicle as control, starting 12 days prior to immunization for EAE. (A) The mean clinical score for each group of mice (n = 8) is shown. Data are representative of three separate experiments. (B) Spleen cell cultures from the probiotic-treated or control mice were restimulated <i>in vitro</i> with MOG_35–55 peptide. MOG specific T cell proliferation was conducted with [³H]thymidine incorporation assay and each bar represents the mean stimulation index (± SEM) from triplicate measurements (n = 3). (C) Immunohistochemical staining for CD4⁺ T cells in sections from spinal cord isolated from <i>L. paracasei</i> DSM 13434-, <i>L. plantarum</i> DSM 15312-treated and control EAE animals at day 25 of immunization. In the histogram, each bar represents the mean percentage of stained area (± SEM). * represents a p-value≤0,05.</p

A synergistic combination of probiotics induces Treg cell expansion and systemic IL-10 production.

<p>Healthy C57BL/6 mice were daily fed with <i>L. paracasei</i> DSM 13434, <i>L. plantarum</i> DSM 15312, <i>L. plantarum</i> DSM 15313, a mixture of all three (Lacto-mix) or saline (control) during 14 days (n = 5). MLNs and spleens isolated from these animals were analysed for expression of CD3, CD4, CD25 and Foxp3 by flow cytometry. (A) The proportion of CD4⁺CD25⁺Foxp3⁺ stained cells was examined on the total gated CD3⁺ lymphocytes from MLNs (top) and spleen cells (bottom). Blood serum from these animals was analysed for different cytokines using ELISA kits. Shown are data from (B) IL-10, (C) TGF-β1 and (D) IL-27 analysis. Data are mean ± SEM and representative of two experiments. * represents a p-value ≤0,05 and ** a p-value ≤0,01.</p