Oral Salmonella expressing Colonization Factor Antigen I stimulates the rapid induction of Galectin-1 on induced Treg cells (P4204)

Abstract Galectin-1 (Gal-1) is one of the 15 Galectin family members that bind to β-galactoside. Gal-1 binding to various cell surface glycoproteins leads to deletion of T effector functions. Gal-1 engagement to CD43 on APCs converts these cells to tolerogenic supporting the generation of Treg cells. Our previous work have shown that oral Salmonella-CFA/I strain H696 (expresses CFA/I fimbriae from enterotoxigenic E. coli) induces a highly suppressive Foxp3+ CD25+CD4+ Treg cells capable of suppressing the development of experimental autoimmune encephalomyelitis (EAE). Gal-1 expression was rapidly induced within 3 days by both Foxp3+ Treg cells obtained from Salmonella-CFA/I-immunized mice unlike mice vaccinated with isogenic Salmonella vaccine (strain H647) control. To assess the function of Gal-1, isolated Treg cells from H696- and H647-vaccinated mice were assessed for their ability to suppress OT-2 OVA-specific effector T cell proliferation following r estimulation with OVA peptide. Upon neutralization of Gal-1, Salmonella-CFA/I-induced Treg cells were unable to suppress OT-2 cell proliferation; similar neutralization of Salmonella vector-derived Treg cells had no effect. Kinetic analysis of Gal-1 expression by dendritic cells (DCs) following H696 imminization also showed early induction of Gal-1 suggesting the importance of Gal-1 to induce tolerogenic T cells and DCs. These results suggest Gal-1 is a major effector molecule for the Salmonella-CFA/I-induced Treg cells.</jats:p

An Oral Bacterial-Based Diarrheal Vaccine Stimulates TGF-β-Producing Regulatory T (Treg) Cells and Confers Protection Against Experimental Autoimmune Encephalomyelitis (EAE) (47.4)

Abstract Oral Salmonella vaccine expressing enterotoxigenic E. coli colonization factor antigen 1 (CFA/I) fimbriae induced production of Treg cells. Adoptive transfer of CD25+ Treg cells obtained from oral Salmonella-CFA/I-vaccinated mice conferred superior protection to proteolipid protein (PLP)139-151-dependent EAE to those from Salmonella vector-immunized mice; this was attributed to their differential cytokine profiles. Salmonella-CFA/I-vaccinated mice produced more TGF-β than Salmonella vector-immunized mice. To test the hypothesis Salmonella-CFA/I-induced Treg cells, independent of PLP-specificity, confers protection via TGF-β, SJL/J mice were immunized with either Salmonella-CFA/I or Salmonella vector. Two wks later, CD25+ and CD25- CD4+ T cells were sorted, adoptively transferred to naïve SJL/J mice, concomitantly treated with anti-TGF-β mAb or isotype IgG, and then induced with EAE. Mice adoptively transferred with Treg cells neutralized of their TGF-β showed earlier disease onset and greater disease severity than mice adoptively transferred with Treg cells treated with IgG control. Moreover, greater proinflammatory cytokine production was observed. This work shows Treg cells can be induced to high potency by simply vaccinating against irrelevant Ags using a live, bacterial vaccine, thus, offering a novel approach to treat autoimmune diseases independent of the auto-Ag. Supported by NIH AI-41123.</jats:p

Vulnerabilities in Yersinia pestis caf Operon Are Unveiled by a Salmonella Vector

During infection, Yersinia pestis uses its F1 capsule to enhance survival and cause virulence to mammalian host. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. To hold this detrimental effect under proper control, Y. pestis expresses the caf operon (encoding the F1 capsule) in a temperaturedependent manner. However, additional properties of the caf operon limit its expression. By overexpressing the caf operon in wild-type Salmonella enterica serovar Typhimurium under a potent promoter, virulence of Salmonella was greatly attenuated both in vitro and in vivo. In contrast, expression of the caf operon under the regulation of its native promoter exhibited negligible impairment of Salmonellae virulence. In-depth investigation revealed all individual genes in the caf operon attenuated Salmonella when overexpressed. The deleterious effects of caf operon and the caf individual genes were further confirmed when they were overexpressed in Y. pestis KIM6+. This study suggests that by using a weak inducible promoter, the detrimental effects of the caf operon are minimally manifested in Y. pestis. Thus, through tight regulation of the caf operon, Y. pestis precisely balances its capsular anti-phagocytic properties with the detrimental effects of caf durin

A Live Diarrheal Vaccine Imprints a Th2 Cell Bias and Acts as an Anti-Inflammatory Vaccine

Abstract An experimental vaccine for enterotoxigenic Escherichia coli (ETEC) composed of a live, attenuated Salmonella vector-expressing enterotoxigenic E. coli fimbriae, colonization factor Ag I (CFA/I), stimulated a biphasic Th cell response when given orally and suppressed the normally produced proinflammatory response. Such suppression was also evident upon the Salmonella-CFA/I infection of macrophages resulting in diminished TNF-α, IL-1, and IL-6 production and suggesting that the CFA/I fimbrial expression by Salmonella may protect against a proinflammatory disease. To test this hypothesis, SJL/J mice were vaccinated with Salmonella-CFA/I construct 1 or 4 wk before induction of experimental autoimmune encephalomyelitis using an encephalitogenic proteolipid protein peptide, PLP139–151. Mice receiving Salmonella-CFA/I vaccine recovered completely from mild acute clinical disease and showed only mild inflammatory infiltrates in the spinal cord white and gray matter. This protective effect was accompanied by a loss of encephalitogenic IFN-γ-secreting Th cells and was replaced with an increase in IL-4, IL-10, and IL-13 secretion. Collectively, these data suggested that Salmonella-CFA/I is an anti-inflammatory vaccine that down-regulates proinflammatory cells and confers protection against a proinflammatory disease, experimental autoimmune encephalomyelitis, via immune deviation.</jats:p

Milk-based nutraceutical for treating autoimmune arthritis via the stimulation of IL-10- and TGF-β-producing CD39+ regulatory T cells.

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39+ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I

Bacterial strains, plasmids, and their characteristics.

<p>Bacterial strains, plasmids, and their characteristics.</p

Two doses of <i>L</i>. <i>lactis</i>-CFA/I fermented milk protect against CIA.

<p>Groups of C57BL/6 mice (n = 8/group) were CII-challenged on day 0, and treated twice on days 18 and 22 with <b>(A)</b><i>L</i>. <i>lactis</i>-CFA/I- or <i>L</i>. <i>lactis</i> vector-fermented milk that contained 2.5×10⁸ CFUs or treated with sterile PBS. Mice were monitored for disease until day 39 measuring <b>(B)</b> average clinical score <b>(C)</b> and incidence of arthritis. Arrows indicate days of fermented milk administration; * <i>p</i> ≤ 0.001 as compared to each control group. <b>(D-G)</b> LN CD4⁺ T cells isolated from each treatment group were restimulated with 50 μg CII in the presence of mitomycin C-treated Ag-presenting cells for 4 days. CIA mice treated with <i>L</i>. <i>lactis</i>-CFA/I fermented milk showed reduced LN <b>(D)</b> IFN-γ and <b>(E)</b> IL-17 production with concomitant increases in <b>(F)</b> IL-10 and <b>(G)</b> TGF-β; * <i>p</i> ≤ 0.001, ** <i>p</i> < 0.05 versus PBS-treated mice; and ^<i>+</i><i>p</i> ≤ 0.001 versus <i>L</i>. <i>lactis</i> vector-treated mice.</p

Effect of overexpression of <i>caf</i> operon or individual <i>caf</i> genes on <i>Yersinia</i> antimicrobial and temperature susceptibilities.

<p>Six strains of KIM6+/pF1, pHF, pSA, pSF1, pSM, and pY were analyzed for their sensitivities to (<b>A</b>) erythromycin, (<b>B</b>) PMB, (<b>C</b>) hydrogen peroxide, (<b>D</b>) bile salt, (<b>E</b>) temperature of 37°C, and (<b>F</b>) bile salt combined with temperature of 37°C. (<b>A</b>, <b>B</b>) The erythromycin and PMB MICs of the 6 strains were respectively determined. (<b>C</b>) The 6 strains were incubated with 2.5 mM H₂O₂ for 1 hr and the survival percentages were determined in comparison with the non-treated controls, respectively. (<b>D</b>) The 6 strains were dropped onto BHI agar containing 1% bile and were incubated at 27°C for 48 hrs for CFU enumeration in comparison with those grown on BHI agar without bile, respectively. (<b>E</b>) The 6 strains were dropped onto BHI agar and were incubated at 37°C for CFU enumeration in comparison with those grown on BHI agar at 27°C, respectively. (<b>F</b>) The 6 strains were dropped onto BHI agar containing 1% bile and were incubated at 37°C for CFU enumeration in comparison with those grown on BHI agar without bile at 27°C, respectively. All experiments (<b>A</b> to <b>F</b>) were statistically analyzed for significant differences among these 6 strains using the Tukey Kramer multiple comparisons test with * <i>P</i><0.05, ** <i>P</i><0.01, and *** <i>P</i><0.001. Depicted (<b>A</b> to <b>F</b>) are the mean ± SEM of three independent experiments.</p