Regulatory T (Treg) cells show promise for treating autoimmune diseases, but their induction to elevated potency has been problematic when the most optimally derived cells are from diseased animals. To circumvent reliance on autoantigen-reactive Treg cells, stimulation to myelin-independent Ags may offer a viable alternative while maintaining potency to treat experimental autoimmune encephalomyelitis (EAE). The experimental Salmonella vaccine expressing colonization factor Ag I possesses anti-inflammatory properties and, when applied therapeutically, reduces further development of EAE in SJL mice. To ascertain Treg cell dependency, a kinetic analysis was performed showing increased levels of FoxP3+CD25+CD4+ T cells. Inactivation of these Treg cells resulted in loss of protection. Adoptive transfer of the vaccine-induced Treg cells protected mice against EAE with greater potency than naive or Salmonella vector-induced Treg cells, and cytokine analysis revealed enhanced production of TGF-β, not IL-10. The development of these Treg cells in conjunction with immune deviation by Th2 cells optimally induced protective Treg cells when compared those induced in the absence of Th2 cells. These data show that Treg cells can be induced to high potency to non-disease-inducing Ags using a bacterial vaccine.

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.

Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE), was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to discern whether flagella overexpression could induce similar attenuation. To test its feasibility, flagellar filament subunit FliC and flagellar regulon master regulator FlhDC were overexpressed in Salmonella enterica serovar Typhimurium wild-type strain H71. The results show that the expression of either FliC or FlhDC alone, and co-expression of the two, significantly attenuates Salmonella. The flagellated bacilli were unable to replicate within macrophages and thus were not lethal to mice. In-depth investigation suggests that flagellum-mediated AGE was due to the disruptive effects of flagella on the bacterial membrane, resulting in heightened susceptibilities to hydrogen peroxide and bile. Furthermore, flagellum-attenuated Salmonella elicited elevated immune responses to Salmonella presumably via FliC’s adjuvant effect and conferred robust protection against wild-type Salmonella challenge.

BackgroundExtracts of leaves from Clerodendrum have been used for centuries to treat a variety of medicinal problems in tropical Africa. However, little is known about the high-molecular weight active components conferring therapeutic properties to these extracts.MethodsPolysaccharides from the leaves of Clerodendrum splendens were extracted and fractionated by ion exchange and size-exclusion chromatography. Molecular weight determination, sugar analysis, degree of methyl esterification, and other chemical characterization of the fractions were performed. Immunomodulatory activity of the fractions was evaluated by determining their ability to induce monocyte/macrophage nitric oxide (NO), cytokine production, and mitogen-activated protein kinase (MAPK) phosphorylation. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice, and severity of EAE was monitored in mice treated with intraperitoneal (i.p.) injections of the most active polysaccharide fraction. Lymph nodes (LN) and spleen were harvested, and levels of cytokines in supernatants from LN cells and splenocytes challenged with myelin oligodendrocyte glycoprotein peptide were determined.ResultsFractions containing type II arabinogalactan had potent immunomodulatory activity. Specifically, the high-molecular weight sub-fraction CSP-AU1 (average of 38.5 kDa) induced NO and cytokine [interleukin (IL)-1α, -1β, -6, -10, tumor necrosis factor (TNF; designated previously as TNF-α), and granulocyte macrophage-colony stimulating factor (GM-CSF)] production by human peripheral blood mononuclear cells (PBMCs) and monocyte/macrophages. CSP-AU1-induced secretion of TNF was prevented by Toll-like receptor 4 (TLR4) antagonist LPS-RS, indicating a role for TLR4 signaling. Treatment with CSP-AU1 also induced phosphorylation of a number of MAPKs in human PBMC and activated AP-1/NF-κB. In vivo treatment of mice with CSP-AU1 and CSP-NU1 resulted in increased serum IL-6, IL-10, TNF, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α/CCL3, and MIP-1β/CCL4. CSP-AU1 treatment of mice with EAE (50 mg/kg, i.p., daily, 13 days) resulted in significantly reduced disease severity in this experimental model of multiple sclerosis. Levels of IL-13, TNF, interferon (IFN)-γ, IL-17, and GM-CSF were also significantly decreased, whereas transforming growth factor (TGF)-β was increased in LN cells from CSP-AU1-treated EAE mice.ConclusionsPolysaccharide CSP-AU1 is a potent natural innate immunomodulator with a broad spectrum of agonist activity in vitro and immunosupressive properties after chronic administration in vivo.