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IL-35, a relatively newly discovered cytokine belonging to the larger IL-12 family, shows unique anti-inflammatory properties, believed to be associated with dedicated receptors and signaling pathways. IL-35 plays a pivotal role in the development and the function of both regulatory B (Bregs) and T cells (Tregs). In order to further its therapeutic potential, a dairy Lactococcus lactis strain was engineered to express murine IL-35 (LL-IL35), and this recombinant strain was applied to suppress collagen-induced arthritis (CIA). Oral administration of LL-IL35 effectively reduced the incidence and disease severity of CIA. When administered therapeutically, LL-IL35 abruptly halted CIA progression with no increase in disease severity by reducing neutrophil influx into the joints. LL-IL35 treatment reduced IFN-γ and IL-17 3.7- and 8.5-fold, respectively, and increased IL-10 production compared to diseased mice. Foxp3 + and Foxp3 - CD39 + CD4 + T cells were previously shown to be the Tregs responsible for conferring protection against CIA. Inquiry into their induction revealed that both CCR6 + and CCR6 - Foxp3 +or- CD39 + CD4 + T cells act as the source of the IL-10 induced by LL-IL35. Thus, this study demonstrates the feasibility and benefits of engineered probiotics for treating autoimmune diseases.

Regulatory T cells (Tregs) induced during autoimmunity often become quiescent and unable to resolve disease, suggesting inadequate activation. Resolution of established experimental autoimmune encephalomyelitis (EAE) can be achieved with myelin oligodendrocyte glycoprotein (MOG) fused to reovirus protein σ1 (MOG-pσ1), which activates Tregs, restoring protection, but requiring other regulatory cells to revitalize them. B cells have a dichotomous role in both the pathogenesis and recovery from EAE. Although inflammatory B cells contribute to EAE's pathogenesis, treatment of EAE mice with MOG-pσ1, but not OVA-pσ1, resulted in an influx of IL-10-producing B220(+)CD5(+) B regulatory cells (Bregs) enabling Tregs to recover their inhibitory activity, and in turn, leading to the rapid amelioration of EAE. These findings implicate direct interactions between Bregs and Tregs to facilitate this recovery. Adoptive transfer of B220(+)CD5(-) B cells from MOG-pσ1-treated EAE or Bregs from PBS-treated EAE mice did not resolve disease, whereas the adoptive transfer of MOG-pσ1-induced B220(+)CD5(+) Bregs greatly ameliorated EAE. MOG-pσ1-, but not OVA-pσ1-induced IL-10-producing Bregs, expressed elevated levels of B and T lymphocyte attenuator (BTLA) relative to CD5(-) B cells, as opposed to Tregs or effector T (Teff) cells, whose BTLA expression was not affected. These induced Bregs restored EAE Treg function in a BTLA-dependent manner. BTLA(-/-) mice showed more pronounced EAE with fewer Tregs, but upon adoptive transfer of MOG-pσ1-induced BTLA(+) Bregs, BTLA(-/-) mice were protected against EAE. Hence, this evidence shows the importance of BTLA in activating Tregs to facilitate recovery from EAE., (Copyright © 2016 by The American Association of Immunologists, Inc.)

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.

To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25(+) Treg cells are stimulated, but for arthritis CD39(+) Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-β and interleukin-10.

To assess the potency of regulatory T (Treg) cells induced against an irrelevant Ag, mice were orally vaccinated with Salmonella expressing Escherichia coli colonization factor antigen I fimbriae. Isolated CD25⁺ and CD25⁻CD4⁺ T cells were adoptively transferred to naive mice, and Treg cells effectively protected against experimental autoimmune encephalomyelitis (EAE), unlike Treg cells from Salmonella vector-immunized mice. This protection was abrogated upon in vivo neutralization of TGF-β, resulting in elevated IL-17 and loss of IL-4 and IL-10 production. Thus, Treg cells induced to irrelevant Ags offer a novel approach to treat autoimmune diseases independent of auto-Ag., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Stimulation of Ag-specific inducible Treg can enhance resolution of autoimmune disease. Conventional methods to induce Treg often require induction of autoimmune disease or subjection to infection. Reovirus adhesin, protein σ1 (pσ1), can successfully facilitate tolerance when fused to a tolerogen. We tested whether myelin oligodendrocyte glycoprotein (MOG) fused to pσ1 (MOG-pσ1) can stimulate Ag-specific Treg. We show that C57BL/6 mice treated nasally with MOG-pσ1 fail to induce MOG-specific Abs and delayed-type hypersensitivity (DTH) responses and resist EAE. Such resistance was attributed to stimulation of Foxp3(+) Treg, as well as Th2 cells. MOG-pσ1's protective capacity was abrogated in IL-10(-/-) mice, but restored when adoptively transferred with MOG-pσ1-induced Treg. As a therapeutic, MOG-pσ1 diminished EAE within 24 h of nasal application, unlike recombinant MOG (rMOG), pσ1, or pσ1+rMOG, implicating the importance of Ag specificity by pσ1-based therapeutics. MOG-pσ1-treated mice showed elevated IL-4, IL-10, and IL-28 production by CD4(+) T cells, unlike rMOG treated or control mice that produced elevated IFN-γ or IL-17, respectively. These data show the feasibility of using pσ1 as a tolerogen platform for Ag-specific tolerance induction and highlight its potential use as an immunotherapeutic for autoimmunity., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

IL-35 is produced by regulatory T cells, and this novel cytokine can downregulate Th17 cell development and inhibit autoimmune inflammation. In this work, an rIL-35, as a single-chain fusion between murine IL-12p35 and EBV-induced gene 3, was expressed in yeast. This rIL-35 inhibited OVA-specific cellular and Ab responses in OVA-challenged recipients of DO11.10 CD4+ T cells. Likewise, IL-35 inhibited clinical manifestation of collagen-induced arthritis or could cease further disease exacerbation upon initiation of IL-35 treatment. Exogenous IL-35 treatments suppressed Th1 and Th17 cells and promoted CD39 expression by CD4+ T cells. Sorted CD25-CD39+CD4+ T cells from IL-35-treated mice produced IL-10 and, upon adoptive transfer, were sufficiently potent to inhibit subsequent development of inflammation in mice with collagen-induced arthritis, whereas sorted CD25+CD39+CD4+ T cells showed reduced potency. IL-35 treatments of IL-10-/- mice failed to induce protective CD39+CD4+ T cells, demonstrating the effector role of IL-10 by IL-35 immunosuppression.

Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen uptake using reovirus adhesin, protein sigma 1 (psigma1), may circumvent these shortcomings based upon the recent finding that when reovirus psigma1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP(130-151)) was genetically fused to OVA to psigma1 (PLP:OVA-psigma1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10(+) forkhead box P3 (FoxP3)(+) CD25(+)CD4(+) T(reg) and IL-4(+)CD25(-)CD4(+) Th2 cells. IL-10R or IL-4 neutralization reversed protection to EAE conferred by PLP:OVA-psigma1, and adoptive transfer of Ag-specific T(reg) or Th2 cells restored protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired PLP:OVA-psigma1's protective capacity, triggering TGF-beta-mediated inflammation; however, concomitant inactivation of TGF-beta and CD25 reestablished PLP:OVA-psigma1-mediated protection by IL-28-producing FoxP3(+)CD25(-)CD4(+) T cells. Thus, psigma1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity.

Anti-inflammation immunotherapy has been successfully applied for the treatment of autoimmune diseases. Mucosal vaccines against autoimmune disorders are beneficial by influencing the regulatory compartment of gut and systemic adaptive immune systems. A Salmonella vector expressing colonization factor Ag I (CFA/I), shown to behave as an anti-inflammatory vaccine, stimulates the production of CD4(+)CD25(+) T cells and regulatory cytokines. In this work, we queried whether Salmonella-CFA/I can protect DBA/1 mice from collagen-induced arthritis. The incidence of arthritis and cartilage loss in vaccinated DBA/1 mice was remarkably lower when compared with unprotected mice. Clinical findings were accompanied by the suppression of inflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-27. Vaccination evoked a multi-tier response consisting of IL-4 producing Th2 cells, an increased production of TGF-beta by CD4(+) T cells, and suppression of collagen II-specific CD4(+) T cell proliferation. To assess the contribution of Salmonella-CFA/I-primed CD4(+) T cells, adoptive transfer studies with total CD4(+), CD4(+)CD25(-), or CD4(+)CD25(+) T cells were performed 15 days postchallenge. Mice receiving either subset showed reduced disease incidence and low clinical scores; however, mice receiving total CD4(+) T cells showed delayed disease onset by 10 days with reduced clinical scores, reduced IL-17 and IL-27, but enhanced IL-4, IL-10, IL-13, and TGF-beta. Inhibition of TGF-beta or IL-4 compromised protective immunity. These data show that Salmonella-CFA/I vaccination of DBA/1 mice protects against collagen-induced arthritis by stimulating TGF-beta- and IL-4-producing regulatory CD4(+) T cells.

Treatment with an anti-inflammatory Salmonella vaccine expressing enterotoxigenic Escherichia coli colonization factor Ag 1 (CFA/I) proved effective in stimulating protective, potent CD25(+)CD4(+) regulatory T (T(reg)) cells in susceptible mice challenged with experimental autoimmune encephalomyelitis (EAE). Because the Salmonella vector was considerably less protective, we questioned whether altering fimbrial subunit expression to resemble conventional Salmonella expression may impact T(reg) cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/I(IC)). SJL mice were challenged with proteolipid protein peptide 139-151 to induce EAE and orally treated with one of three Salmonella vaccines 6 days postchallenge. Treatment with Salmonella-CFA/I(IC) greatly reduced clinical disease, similarly as Salmonella-CFA/I, by subduing IL-17 and IL-21; however, mechanisms of protection differed as evident by increased IL-13 and IFN-gamma but diminished TGF-beta production by T(reg) cells from Salmonella-CFA/I(IC)-treated mice. Adoptive transfer of T(reg) cells from both CFA/I-expressing constructs was equivalent in protecting against EAE, showing minimal disease. Although not as potent in its protection, CD25(-)CD4(+) T cells from Salmonella-CFA/I(IC) showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN-gamma neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/I(IC)-induced T(reg) cells. Thus, the Salmonella-CFA/I(IC) vaccine elicits T(reg) cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these T(reg) cells can be induced to high potency by simply vaccinating against irrelevant Ags, offering a novel approach to treat autoimmune diseases independently of the autoantigen.

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein sigma1 (psigma1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via psigma1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus psigma1, termed OVA-psigma1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-psigma1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-psigma1 could be adoptively transferred using cervical lymph node CD4(+) T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-beta1, with greatly reduced IL-17 and IFN-gamma. The induced IL-10 was derived predominantly from FoxP3(+)CD25(+)CD4(+) T cells. No FoxP3(+)CD25(+)CD4(+) T cells were induced in OVA-psigma1-dosed IL-10-deficient (IL-10(-/-)) mice, and despite showing increased TGF-beta1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using psigma1 as a mucosal delivery platform specifically for low-dose tolerance induction.

Regulatory T (T(reg)) 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 T(reg) 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 T(reg) cell dependency, a kinetic analysis was performed showing increased levels of FoxP3(+)CD25(+)CD4(+) T cells. Inactivation of these T(reg) cells resulted in loss of protection. Adoptive transfer of the vaccine-induced T(reg) cells protected mice against EAE with greater potency than naive or Salmonella vector-induced T(reg) cells, and cytokine analysis revealed enhanced production of TGF-beta, not IL-10. The development of these T(reg) cells in conjunction with immune deviation by Th2 cells optimally induced protective T(reg) cells when compared those induced in the absence of Th2 cells. These data show that T(reg) cells can be induced to high potency to non-disease-inducing Ags using a bacterial vaccine.