Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, for which there is a lack of a clinically approved vaccine in humans. As an intracellular pathogen, Salmonella impacts many cellular pathways. However, the intercellular communication mechanism facilitated by host-derived small extracellular vesicles (EVs), such as exosomes, is an overlooked aspect of the host responses to this infection. We used a comprehensive proteome-based network analysis of exosomes derived from Salmonella-infected macrophages to identify host molecules that are trafficked via these EVs. This analysis predicted that the host-derived small EVs generated during macrophage infection stimulate macrophages and promote activation of T helper 1 (Th1) cells. We identified that exosomes generated during infection contain Salmonella proteins, including unique antigens previously shown to stimulate protective immune responses against Salmonella in murine studies. Furthermore, we showed that host EVs formed upon infection stimulate a mucosal immune response against Salmonella infection when delivered intranasally to BALB/c mice, a route of antigen administration known to initiate mucosal immunity. Specifically, the administration of these vesicles to animals stimulated the production of anti-Salmonella IgG antibodies, such as anti-OmpA antibodies. Exosomes also stimulated antigen-specific cell-mediated immunity. In particular, splenic mononuclear cells isolated from mice administered with exosomes derived from Salmonella-infected antigen-presenting cells increased CD4+ T cells secreting Th1-type cytokines in response to Salmonella antigens. These results demonstrate that small EVs, formed during infection, contribute to Th1 cell bias in the anti-Salmonella responses. Collectively, this study helps to unravel the role of host-derived small EVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria. Understanding the EV-mediated defense mechanisms will allow the development of future approaches to combat bacterial infections.

Brucellosis remains the most common zoonotic disease globally. Currently no vaccines for humans exist, and conventional brucellosis vaccines for livestock fail to confer complete protection; hence, an improved vaccine is needed. Although Brucella infections primarily occur following a mucosal exposure, vaccines are administered parenterally. Few studies have considered mucosal vaccinations, or even targeting of tissue-resident memory T (T_RM) cells. T_RM cells protect against viral infections, but less is known of their role in bacterial infections, and even less for brucellosis. Oral prime, nasal boost with a newly developed Brucella abortus double mutant (znBAZ) confers nearly complete protection against pulmonary challenge with wild-type (wt) B. abortus 2308, and its protective efficacy is >2800-fold better than the RB51 vaccine. Vaccination with znBAZ potently stimulated CD8⁺ T cells, whereas mucosal vaccination with RB51 induced mostly CD4⁺ T cells. Subsequent analysis revealed these pulmonary CD44⁺ CD69⁺ CD8⁺ T cells to be either CD103⁺ or CD103^- T_RM cells, and these sequestered to the lung parenchyma as CXCR3^lo and to the airways as CXCR3^hi. Both CD8⁺ T_RM subsets contained single-positive IFN-γ and TNF-α, as well as, polyfunctional cells. IL-17-producing CD8⁺ T_RM cells were also induced by znBAZ vaccination, but in vivo IL-17 neutralization had no impact upon protection. In vivo depletion of CD4⁺ T cells had no impact upon protection in znBAZ-vaccinated mice. In contrast, CD4⁺ T cell depletion reduced RB51’s protective efficacy in spleens and lungs by two- and three-logs, respectively. Although anti-CD8 mAb-treated znBAZ-vaccinated mice showed a significantly reduced pulmonary efficacy, this treatment failed to completely deplete the lung CD8⁺ T cells, leaving the CD103⁺ and CD103^- CD8⁺ T_RM cell ratios intact. Only znBAZ-vaccinated CD8^-/- mice were fully sensitive to pulmonary challenge with virulent wt B. abortus 2308 since CD8⁺ T_RM cells could not be induced. Collectively, these data demonstrate the key role of mucosal vaccination for the generation of CD8⁺ T_RM cells in protecting against pulmonary challenge with virulent B. abortus. Brucellosis is the most common zoonotic disease worldwide and is transmitted via the consumption of unpasteurized dairy products or exposure to Brucella-laden aerosols. In fact, mucosal exposure is the most common route of infection for humans and animals, yet parenteral vaccination of livestock remains the preferred route of immunization. To determine whether development of a mucosal vaccination regimen could effectively generate immunity against pulmonary challenge with virulent B. abortus, a double-mutant B. abortus vaccine was administered mucosally, and found to induce CD8⁺ T_RM cells. These conferred complete protection against pulmonary infection and prevented systemic brucellae spread even in the absence of immune recirculating CD8⁺ T cells. These data show that mucosal vaccination can stimulate the induction of T_RM cells, which should be considered as a more effective means to protect against brucellosis. Furthermore, brucellosis needs to be considered a mucosal pathogen to warrant development of approaches different from conventional methods to protect humans and livestock against this disease.

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.

Background: Extracts of leaves fromClerodendrumhave 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.Methods: Polysaccharides from the leaves ofClerodendrum splendenswere 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.Results: Fractions containing type II arabinogalactan had potent immunomodulatory activity. Specifically, the high-molecular weight sub-fractionCSP-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 withCSP-AU1also induced phosphorylation of a number of MAPKs in human PBMC and activated AP-1/NF-κB.In vivotreatment of mice withCSP-AU1andCSP-NU1resulted 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-AU1treatment 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 fromCSP-AU1-treated EAE mice.Conclusions: PolysaccharideCSP-AU1is a potent natural innate immunomodulator with a broad spectrum of agonist activityin vitroand immunosupressive properties after chronic administrationin vivo.

Brucellosis remains a significant zoonotic threat worldwide. Humans and animals acquire infection via their oropharynx and upper respiratory tract following oral or aerosol exposure. After mucosal infection, brucellosis develops into a systemic disease. Mucosal vaccination could offer a viable alternative to conventional injection practices to deter disease. Using a nasal vaccination approach, the ΔznuA B. melitensis was found to confer potent protection against pulmonary Brucella challenge, and reduce colonization of spleens and lungs by more than 2500‐fold, with >50% of vaccinated mice showing no detectable brucellae. Furthermore, 10‐fold more brucellae‐specific, interferon‐γ (IFN‐γ)‐producing CD8⁺ T cells than CD4⁺ T cells were induced in the spleen and respiratory lymph nodes. Evaluation of pulmonary and splenic CD8⁺ T cells from mice vaccinated with ΔznuA B. melitensis revealed that these expressed an activated effector memory (CD44^hiCD62L^loCCR7^lo) T cells producing elevated levels of IFN‐γ, tumor necrosis factor‐α, perforin and granzyme B. To assess the relative importance of these increased numbers of CD8⁺ T cells, CD8^−/− mice were challenged with virulent B. melitensis, and they showed markedly increased bacterial loads in organs in contrast to similarly challenged CD4^−/− mice. Only ΔznuA B. melitensis ‐ and Rev‐1‐vaccinated CD4^−/− and wild‐type mice, not CD8^−/− mice, were completely protected against Brucella challenge. Determination of cytokines responsible for conferring protection showed the relative importance of IFN‐γ, but not interleukin‐17 (IL‐17). Unlike wild‐type (wt) mice, IL‐17 was greatly induced in IFN‐γ^−/− mice, but IL‐17 could not substitute for IFN‐γ's protection, although an increase in brucellae dissemination was observed upon in vivo IL‐17 neutralization. These results show that nasal ΔznuA B. melitensis vaccination represents an attractive means to stimulate systemic and mucosal immune protection via CD8⁺ T‐cell engagement.