La percepción australiana de sus núcleos despoblados se percibe allí como un reto lleno de potencialidades, ya que históricamente se han considerado sus desérticas llanuras una tierra hostil, donde sin embargo se podía empezar una nueva y mejor vida con esfuerzo. En el Viejo Continente, nuestras zonas despobladas se miran con recelo, como si una decadencia inevitable se fuera a apoderar de ellas e incluso de forma inconsciente se tiende a denostar a sus habitantes por falta de ambición. La idea que se plantea es la liberación de esa percepción negativista y denostadora para poder llegar a una apreciación del estilo de vida libre y sostenible que se puede tener en nuestros núcleos despoblados. Este cambio de paradigma puede destapar el enorme poder de transformación social de estos territorios.

Mucosal vaccination with a live attenuated B. melitensis double-mutant (BMDM) confers sterilizing protection against pulmonary challenge with wild-type (wt) BM 16M. Although T cell-dependent immunity is essential for protection, we queried the role of B cells in this response. We hypothesized that B cells enable BMDM to persist in the lungs for stimulation of robust adaptive responses. Using BALB/c B cell-deficient (μMT) mice, early and long-term immunity subsequent mucosal vaccination was examined. Groups of BALB/c and μMT mice were orally primed, nasally boosted, and 4 wks later, splenic and lung lymphocytes were antigen-pulsed to determine cytokine responses. μMT mice showed significantly reduced pulmonary proinflammatory responses by three-fold relative to BALB/c mice. Nasal challenge with wt BM 16M of BMDM-vaccinated mice resulted in impaired protective immunity in μMT mice evidenced by the significantly increased brucellae colonization of lungs and spleens. To determine the types of myeloid cells involved in brucellae recognition, μMT mice nasally vaccinated with a mCherry variant of BMDM showed a 23-fold reduction in lung mCherry expression relative to similarly vaccinated BALB/c mice at 5 days post-immunization. mCherry expression associated with various myeloid and B cells. Interestingly, although antigen-presenting cells (APCs) increased similarly in both groups of mice, mCherry expression in lung neutrophils and monocyte-derived macrophages in BALB/c mice was 23- and 4-fold enhanced, respectively, to μMT mice. We believe B cell retention of BMDM mutant enhances APC activity within the lungs facilitating the development of potent local T cell responses. Work supported by NIH R01 AI123244 and AI124244-03S1.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing pancreatic β cells are destroyed by auto-aggressive T cells. Tregs in T1D have been shown to lose their suppressive function and/or become inflammatory, complicating Treg immunotherapy. Oral treatment with the adhesin from enterotoxigenic E. coli, CFA/I fimbriae, protects against autoimmunity. Although CFA/I fimbriae act in a bystander or in an antigen (Ag)-independent fashion, protection is ultimately dependent upon the induction and/or activation of auto-Ag-specific Tregs. Our initial findings showed that oral dosing with a Lactococcus lactis vector expressing CFA/I fimbriae (LL-CFA/I) reduces incidence of T1D in NOD mice by 45%. We hypothesized that dosing NOD mice more frequently would induce Foxp3+ Tregs in the pancreas and PaLNs to provide more robust protection. To test this, 4 wk-old NOD mice were orally dosed with LL-CFA/I varying the dosage and frequency of treatment. When dosed every 2 wks, the frequency of insulitis was reduced by more than half at 11 wks of age, and tetramer staining revealed a 2-fold reduction of insulin-specific CD4+ and CD8+ T cells within the PaLNs. Additional groups were followed until 16 wks of age, and examined for Treg and inflammatory T cell phenotypes. Treatment with LL-CFA/I reduced 2-fold IFN-γ-producing CD8+ T cells in the spleen and PaLNs. PaLN Foxp3+ T cells from the PBS and vector control groups, but not the LL-CFA/I-treated mice, co-expressed Tbet and IFN-γ, suggesting that Tregs from LL-CFA/I treated mice maintained their regulatory function at 16 wks. These data show that orally dosing with LL-CFA/I diminishes or halts T1D progression via activation of stably functional Foxp3+ Tregs. Work supported by AI121745.

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.

Interleukin-35 (IL-35), consisting of two subunits, Epstein-Barr virus-induced gene 3 (EBI3) and p35, is a novel anti-inflammatory responsive cytokine in IL-12 cytokine family. IL-35 is produced by...

Orally delivered colonization factor antigen I (CFA/I) fimbriae stimulate IL-35 producing Foxp3+CD39+CD4+ regulatory T cells (Tregs) essential against CIA in C57BL/6 mice. To begin to understand how CFA/I-induced IL-35 mediates protection, recent studies have begun to examine immunosuppressive mediators by dendritic cells (DCs). It was found that IDO was induced in splenic, mesenteric lymph nodes (MLNs), and inguinal, axillary, and iliac lymph nodes (LNs) DCs in protected mice. We hypothesize that CFA/I-induced IL-35 could directly regulate development of IDO+ DCs to sustain anti-inflammatory responses. By flow cytometry, recombinant IL-35 was found capable of binding DCs in vitro suggesting direct action of IL-35 upon DCs. Mice deficient in IL-35 (EBI3-/-) were unresponsive to CFA/I fimbriae and unable to induce IDO. Similar results were observed in CD39-/- mice correlating with the lack of clinical interventions. To inhibit IL-35's impact, CFA/I- or PBS-dosed C57BL/6 mice were treated in vivo with rabbit IgG anti-mouse IL-35 during arthritis manifestation resulting in 50% suppression of intracellular IDO expression along with significantly enhanced CIA severity when compared to nonimmune IgG-treated mice. Adoptive transfer of CFA/I-induced CD39+CD4+ Tregs to mice induced with CIA elevated IDO expression by DCs. Thus, our findings demonstrate the effect of induced IL-35 by Tregs upon IDO+ DCs in conferring protection against CIA. Supported by NIH P01 AT-04986.

Immune responses in the aerodigestive tract are characterized by production and transport of specific IgA antibodies across the epithelium to act as a first line of defense against pathogens in the external environment. To sample antigens on mucosal surfaces in the intestine and upper respiratory tract, the immune system relies on a close collaboration between specialized antigen-sampling epithelial M cells and lymphoid cells. Depending on various factors, local antigen presentation in the mucosal tissue leads to tolerance or initiation of an active immune response. Recently, molecules that could be used to target vaccine antigens to apical M cell surfaces have been identified. Here we review the M cell-targeted vaccine strategy, an approach that could be used to enhance uptake and efficacy of vaccines delivered in the nasal cavity or intestine.

Abstract Mucosal intervention with CFA/I fimbriae effectively diminishes CIA by stimulation of TGF-β+ and IL-10+ regulatory CD39+CD4+ T cells (Tregs), resulting in suppression of Th1 and Th17 cells and IL-27. CFA/I also stimulates Tregs expressing p35 and EBI3 subunits simultaneously, suggesting an essential role of IL-35 in CFA/I-mediated protection. We hypothesize CFA/I fimbriae stimulate endogenous IL-35 production to facilitate protection against autoimmune disease. In testing the relevance of endogenous IL-35 for protection, oral delivery of recombinant CFA/I into EBI3-/- mice on day 14 after CIA induction was found ineffective against CIA compared to similarly treated WT mice, as evidenced by the lack of suppression of IL-17 and IFN-γ (the latter increased 5-fold ). CFA/I-treated EBI3-/- mice lacked enhanced CREB phosphorylation that, in turn, failed to stimulate the transcription of CD39 (apyrase). In fact, surface apyrase activity by EBI3-/- CD4+ T cells was uninduced. Because of the lack of induced CD39 expression, IL-10 production was greatly diminished. Clinical improvement of CIA in CFA/I-treated EBI3-/- mice required further intervention with recombinant IL-35, resulting in the induction of CD39+CD4+ T cells, enhanced TGF-β production, and reduction in IFN-γ and IL-17. Thus, in the absence of endogenous IL-35, mucosal CFA/I fimbriae intervention is ineffective against autoimmune arthritis. Supported by AT-004986.

Infection of the joints is the most frequent localized manifestation of brucellosis, which is a common cause of infectious arthritis. However, no experimental murine model of Brucella-induced arthritis has been reported. Here we report that IFN-γ-/- mice develop joint inflammation following oral, nasal, or parenteral infection with B. abortus or B. melitensis. Joints from Brucella-infected IFN-γ-/-, but not wild-type mice, were found to contain extensive inflammatory infiltrates and debris within the joint space which co-localized with brucellae. Osteoarthritis, joint space narrowing, necrosis, soft tissue inflammation, and substantial Brucella burdens were also observed, although antibody or cytokine responses against collagen were not detected. Elevated IL-1β, but not TNF-α, IL-6, nor IL-17, was detected in the joints of Brucella-infected IFN-γ-/- mice. A six-week regimen of rifampicin effectively cleared infection and halted further progression of inflammation, although some symptoms and swelling remained. However, administration of an adenovirus expressing the IL-1receptor antagonist augmented antibiotic resolution of joint swelling by >50%. These results show that the IFN-γ-/- mouse represents a useful model to study the pathogenesis of joint inflammation due to Brucella infection, and that intervention strategies targeting IL-1 can complement antibiotic treatment of Brucella-induced inflammation. Supported by USDA 2007-01612 and USDA 2009-34397-20133.

Abstract Salmonella vaccine vector expressing enterotoxigenic E. coli CFA/I fimbriae has been shown to protect against EAE and CIA. Vaccine-induced CD25+CD4+ and CD25-CD4+ T cells were potent against CIA, and such protection was reversed by anti-TGF-β treatment. We demonstrate, Salmonella-CFA/I-stimulated CD39+CD4+ T cells correlate with accelerated hydrolysis of extracellular ATP, a mechanism of immune suppression. Since vaccine-induced FoxP3-CD39+CD25-CD4+ T cells are shown to be the source of TGF-β, we hypothesized TGF-β could be important in regulating CD39 expression and consequential suppression of CIA. Salmonella-CFA/I-induced CD4+ T cells featured enhanced activation of cAMP-response element-binding protein (CREB), essential for transcriptional regulation of CD39. CREB phosphorylation was suppressed by in vivo treatment of Salmonella-CFA/I-immunized mice with anti-TGF-β mAb. Decrease in phosphorylated CREB correlated with 2-fold less CD39+CD4+ T cells. Although Salmonella-CFA/I-induced FoxP3-CD4+ T cells protected against CIA, as well as CD39+CD4+ T cells upon adoptive transfer, subsequent FACS analysis of recipients’ CD4+ T cells revealed conversion of FoxP3-(GFP-) cells to FoxP3+. Thus, Salmonella-CFA/I stimulates TGF-β production by FoxP3-CD39+CD4+ T cells, promoting the CREB-dependent expansion of CD39+CD4+ T cells becoming regulatory FoxP3+CD4+ T cells. Supported by AT004312.

Abstract As a novel heterodimeric cytokine and one of the IL-12 family members, IL-35 is composed of IL-12p35 subunit and Epstein-Barr virus-induced gene 3 (EBI3) protein. IL-35 is known to play an essential role in immune regulation of the CD4+CD25+ Treg cells, alleviating inflammatory responses. We have developed the recombinant fusion construct of mouse IL-35 (mIL-35)-hIgG1 Fc (hFc), in which EBI3 and IL-12p35 are joined by a flexible linker of (Gly4Ser)3. The mIg k-chain leader sequence at the amino terminus also allows secretion of the fusion protein. The mIL-35 construct was stably expressed in HEK 293 cells, and culture supernatants of single clones were screened by ELISA using a combination of anti-hFc and anti-mEBI3 antibodies, among which the most positive clone 35-5 was selected and adapted in a serum-free culture system. Functional activity of the secreted mIL-35 from this clone was tested using the [3H]thymidine-based cell proliferation assay. When CD4+ T cells purified from TCR-transgenic DO11.10 mice were stimulated with a combination of syngeneic irradiated APCs and OVA peptide in the presence of the mIL-35 culture supernatants, the mIL-35 significantly suppressed CD4+ T cell proliferation. Furthermore, the mIL-35-mediated inhibition of CD4+ T cell proliferation was reversed by anti-mIL-35 antibody. Taken together, these results demonstrate that the recombinant mIL-35 is functionally active and can be a useful research tool to study T cell-based immune regulations.

Reovirus protein sigma 1 (pσ1), engineered to deliver OVA mucosally, induced tolerance in mice even with a single, low dose. To test the efficacy of pσ1-based therapy, the extracellular domain of myelin oligodendrocyte glycoprotein (MOG29-146) was genetically fused to pσ1 (MOG-pσ1). Susceptible C57BL/6 mice were nasally dosed with PBS, MOG-pσ1, or recombinant (r)MOG and 3 wks later were challenged with MOG35-55-induced EAE. MOG-pσ1, not rMOG or PBS, abrogated EAE incidence evidenced by >8-fold reductions in IFN-γ, IL-17, and IL-21, but greatly elevated IL-4 and IL-10. To test its therapeutic potential at the peak of MOG35-55-induced EAE (around day 15), mice were nasally treated with 50 μg of MOG-pσ1 or PBS. Strikingly, within 24 hrs of treatment with MOG-pσ1, >2-fold reduction in clinical symptoms were obtained. This improved clinical response was accompanied by a significant increase of at least 4-fold in FoxP3+ Treg cells in the spinal cords of treated mice; however, such increases were not evident in head and neck lymph nodes or the spleen. This increase in Treg cell infiltration into the CNS appears to be NK cell-dependent since this infiltration was abated in NK cell-depleted mice. Finally, enhanced apoptosis (30%) of infiltrating CD11c+ CD11b+ DCs was observed, underscoring the complexity of this system. * Authors contributed equally to the work; supported by NIH AI-78938.

Abstract Pulmonary F. tularensis infections are highly lethal in untreated patients, and current antibiotic regimens are not always effective. Thus, we hypothesize that innate immunity augmentation with natural compounds could improve protective immunity to Francisella and complement antibiotic therapies. Screening of a natural compound library revealed a plant polysaccharide with the ability to activate human γδ T cells. The addition of this plant polysaccharide to both mock- and Francisella-infected murine macrophages enhanced surface expression of CD11b, CD40, CD80 and CD86 along with augmenting production of IL-6, TNF-α, and IL-1β. Plant polysaccharide treatment also augmented clearance of F. tularensis from infected murine macrophages, an effect that was time-, dose-, and nitric oxide-dependent, but independent of MyD88. In addition, this plant polysaccharide could enhance clearance of Francisella from infected primary human macrophages when co-cultured with autologous NK cells, which were found to have elevated expression of IFN-γ mRNA. Therefore, this plant polysaccharide holds potential as a therapeutic agonist that can enhance resistance to F. tularensis infection. This work supported by NIH U54AI-65357, P01AT-04986 and NIH Contract HHSN2662004000009/N01-AI40009.