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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 (Treg) 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 Treg cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/IIC). 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/IIC 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-γ but diminished TGF-β production by Treg cells from Salmonella-CFA/IIC-treated mice. Adoptive transfer of Treg 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/IIC showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN-γ neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/IIC-induced Treg cells. Thus, the Salmonella-CFA/IIC vaccine elicits Treg cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these Treg 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.

Intratracheal instillation of L-selectin-deficient (L-Sel−/−) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or β-galactosidase-specific CTLs with concomitant long-lived β-galactosidase transgene expression in the lungs. The absence of Ag-specific CTLs was attributed to a deficiency in lymphoid CD11c+CD8+ dendritic cells (DCs) in the lower respiratory lymph nodes (LRLNs). To enable L-Sel−/− CTL activity, cell-sorted L-Sel−/−CD8+ T cells were cocultured with cell-sorted L-Sel+/+CD8+ or CD8− DCs or L-Sel−/−CD8− DCs. Only the CD8+ DCs restored CTL activity; L-Sel−/−CD8− DCs failed to support L-Sel+/+ CTLs because these remained immature, lacking the ability to express costimulatory molecules CD40, CD80, or CD86. Although no lung CD8+ DCs were detected, the DC environment remained suppressive in L-Sel−/− mice evident by the lack of CTL responses following adenoviral challenge with OVA in recipient L-Sel−/− adoptively transferred with OT-1 CD8+ T cells. To assess whether the L-Sel−/−CD8− DCs could be induced into maturity, microbial stimulation studies were performed showing the failure of L-Sel−/− LRLN to make matured DCs. When L-Sel−/− mice were subjected in vivo to microbial activation before Ad2 vector dosing, CTL activity was restored stimulating the renewed presence of LRLN CD8+ DCs in L-Sel−/− mice. These studies show that impairment of L-Sel−/− DC maturation results in insufficient mature DCs that require microbial activation to restore increases in respiratory CD8+ DCs to support CTL responses.

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 σ1 (pσ1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via pσ1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus pσ1, termed OVA-pσ1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-pσ1 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-pσ1 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-β1, with greatly reduced IL-17 and IFN-γ. The induced IL-10 was derived predominantly from FoxP3+CD25+CD4+ T cells. No FoxP3+CD25+CD4+ T cells were induced in OVA-pσ1-dosed IL-10-deficient (IL-10−/−) mice, and despite showing increased TGF-β1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using pσ1 as a mucosal delivery platform specifically for low-dose tolerance induction.

Osteoarticular and soft tissue inflammation occur in subsequent infection with wild-type Brucella in mice deficient of IFN-γ, resembling aspects of human disease. Human brucellosis exhibits diverse pathological manifestations that can affect almost any organ. In particular, osteoarticular complications are the most common focal manifestation of brucellosis and occur in 40–80% of patients. In immunocompetent mice, Brucella replication is generally restricted to the spleen, liver, and to a lesser extent, LNs, thereby limiting their use for study of focal inflammation often found in brucellosis. Here, we report that nasal, oral, or peritoneal infection of IFN-γ−/− mice with WT Brucella melitensis or Brucella abortus results in joint and periarticular tissue inflammation. Histological analysis of the affected joints revealed inflammatory infiltrates and debris within the joint space colocalizing with Brucella antigen. Osteoarthritis, necrosis, periarticular soft tissue inflammation, and substantial brucellae burdens were observed. Oral rifampicin was effective in clearing infection and halting further progression of focal inflammation from infected IFN-γ−/− mice, although some symptoms and swelling remained. Elevated IL-1β, but not TNF-α, IL-6, or IL-17, was detected in joint homogenates from infected IFN-γ−/− mice. Whereas more susceptible to systemic infection, IL-1R−/− mice depleted of IFN-γ were more resistant to focal inflammation than WT mice similarly depleted of IFN-γ. Collectively, these results show IFN-γ−/− mice represent a potential model for study of focal inflammation attributed to Brucella infection and will allow evaluation of intervention strategies targeting IL-1, IL-1R, or other inflammatory mediators, with the potential to complement antibiotic-based therapies.

Natural killer (NK) cells and dendritic cells (DCs) have been shown to link the innate and adaptive immune systems. Likewise, a new innate cell subset, interferon-producing killer DCs (IKDCs), shares phenotypic and functional characteristics with both DCs and NK cells. Here, we show IKDCs play an essential role in the resolution of experimental autoimmune encephalomyelitis (EAE) upon treatment with the tolerizing agent, myelin oligodendrocyte glycoprotein (MOG), genetically fused to reovirus protein σ1 (termed MOG-pσ1). Activated IKDCs were recruited subsequent MOG-pσ1 treatment of EAE, and disease resolution was abated upon NK1.1 cell depletion. These IKDCs were able to kill activated CD4(+) T cells and mature dendritic DCs, thus, contributing to EAE remission. In addition, IKDCs were responsible for MOG-pσ1-mediated MOG-specific regulatory T cell recruitment to the CNS. The IKDCs induced by MOG-pσ1 expressed elevated levels of HVEM for interactions with cognate ligand-positive cells: LIGHT(+) NK and T(eff) cells and BTLA(+) B cells. Further characterization revealed these activated IKDCs being MHC class II(high), and upon their adoptive transfer (CD11c(+)NK1.1(+)MHC class II(high)), IKDCs, but not CD11c(+)NK1.1(+)MHC class II(intermediate/low) (unactivated) cells, conferred protection against EAE. These activated IKDCs showed enhanced CD107a, PD-L1, and granzyme B expression and could present OVA, unlike unactivated IKDCs. Thus, these results demonstrate the interventional potency induced HVEM(+) IKDCs to resolve autoimmune disease.

Coxiella burnetiiis a highly infectious obligate intracellular bacterium. The phase I form is responsible for Q fever, a febrile illness with flu-like symptoms that often goes undiagnosed. The attenuatedC. burnetiiphase II (having a truncated “O” chain of its lipopolysaccharide) does not cause disease in immunocompetent animals; however, phase II organisms remain infectious, and we questioned whether disease could be produced in immunodeficient mice. To studyC. burnetiiphase II infections, febrile responses in gamma interferon knockout (IFN-γ−/−), BALB/c, Toll-like receptor 2 knockout (TLR2−/−), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain ofC. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-γ−/−and TLR2−/−mice, which showed elevated rectal temperatures that were sustained for ∼15 days with transient increases in splenic weights. Reinfection of IFN-γ−/−and TLR2−/−mice withC. burnetiiNMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection ofCoxiellain TLR2−/−mouse spleens was observed, greater colonization was evident in the IFN-γ−/−mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-γ, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-γ−/−macrophages produced elevated levels of IL-6, IL-10, and IL-12, while TLR2−/−macrophages produced GM-CSF, IL-12, and minimal IL-10. To distinguish immunity conferred by innate or adaptive systems, adoptive transfer studies were performed and showed that immune lymphocytes obtained from immunocompetent mice protected against a subsequent challenge with NMII, indicating that adaptive immunity mediates the observed protection. Thus, our data show that NMII is capable of eliciting disease in immunocompromised mice, which may help in evaluation of vaccine candidates as well as the study of host-pathogen interactions.

Nasal immunization stimulates robust Ab responses in the head and neck lymph nodes (HNLN) and nasal-associated lymphoid tissue (NALT). Studies showed that B lymphocyte trafficking in the HNLN and NALT is both L-selectin- and β7-dependent. β7 integrin generally associates with αE or α4 integrins to mediate lymphocyte retention in the epithelium or the mucosa, respectively. To study the contribution of β7 integrin to upper respiratory immunity, β7−/− mice were nasally dosed with CT on days 0, 7, and 14. Mucosal IgA or serum IgG Ab titers were not different from B6 β7+/+ mice at any of the time points examined til day 42. Vaginal IgG responses were slightly enhanced in β7−/− mice, and absent in L-selectin−/− mice. However, the absence of β7 did alter the types of supporting B cells found in the NP. Increased expression of αE was observed on day 42 despite the absence of its co-receptor, β7. This increased αE expression was not observed in B6 β7+/+ mice. αE+ B cells continued to be found in the β7−/− small intestinal lamina propria. Minimal αE+ B cells were found in the HNLN. From these studies, although mucosal Ab responses remain intact in β7−/− mice, it does appear that different homing receptor-dependent mechanisms are employed. This work is supported by NIH AI-55563.

Enterotoxigenic Escherichia coli CFA/I is a protective antigen and has been overexpressed in bacterial vectors, such as Salmonella Typhimurium H683, to generate vaccines. Effects that overexpressed CFA/I may engender on the bacterial host remain largely unexplored. To investigate, we constructed a high CFA/I expression strain, H683-pC2, and compared it to a low CFA/I expression strain, H683-pC, and to a non-CFA/I expression strain, H683-pY. The results showed that H683-pC2 was less able to migrate into semisolid agar (0.35%) than either H683-pC or H683-pY. Bacteria that migrated showed motility halo sizes of H683-pC2 < H683-pC < H683-pY. In the liquid culture media, H683-pC2 cells precipitated to the bottom of the tube, while those of H683-pY did not. In situ imaging revealed that H683-pC2 bacilli tended to auto-agglutinate within the semisolid agar, while H683-pY bacilli did not. When the cfaBE fimbrial fiber encoding genes were deleted from pC2, the new plasmid, pC2(−), significantly recovered bacterial swimming capability. Our study highlights the negative impact of overexpressed CFA/I fimbriae on bacterial swimming motility.

Ovalbumin (OVA) genetically fused to protein sigma 1 (pσ1) results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-pσ1, termed OVA-pσ1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4(+) and CD8(+) T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s) can be fused to vaccines to effectively elicit improved SIgA responses.

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.

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.

Oral immunization with soluble protein plus adjuvant stimulates excellent regional, but not distal Ab responses. Although low level expression of distal immune B cells is observed, these wane with time. Studies examining homing receptor expression should provide insight into understanding how distal mucosal immunity is maintained. Studies showed that B lymphocyte trafficking in the HNLN and NALT is L-Sel-dependent. However, the absence of L-Sel enhanced distal B cell maintenance following oral CT immunization of L-Sel−/− mice and was partly compensated by increased αEβ7+ B cells, as evident by the significantly increased IgA antibody-forming cells (AFC) in L-Sel−/− nasal passages (NP) when compared to B6 NP. These IgA responses were supported by elevated HNLN IgA AFC responses (10- to 100-fold greater), and these AFC endured beyond 42 days, but not in B6 mice. To discern whether αEβ7 expression was influenced by CT directly, naive HNLN, mesenteric LN (MLN), and Peyer’s patches (PP) were isolated and stimulated in vitro with LPS, LPS + CT, Con A, or media. The mitogen-stimulated L-Sel−/− HNLN, MLN, and PP B cells showed early expression of αEβ7 at 48 hrs, but by 96 hrs, the L-Sel−/− HNLN clearly showed the greatest levels (30–35%) of αEβ7+ B cells. Both unstimulated and stimulated HNLN B cells showed similar levels of αEβ7 expression, and CT did not enhance αEβ7 expression. MLN and PP showed fewer (~10%) αEβ7+ B cells, and B6 B cells from each tissue showed Supported by NIH AI-55563.

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.