Role of overexpressed CFA/I fimbriae in bacterial swimming.

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.

Tolerogen-induced interferon-producing killer dendritic cells (IKDCs) protect against EAE.

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.

The absence of lymphoid CD8+ dendritic cell maturation in L-selectin-/- respiratory compartment attenuates antiviral immunity.

Intratracheal instillation of L-selectin-deficient (L-Sel(-/-)) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or beta-galactosidase-specific CTLs with concomitant long-lived beta-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.

IL-13 production by regulatory T cells protects against experimental autoimmune encephalomyelitis independently of autoantigen.

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.

Attenuated Coxiella burnetii phase II causes a febrile response in gamma interferon knockout and Toll-like receptor 2 knockout mice and protects against reinfection.

Coxiella burnetii is 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 attenuated C. burnetii phase 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 study C. burnetii phase II infections, febrile responses in gamma interferon knockout (IFN-gamma(-/-)), BALB/c, Toll-like receptor 2 knockout (TLR2(-/-)), and C57BL/6 mice were measured using the Nine Mile phase II (NMII) strain of C. burnetii. Immunocompetent mice showed minimal febrile responses, unlike those obtained with IFN-gamma(-/-) and TLR2(-/-) mice, which showed elevated rectal temperatures that were sustained for approximately 15 days with transient increases in splenic weights. Reinfection of IFN-gamma(-/-) and TLR2(-/-) mice with C. burnetii NMII 30 days after primary infection protected mice as evident by reduced febrile responses and a lack of splenic inflammation. Although minimal detection of Coxiella in TLR2(-/-) mouse spleens was observed, greater colonization was evident in the IFN-gamma(-/-) mice. Cytokine analysis was performed on infected peritoneal macrophages isolated from these mice, and immunocompetent macrophages showed robust tumor necrosis factor alpha, IFN-gamma, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but no interleukin-12 (IL-12) responses. IFN-gamma(-/-) 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.

Loss of sialic acid binding domain redirects protein σ1 to enhance M cell-directed vaccination.

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.

IFN-γ-deficient mice develop IL-1-dependent cutaneous and musculoskeletal inflammation during experimental brucellosis.

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-1 ß, 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.

Low-dose tolerance is mediated by the microfold cell ligand, reovirus protein sigma1.

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 cell vaccination without autoantigen protects against experimental autoimmune encephalomyelitis.

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.