The Delta fbpA mutant derived from Mycobacterium tuberculosis H37Rv has an enhanced susceptibility to intracellular antimicrobial oxidative mechanisms, undergoes limited phagosome maturation and activates macrophages and dendritic cells.

Mycobacterium tuberculosis H37Rv (Mtb) excludes phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS) while preventing lysosomal fusion in macrophages (MPhis). The antigen 85A deficient (Delta fbpA) mutant of Mtb was vaccinogenic in mice and the mechanisms of attenuation were compared with MPhis infected with H37Rv and BCG. Delta fbpA contained reduced amounts of trehalose 6, 6, dimycolate and induced minimal levels of SOCS-1 in MPhis. Blockade of oxidants enhanced the growth of Delta fbpA in MPhis that correlated with increased colocalization with phox and iNOS. Green fluorescent protein-expressing strains within MPhis or purified phagosomes were analysed for endosomal traffick with immunofluorescence and Western blot. Delta fbpA phagosomes were enriched for rab5, rab11, LAMP-1 and Hck suggesting enhanced fusion with early, recycling and late endosomes in MPhis compared with BCG or H37Rv. Delta fbpA phagosomes were thus more mature than H37Rv or BCG although, they failed to acquire rab7 and CD63 preventing lysosomal fusion. Finally, Delta fbpA infected MPhis and dendritic cells (DCs) showed an enhanced MHC-II and CD1d expression and primed immune T cells to release more IFN-gamma compared with those infected with BCG and H37Rv. Delta fbpA was thus more immunogenic in MPhis and DCs because of an enhanced susceptibility to oxidants and increased maturation.

Mycobacteria-primed macrophages and dendritic cells induce an up-regulation of complement C5a anaphylatoxin receptor (CD88) in CD3+ murine T cells.

Complement C5a anaphylatoxin is a potent activator of macrophages, neutrophils, and dendritic cells (DC) and binds the C5a receptor (C5a-R; CD88). Although C5a is chemotactic for T cells, expression of C5a-R on murine T cells has been disputed. We report here that naïve, Con A-activated, and cytokine (IL-12, IL-18)-stimulated murine CD3+ T cells from three strains of mice [C57Bl/6, B10.nSn (C5+/+), B10.on (C5-/-)] lacked C5a-R, as evaluated by immunophenotyping with an anti-C5a-R mAb. Ligation of CD3 induced a modest up-regulation with 3% of CD3+ T cells expressing cell surface C5a-R. T cells primed by APC differentiate into effector T cells. Activation of mycobacteria [bacillus Calmette-Guerin (BCG)]-sensitized T cells through MHC II and TCR interactions via BCG-infected macrophages enhanced the expression of C5a-R with approximately 14% of CD3+ T cells positive for C5a-R. Comparable expression was found in C5+/+ as well as C5-/- strains of mice (14% and 15%, respectively). Furthermore, anti-CD3-activated T cells were primed by BCG-infected DC, and a larger proportion of the primed T cells expressed C5a-R (30-40%). Finally, mice infected with BCG showed significant numbers of CD3+ T cells expressing C5a-R in the spleens during infection. As APC, such as macrophages and DC, can secrete C5 and cleave C5 to C5a and C5b through a peptidase, we suggest that macrophage and DC-T cell interactions can up-regulate C5a-R on T cells through MHC II-TCR and provide a C5a peptide for additional local activation of T cells via C5a-R.

Complement C5a anaphylatoxin is an innate determinant of dendritic cell-induced Th1 immunity to Mycobacterium bovis BCG infection in mice.

During acquired immunity to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in mice, dendritic cells (DCs) present mycobacterial antigens to naive T cells to prime an immune response. Complement C5a (anaphylatoxin) secreted by mycobacteria-infected macrophages regulates IL-12p70 production. As IL-12p70 regulates Th1 immunity against mycobacteria in mice, we examined the effects of C5a on IL-12p70 secretion by murine DCs and Th1 immunity. DCs cultured from C5-deficient (C5(-/-)) and -sufficient (C5(+/+)) mice were infected with BCG in the presence or absence of the C5a peptide. ELISA showed that C5(-/-) DCs secreted less IL-12p70 (600 pg/mL vs. 100 pg/mL) than C5(+/+) DCs, and they secreted more IL-10. Using immunophenotyping, reduced CD40 expression was found on C5(-/-) DCs after BCG infection. BCG-primed DCs were then cocultured with naive or BCG-immune T cells to differentiate them into IFN-gamma-secreting Th1 T cells. Coincident with increased IL-12p70 levels, BCG-primed C5(+/+) DCs cocultured with naive or immune C5(+/+) T cells showed a larger increase in CD4+ IFN-gamma/CD8+ IFN-gamma+ T cells compared with cocultured DCs and T cells from C5(-/-) mice. Thus, BCG-primed C5(+/+) DCs were better able to drive a Th1 response. Furthermore, BCG aerosol-infected C5(-/-) mice showed reduced CD4 and CD8 IFN-gamma-secreting T cells in the lungs, concurrent with an increased growth of BCG. Thus, C5a, an innate peptide, appears to play an important role in the generation of acquired immune responses in mice by regulating the Th1 response through modulation of IL-12p70 secretion from DCs.

The ΔfbpA attenuated candidate vaccine from Mycobacterium tuberculosis, H37Rv primes for a stronger T-bet dependent Th1 immunity in mice.

The ΔfbpA candidate vaccine derived from Mycobacterium tuberculosis (H37Rv) (Mtb) protects mice better than BCG against tuberculosis, and we investigated the hypothesis that ΔfbpA may induce a stronger Th1 immunity. Since T-bet transcription factor regulates Th1 immunity, mice infected with ΔfbpA, BCG vaccine and related mycobacteria were analyzed for T-bet positive T cells. Mouse dendritic cells (DCs) or macrophages were also pulsed with excretory-secreted antigens (ES; Antigen-85B, ESAT-6 and CFP10) and cocultured with T cells from immunized or naïve mice and tested for in vitro induction of T-bet and IFN-γ. In both models, ΔfbpA mutant induced a stronger response of T-bet(+)CD4 T cells, which correlated with an increased expansion of IFN-γ(+)CD4 T cells in vivo and in vitro. When DCs pulsed with ES antigens were allowed to stimulate T cells, ESAT-6 and CFP-10 failed to induce a recall expansion of T-bet(+)IFN-γ(+)CD4 T cells from BCG vaccinated mice. Thus, deletion of RD1 in BCG seems to reduce its ability to induce T-bet and induce stronger Th1 immunity. Finally, mice were vaccinated with ΔfbpA and BCG and challenged with virulent Mtb for evaluation of protection and T cell expansion. ΔfbpA vaccinated mice showed a rapid and stronger expansion of CD4(+)CXCR3(+) IFN-γ(+) T cells in the lungs of Mtb challenged mice, compared to those which had BCG vaccine. ΔfbpA immunized mice also showed a better decline of the Mtb bacterial counts of the lungs. Mtb derived ΔfbpA candidate vaccine therefore induces qualitatively better T-bet dependent Th1 immunity than BCG vaccine.

A defect in the synthesis of Interferon-γ by the T cells of Complement-C5 deficient mice leads to enhanced susceptibility for tuberculosis.

Interferon-γ (IFNγ) plays a major role during host defense against Mycobacterium tuberculosis (Mtb). T cells produce IFNγ in response to IL-12 and IL-18 secreted from Mtb infected macrophages. IFNγ in turn, induces nitric oxide secretion in macrophages that kills Mtb. IFNγ knockout mice are thus hyper-susceptible to tuberculosis. We reported earlier that Complement-C5 deficient (C5(-/-)) congenic mice are more susceptible to tuberculosis and showed reduced IL-12 synthesis in their macrophages. Using C5(-/-) congenic mice that carry a deletion in the C5 gene and the wild type C5(+/+) mice, we demonstrate here that, the C5(-/-) derived CD3(+) T cells, have an additional defect in the synthesis of IFNγ. C5(-/-) T cells produced lower levels of IFNγ upon stimulation by antigen presenting cells (APCs) infected with Mtb or when stimulated directly with a combination of IL-12 and IL-18. The latter was in part due to a reduced phosphorylation of STAT4 following IL-12/IL-18 stimulation. Addition of C5a peptide to IL-12/IL-18 partially restored STAT4 phosphorylation and IFNγ synthesis in C5(-/-) T cells indicating that IL-12/IL-18 mediated signaling within CD3(+) T cells involves C5a peptide. Finally, C5(-/-) T cells derived from M. bovis BCG or Mtb infected mice showed a reduced expression of T-bet (T-box expressed in T cells) transcription factor, which correlated well with a reduced T cell secretion of IFNγ. Since T-bet mediated IFNγ synthesis facilitates Th1 expansion, C5(-/-) mouse derived T cells appear to have an intrinsic defect in the production of IFNγ, which is related to C5 deficiency and this may explain their increased susceptibility to infection with Mtb and BCG.

Anti-tuberculosis immunity induced in mice by vaccination with Mycobacterium smegmatis over-expressing Antigen 85B is due to the increased influx of IFNgamma-positive CD4 T cells into the lungs.

BCG vaccine is unsafe for use in patients with AIDS. Mycobacterium smegmatis (Msm), an avirulent species unlike virulent Mycobacterium tuberculosis (H37Rv, Mtb) has been used as a carrier vaccine with ambiguous results due to the elicitation of poor immune responses to antigens in mice. In this study, we over-expressed the immunodominant antigen 85B in M. smegmatis (Msm-OEAg85B) and compared the immunogenicity of Msm-OEAg85B with that of wild-type Msm. Mice which were vaccinated with either Msm or Msm-OEAg85B and challenged 2 weeks later with Mtb. Vaccine-induced protection and lung T cell responses were evaluated post vaccination and post challenge. Unlike wild-type Msm that elicited minimal T cell responses in mice, MsmOE-Ag85B induced enhanced CD4+IFNgamma+ T cell responses that leveled off over 2 weeks. After virulent challenge at 2 weeks, Mtb grew progressively in the lungs of naive mice and mice vaccinated with wild-type Msm, but showed reduced growth (<0.6 log(10)) and therefore protection in Msm-OEAg85B-vaccinated mice. Lungs of Msm-OEAg85B-vaccinated mice showed increased numbers of CD4+IFNgamma+ T cells suggesting that the reduced bacterial growth was likely due to the enhanced T cell response in lungs. Since wild-type Msm was unable to protect but Msm-OEAg85B was, we suggest that Msm can be genetically manipulated to over-express selected Mtb antigens, thereby paving the way for safer vaccines that can be used in immunodeficient patients.

Autophagy enhances the efficacy of BCG vaccine by increasing peptide presentation in mouse dendritic cells.

The variable efficacy of Bacille Calmette Guerin (BCG) vaccination against tuberculosis has prompted efforts to improve the vaccine. In this study, we used autophagy to enhance vaccine efficacy against tuberculosis in a mouse model. We examined the effect of autophagy on the processing of the immunodominant mycobacterial antigen Ag85B by antigen presenting cells (APCs), macrophages and dendritic cells (DCs). We found that rapamycin-induced autophagy enhanced Ag85B presentation by APCs infected with wild-type Mycobacterium tuberculosis H37Rv, H37Rv-derived DeltafbpA attenuated candidate vaccine or BCG. Furthermore, rapamycin enhanced localization of mycobacteria with autophagosomes and lysosomes. Rapamycin-enhanced antigen presentation was attenuated when autophagy was suppressed by 3-methyladenine or by small interfering RNA against beclin-1. Notably, mice immunized with rapamycin-treated DCs infected with either DeltafbpA or BCG showed enhanced T helper type 1-mediated protection when challenged with virulent Mycobacterium tuberculosis. Finally, overexpression of Ag85B in BCG induced autophagy in APCs and enhanced immunogenicity in mice, suggesting that vaccine efficacy can be enhanced by augmenting autophagy-mediated antigen presentation.

The reduced bactericidal function of complement C5-deficient murine macrophages is associated with defects in the synthesis and delivery of reactive oxygen radicals to mycobacterial phagosomes.

Complement C5-deficient (C5(-/-)) macrophages derived from B.10 congenic mice were found to be defective in killing intracellular Mycobacterium tuberculosis (MTB). They were bacteriostatic after activation with IFN-gamma alone but bactericidal in the combined presence of IFN-gamma and C5-derived C5a anaphylatoxin that was deficient among these macrophages. Reduced killing correlated with a decreased production of reactive oxygen species (ROS) in the C5(-/-) macrophages measured using fluorescent probes. Furthermore, a lack of colocalization of p47(phox) protein of the NADPH oxidase (phox) complex with GFP-expressing MTB (gfpMTB) indicated a defective assembly of the phox complex on phagosomes. Reconstitution with C5a, a known ROS activator, enhanced the assembly of phox complex on the phagosomes as well as the production of ROS that inhibited the growth of MTB. Protein kinase C (PKC) isoforms are involved in the phosphorylation and translocation of p47(phox) onto bacterial phagosomes. Western blot analysis demonstrated a defective phosphorylation of PKC (alpha, beta, delta) and PKC-zeta in the cytosol of C5(-/-) macrophages compared with C5 intact (C5(+/+)) macrophages. Furthermore, in situ fluorescent labeling of phagosomes indicated that PKC-beta and PKC-zeta were the isoforms that are not phosphorylated in C5(-/-) macrophages. Because Fc receptor-mediated phox assembly was normal in both C5(-/-) and C5(+/+) macrophages, the defect in phox assembly around MTB phagosomes was specific to C5 deficiency. Reduced bactericidal function of C5(-/-) macrophages thus appears to be due to a defective assembly and production of ROS that prevents effective killing of intracellular MTB.