Functional transitions in macrophages during in vivo infection with Mycobacterium bovis bacillus Calmette-Guérin

Macrophage activation during the immune response to intracellular bacteria is critical for resolution of the infection. We have investigated the pathway of macrophage activation during murine Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. Three distinct phenotypes of macrophages were identified and compared: resident peritoneal macrophages, day 2 postinfection macrophages, and 12-day postinfection macrophages. Compared with resident peritoneal macrophages, day 2 BCG macrophages expressed intermediate levels of the cell surface receptors Mac1 and F4/80 and low levels of MHC class II molecules. These cells were highly phagocytic and produced large amounts of mRNA encoding the chemokine IP-10. In addition, day 2 BCG macrophages did not generate reactive nitrogen intermediates, though they were primed to do so, and did not have increased levels of TNF-alpha mRNA. Blockade of monocyte influx into the peritoneal cavity using Abs to platelet endothelial cell adhesion molecule 1 had no effect on the appearance of day 2 BCG macrophages, suggesting this cell can differentiate from resident peritoneal macrophages. In contrast to day 2 BCG macrophages, day 12 BCG macrophages were poorly phagocytic, but produced high levels of reactive nitrogen intermediates, IP-10 and TNF-alpha mRNA, and class II MHC molecules. We propose that day 2 BCG macrophages are specialized for phagocytic uptake of pathogens from the extracellular space, whereas day 12 BCG macrophages are specialized for killing of the internalized pathogens. This functional transition during activation is reminiscent of that seen during maturation/activation of the related dendritic cell lineage induced by bacterial or inflammatory stimuli.     

The shift of Th1 to Th2 immunodominance associated with the chronicity of Mycobacterium bovis bacille Calmette-Guérin infection does not affect the memory response

In the present study we investigated the shaping and evolution of the immunodominance of the T cell response during a chronic mycobacterial infection. Using a recombinant bacille Calmette-Guérin expressing a reporter Ag, the Escherichia coli MalE protein, we analyzed the peptide specificity and the cytokine profile of the T cell response to the reporter Ag by ELISPOT. During the early steps of infection, the T cell response was focused on two dominant MalE epitopes and was characterized by a pure IFN-gamma response. Then, in the course of infection the initial IFN-gamma response to these two epitopes shifted to a mixed IFN-gamma/IL-4 response. At the same time, the peptide specificity of the T cell response was broadened to two additional MalE epitopes characterized by a unique IL-4 response resulting in the establishment of a dominant IL-4 response to the MalE protein at 16 wk postinfection. However, this phenomenon did not impair the outcome of a predominant IFN-gamma response upon subsequent MalE recall in vivo performed in the presence of CFA, a Th1-driving adjuvant. These results indicate that the Th2 nature of the immune response established during a chronic infection, which most likely reflects regulatory mechanisms to allow the return to T cell homeostasis, does not shape the Th1/Th2 nature of the memory response.     

Transmembrane TNF induces an efficient cell-mediated immunity and resistance to Mycobacterium bovis bacillus Calmette-Guérin infection in the absence of secreted TNF and lymphotoxin-alpha

The contribution of a transmembrane (Tm) form of TNF to protective immunity against Mycobacterium bovis bacillus Calmette-Guérin (BCG) was studied in transgenic (tg) mice expressing a noncleavable Tm TNF but lacking the TNF/lymphotoxin-alpha (LT-alpha) locus (Tm TNF tg mice). These mice were as resistant to BCG infection as wild-type mice, whereas TNF/LT-alpha(-/-), TNF(-/-), and LT-alpha(-/-) mice succumbed. Tm TNF tg mice developed granulomas of smaller size but at 2- to 4-fold increased frequencies compared with wild-type mice. Granulomas were mainly formed by monocytes and activated macrophages expressing Tm TNF mRNA and accumulating acid phosphatase. NO synthase 2 activation as a key macrophage bactericidal mechanism was low during the acute phase of infection in Tm TNF tg mice but was still sufficient to limit bacterial growth and increased in late infection. While infection with virulent Mycobacterium tuberculosis resulted in very rapid death of TNF/LT-alpha(-/-) mice, it also resulted in survival of Tm TNF tg mice which presented an increase in the number of CFU in spleen (5-fold) and lungs (10-fold) as compared with bacterial load of wild-type mice. In conclusion, the Tm form of TNF induces an efficient cell-mediated immunity and total resistance against BCG even in the absence of LT-alpha and secreted TNF. However, Tm TNF-mediated protection against virulent M. tuberculosis infection can also be efficient but not as strong as in BCG infection, in which cognate cellular interactions may play a more predominant role in providing long-term surveillance and containment of BCG-infected macrophages.     

Role of apoptosis-regulating signal kinase 1 in innate immune responses by Mycobacterium bovis bacillus Calmette-Guérin

Mycobacterium bovis bacillus Calmette-Guérin (BCG) induces innate immune responses through Toll-like receptor (TLR) 2 and TLR4. We investigated the role of apoptosis-regulating signal kinase (ASK) 1 in reactive oxygen species (ROS)-mediated innate immune responses induced by BCG mycobacterial infection. In macrophages, M. bovis BCG stimulation resulted in rapid activation of mitogen-activated protein kinases (MAPKs), secretion of inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, and ROS generation in a TLR2- and TLR4-dependent manner. M. bovis BCG-induced ROS production led to robust activation of ASK1 upstream of the c-jun-N-terminal kinase and p38 MAPK, but not extracellular-regulated kinase 1/2. Blocking ASK1 activity markedly attenuated M. bovis BCG-induced TNF-alpha and IL-6 production by macrophages. Both TLR2 and TLR4 were required for optimal activation of ASK1 in response to M. bovis BCG. Furthermore, we present evidence that TNF receptor-associated factor (TRAF) 6 activities were essential for ROS-mediated ASK1 activation by M. bovis BCG. Finally, ASK1 activities were required for effective control of intracellular mycobacterial survival. Thus, the results of this study suggest a novel role of the TLR-ROS-TRAF6-ASK1 axis in the innate immune response to mycobacteria as a signaling intermediate.     

Mycobacterial codon optimization enhances antigen expression and virus-specific immune responses in recombinant Mycobacterium bovis bacille Calmette-Guérin expressing human immunodeficiency virus type 1 Gag

Although its potential for vaccine development is already known, the introduction of recombinant human immunodeficiency virus (HIV) genes to Mycobacterium bovis bacille Calmette-Guérin (BCG) has thus far elicited only limited responses. In order to improve the expression levels, we optimized the codon usage of the HIV type 1 (HIV-1) p24 antigen gene of gag (p24 gag) and established a codon-optimized recombinant BCG (rBCG)-p24 Gag which expressed a 40-fold-higher level of p24 Gag than did that of nonoptimized rBCG-p24 Gag. Inoculation of mice with the codon-optimized rBCG-p24 Gag elicited effective immunity, as evidenced by virus-specific lymphocyte proliferation, gamma interferon ELISPOT cell induction, and antibody production. In contrast, inoculation of animals with the nonoptimized rBCG-p24 Gag induced only low levels of immune responses. Furthermore, a dose as small as 0.01 mg of the codon-optimized rBCG per animal proved capable of eliciting immune responses, suggesting that even low doses of a codon-optimized rBCG-based vaccine could effectively elicit HIV-1-specific immune responses.     

Human phagocytic cell responses to Mycobacterium leprae and Mycobacterium bovis Bacillus Calmette-Guérin. An in vitro comparison of leprosy vaccine components

Components of current vaccines for Hansen's disease include Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and killed Mycobacterium leprae. BCG infections in humans are rare and most often occur in immune-compromised individuals. M. leprae on the other hand, although not causing clinical disease in most exposed individuals, is capable of infecting and replicating within mononuclear phagocytes. Lymphocytes from patients with the lepromatous form of Hansen's disease exhibit defective lymphokine production when challenged in vitro with M. leprae. This may result in inefficient mononuclear phagocyte activation for oxidative killing. To study the ability of normal phagocytes to ingest and respond oxidatively to BCG and M. leprae, we measured phagocytic cell O2- release and fluorescent oxidative product formation and visually confirmed the ingestion of the organisms. BCG stimulated a vigorous O2- generation in neutrophils and monocytes and flow cytometric oxidative product generation by neutrophils occurred in the majority of cells. M. leprae, stimulated a weak but significant O2- release requiring a high concentration of organisms and long exposure. By flow cytometric analysis, most neutrophils were able to respond to both organisms with the generation of fluorescent oxidative products. Neutrophil oxidative responses to M. leprae were substantially less than responses seen from neutrophils exposed to BCG. By microscopic examination of neutrophils phagocytizing FITC-labeled bacteria, it was shown that both M. leprae and BCG were slowly ingested but that more BCG appeared to be associated with the cell membrane of more of the cells. When phagocytic cells were incubated with BCG and M. leprae for 30 min and subsequently examined by electron microscopy, few organisms were seen in either neutrophils or monocytes. This suggests that BCG are easily recognized and slowly ingested by normal phagocytic cells, the majority of which respond with a strong oxidative burst. M. leprae appeared to only weakly stimulate phagocyte oxidative responses and were also slowly phagocytized.     

Influence of Mycobacterium bovis bacillus Calmette-Guérin on antibody and cytokine responses to human neonatal vaccination.

The immaturity of the immune system increases the susceptibility of young infants to infectious diseases and prevents the induction of protective immune responses by vaccines. We previously reported that Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination induces a potent Th1 response to mycobacterial Ags in newborns. In this study, we evaluated the influence of BCG on the response to unrelated vaccines given in early life. Newborns were randomly allocated to one of three study groups receiving BCG at birth, when infants received their first dose of hepatitis B and oral polio vaccines; at 2 mo of age, when infants received their first dose of diphtheria and tetanus vaccines; or at 4.5 mo of age, when immune responses to vaccines were measured. Administration of BCG at the time of priming markedly increased the cellular and Ab responses to the hepatitis B vaccine, but had only a limited influence on the cytokine response to tetanus toxoid and no effect on the Ab responses to tetanus and diphtheria toxoids. Although BCG induced a potent Th1-type response to mycobacterial Ags, it promoted the production of both Th1- and Th2-type cytokines in response to unrelated vaccines. The effect of BCG was apparent at the systemic level, as it increased the Ab response to oral polio vaccine. These results demonstrate that BCG influences the immune response to unrelated Ags in early life, likely through its influence on the maturation of dendritic cells.     

ATP-mediated killing of Mycobacterium bovis bacille Calmette-Guérin within human macrophages is calcium dependent and associated with the acidification of mycobacteria-containing phagosomes

We previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca(2+) concentration in macrophages by mobilizing intracellular Ca(2+) via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca(2+) via P2X purinoceptors. The relative contribution of these receptors and Ca(2+) sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca(2+) in UTP-desensitized macrophages (in Ca(2+)-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca(2+) store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca(2+)-mobilizing P2Y purinoceptor in addition to the P2Y(2) subtype is expressed on human macrophages. ATP and the Ca(2+) ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca(2+)-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N',N'-tetraacetic acid acetoxymethyl ester (an intracellular Ca(2+) chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3'-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca(2+) mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.     

Overexpression of IL-15 in vivo enhances protection against Mycobacterium bovis bacillus Calmette-Guérin infection via augmentation of NK and T cytotoxic 1 responses.

To investigate the immunomodulating effects of IL-15 in vivo on mycobacterial infection, we used IL-15-transgenic (Tg) mice, which were recently constructed with cDNA-encoding secretable isoform of IL-15 precursor protein under the control of a MHC class I promoter. The IL-15-Tg mice exhibited resistance against infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), as assessed by bacteria growth. IFN-gamma level in serum was significantly higher in IL-15-Tg mice than in non-Tg mice after BCG infection. NK cells were remarkably increased, and Ag-specific T cytotoxic 1 response mediated by CD8+ T cells producing IFN-gamma was significantly augmented in the IL-15-Tg mice following BCG infection. Neutralization of endogenous IFN-gamma by in vivo administration of anti-IFN-gamma mAb deteriorated the clearance of the bacteria. Depletion of of NK cells or CD8+ T cells by in vivo administration of anti-asialo-GM(1) Ab or anti-CD8 mAb hampered the exclusion of bacteria. Thus, overexpression of IL-15 in vivo enhanced protection against BCG infection via augmentation of NK and T cytotoxic 1 responses.     

Characterization of lung gamma delta T cells following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin

The lungs are considered to have an impaired capacity to contain infection by pathogenic mycobacteria, even in the presence of effective systemic immunity. In an attempt to understand the underlying cellular mechanisms, we characterized the gammadelta T cell population following intranasal infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). The peak of gammadelta T cell expansion at 7 days postinfection preceded the 30 day peak of alphabeta T cell expansion and bacterial count. The expanded population of gammadelta T cells in the lungs of BCG-infected mice represents an expansion of the resident Vgamma2 T cell subset as well as an influx of Vgamma1 and of four different Vdelta gene-bearing T cell subsets. The gammadelta T cells in the lungs of BCG-infected mice secreted IFN-gamma following in vitro stimulation with ionomycin and PMA and were cytotoxic against BCG-infected peritoneal macrophages as well as against the uninfected J774 macrophage cell line. The cytotoxicity was selectively blocked by anti-gammadelta TCR mAb and strontium ions, suggesting a granule-exocytosis killing pathway. Depletion of gammadelta T cells by injection of specific mAb had no effect on the subsequent developing CD4 T cell response in the lungs of BCG-infected mice, but significantly reduced cytotoxic activity and IFN-gamma production by lung CD8 T cells. Thus, gammadelta T cells in the lungs might help to control mycobacterial infection in the period between innate and classical adaptive immunity and may also play an important regulatory role in the subsequent onset of alphabeta T lymphocytes.     

Newborns develop a Th1-type immune response to Mycobacterium bovis bacillus Calmette-Guérin vaccination.

Data obtained in animals indicate that neonatal immune responses are biased toward Th2. This could reduce the efficacy of vaccines against viral and mycobacterial diseases. The ability of human newborns to develop a Th1 immune response upon immunization has not been studied. Since the vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) triggers a Th1-type response in adults, we investigated whether it induces a similar response in newborns and whether age at vaccination influences immunogenicity. We found that BCG vaccination at birth induces a memory Th1-type response of similar magnitude to that when given later in life. This study demonstrates that human newborns can be immunized against pathogens controlled by a Th1 immune response.     

Ion-exchange chromatography method for the purification of genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin

The goal of this study was to provide practical strategies for purifying genomic DNA fraction from Mycobacterium bovis Bacillus Calmette-Guérin (BCG-DNA) by ion-exchange chromatography. A multistep process was developed to purify BCG-DNA. The process consisted of sonication, heating, trypsin digestion, ion-exchange chromatography, gel-filter chromatography, and lyophilization. After ion-exchange chromatography, BCG-DNA was highly purified and possessed potent biological effects. The methods described were efficient and had good reproducibility. Further, this was the first reported chromatography method to purify BCG-DNA.     

Identification of novel proteins in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin in the isoelectric point range 6-11

Two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins in the isoelectric point range 6-11 in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Twelve proteins were identified, three of which had not been described previously. The expression of the identified proteins was comparatively analyzed in culture filtrates of BCG in different growth phases and culture conditions. For some of these proteins, the relative protein abundance in the different culture filtrate preparations was significantly different. The differential expression of the identified proteins is discussed in relation to their putative localization and/or biological function.     

The secretion antigen SA5K has a role in the adaptation of Mycobacterium bovis bacillus Calmette-Guérin to intracellular stress and hypoxia

The Mycobacterium tuberculosis TB8.4 (Rv1174c) gene encodes a secreted protein of 8.4 kDa (TB8.4) which has been suggested to be involved in reactivation of dormant mycobacteria. We have previously reported that inactivation of an identical gene (sa5k) in Mycobacterium bovis BCG causes impaired ability of the mutant strain (BCGsa5k::aph) to grow inside human macrophages. This study aimed to investigate the role of TB8.4 in the reactivation of aged cultures of BCG as well as the role of the sa5k gene in the resistance of BCG to intracellular stress conditions and adaptation to hypoxia. Although when added to aged cultures of BCG, TB8.4 caused a statistically significant increase in the number of colony-forming units, a similar effect was obtained in cultures incubated with BSA, suggesting a non-specific growth stimulation by TB8.4. Compared to parental BCG, the BCGsa5k::aph strain showed an increased susceptibility to reactive oxygen and nitrogen intermediates and to acid stress and an impaired ability to adapt to reduced O2 concentrations, when tested in the oxygen-limited Wayne culture system. These results suggest that the product of the sa5k gene (SA5K protein) has a role in both resistance of BCG to intracellular stress and in its adaptation to hypoxia.     

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.     

Estrogen receptor α signaling in T lymphocytes is required for estradiol-mediated inhibition of Th1 and Th17 cell differentiation and protection against experimental autoimmune encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17β-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) α-deficient mice and bone marrow chimera experiments, we show that expression of ERα is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ERα-deficient mice, we demonstrate that ERα is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ERα in host nonhematopoietic tissues, we further show that ERα signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.     

Mycobacterium bovis bacillus Calmette-Guérin secreting active cathepsin S stimulates expression of mature MHC class II molecules and antigen presentation in human macrophages

A successful Th cell response to bacterial infections is induced by mature MHC class II molecules presenting specific Ag peptides on the surface of macrophages. In recent studies, we demonstrated that infection with the conventional vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) specifically blocks the surface export of mature class II molecules in human macrophages by a mechanism dependent on inhibition of cathepsin S (Cat S) expression. The present study examined class II expression in macrophages infected with a rBCG strain engineered to express and secrete biologically active human Cat S (rBCG-hcs). Cat S activity was completely restored in cells ingesting rBCG-hcs, which secreted substantial levels of Cat S intracellularly. Thus, infection with rBCG-hcs, but not parental BCG, restored surface expression of mature MHC class II molecules in response to IFN-gamma, presumably as result of MHC class II invariant chain degradation dependent on active Cat S secreted by the bacterium. These events correlated with increased class II-directed presentation of mycobacterial Ag85B to a specific CD4(+) T cell hybridoma by rBCG-hcs-infected macrophages. Consistent with these findings, rBCG-hcs was found to accelerate the fusion of its phagosome with lysosomes, a process that optimizes Ag processing in infected macrophages. These data demonstrated that intracellular restoration of Cat S activity improves the capacity of BCG-infected macrophages to stimulate CD4(+) Th cells. Given that Th cells play a major role in protection against tuberculosis, rBCG-hcs would be a valuable tuberculosis vaccine candidate.     

The impact of maternal infection with Mycobacterium tuberculosis on the infant response to bacille Calmette–Guérin immunization

Bacille Calmette–Guérin (BCG) immunization provides variable protection against tuberculosis. Prenatal antigen exposure may have lifelong effects on responses to related antigens and pathogens. We therefore hypothesized that maternal latent Mycobacterium tuberculosis infection (LTBI) influences infant responses to BCG immunization at birth. We measured antibody (n = 53) and cellular (n = 31) responses to M. tuberculosis purified protein derivative (PPD) in infants of mothers with and without LTBI, in cord blood and at one and six weeks after BCG. The concentrations of PPD-specific antibodies declined between birth (median [interquartile range (IQR)]) 5600 ng ml⁻¹ [3300–11 050] in cord blood) and six weeks (0.00 ng ml⁻¹ [0–288]). Frequencies of PPD-specific IFN-γ-expressing CD4⁺T cells increased at one week and declined between one and six weeks (p = 0.031). Frequencies of IL-2- and TNF-α-expressing PPD-specific CD4⁺T cells increased between one and six weeks (p = 0.019, p = 0.009, respectively). At one week, the frequency of PPD-specific CD4⁺T cells expressing any of the three cytokines, combined, was lower among infants of mothers with LTBI, in crude analyses (p = 0.002) and after adjusting for confounders (mean difference, 95% CI −0.041% (−0.082, −0.001)). In conclusion, maternal LTBI was associated with lower infant anti-mycobacterial T-cell responses immediately following BCG immunization. These findings are being explored further in a larger study.