Silvia de la Barrera

A DNA Vaccine Coding for the Brucella Outer Membrane Protein 31 Confers Protection against B. melitensis and B. ovis Infection by Eliciting a Specific Cytotoxic Response

ABSTRACT The development of an effective subunit vaccine against brucellosis is a research area of intense interest. The outer membrane proteins (Omps) of Brucella spp. have been extensively characterized as potential immunogenic and protective antigens. This study was conducted to evaluate the immunogenicity and protective efficacy of the B. melitensis Omp31 gene cloned in the pCI plasmid (pCIOmp31). Immunization of BALB/c mice with pCIOmp31 conferred protection against B. ovis and B. melitensis infection. Mice vaccinated with pCIOmp31 developed a very weak humoral response, and in vitro stimulation of their splenocytes with recombinant Omp31 did not induced the secretion of gamma interferon. Splenocytes from Omp31-vaccinated animals induced a specific cytotoxic-T-lymphocyte activity, which leads to the in vitro lysis of Brucella-infected macrophages. pCIOmp31 immunization elicited mainly CD8+ T cells, which mediate cytotoxicity via perforins, but also CD4+ T cells, which mediate lysis via the Fas-FasL pathway. In vivo depletion of T-cell subsets showed that the pCIOmp31-induced protection against Brucella infection is mediated predominantly by CD8+ T cells, although CD4+T cells also contribute. Our results demonstrate that the Omp31 DNA vaccine induces cytotoxic responses that have the potential to contribute to protection against Brucella infection. The protective response could be related to the induction of CD8+ T cells that eliminate Brucella-infected cells via the perforin pathway.

Role Played by the Programmed Death-1-Programmed Death Ligand Pathway during Innate Immunity against Mycobacterium tuberculosis

Tuberculous pleurisy allows the study of specific cells at the site of Mycobacterium tuberculosis infection. Among pleural lymphocytes, natural killer (NK) cells are a major source of interferon gamma (IFN-gamma), and their functions are regulated by activating and inhibitory receptors. Programmed death-1 (PD-1), programmed death ligand 1 (PD-L1), and programmed death ligand 2 (PD-L2) are recognized inhibitory receptors in adaptive immunity, but their role during innate immunity remains poorly understood. We investigated the PD-1:PD-L1/PD-L2 pathway on NK cell effector functions in peripheral blood and pleural fluid from patients with tuberculosis. M. tuberculosis stimulation significantly up-regulated PD-1, PD-L1, and PD-L2 levels on NK cells. Interestingly, a direct correlation between PD-1 and IFN-gamma expression on NK cells was observed. Moreover, blockade of the PD-1 pathway markedly augmented lytic degranulation and IFN-gamma production of NK cells against M. tuberculosis. Furthermore, PD-1(+) NK cells displayed a diminished IFN-gamma mean fluorescence intensity, denoting the relevance of PD-1 on IFN-gamma regulation. Together, we described a novel inhibitory role played by PD-1:PD-L interactions in innate immunity in tuberculosis.

Vaccination with the Recombinant Brucella Outer Membrane Protein 31 or a Derived 27-Amino-Acid Synthetic Peptide Elicits a CD4+ T Helper 1 Response That Protects against Brucella melitensis Infection

ABSTRACT The immunogenicity and protective efficacy of the recombinant 31-kDa outer membrane protein from Brucella melitensis (rOmp31), administered with incomplete Freunds adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp31 conferred protection against B. ovis and B. melitensis infection. rOmp31 induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp31, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp31-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis of Brucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp31 immunization elicited specific CD4+ T cells that secrete IL-2 and gamma interferon, while CD8+ T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp31-elicited protection against B. melitensis infection is mediated by CD4+ T cells while the contribution of CD8+ T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp31 on the Brucella membrane, a peptide that contains amino acids 48 to 74 of Omp31. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+ T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp31 against B. melitensis but less protection than was induced by rOmp31 against B. ovis. Our results indicate that rOmp31 could be a useful candidate for the development of subunit vaccines against B. melitensis and B. ovis.

Mycobacterium tuberculosis Triggers Apoptosis in Peripheral Neutrophils Involving Toll-Like Receptor 2 and p38 Mitogen Protein Kinase in Tuberculosis Patients

ABSTRACT Polymorphonuclear neutrophils (PMN) exposed to Mycobacterium tuberculosis display bactericidal responses and produce inflammatory proteins. This PMN-mediated inflammatory response is regulated by an activation of the apoptotic program, which collaborates to avoid tissue injury. In vitro, circulating PMN from patients with tuberculosis (TB) show an increased spontaneous apoptosis, and M. tuberculosis-induced activation accelerates the PMN apoptosis. In this study, we evaluated the mechanisms involved in spontaneous and M. tuberculosis-induced apoptosis. We demonstrate that apoptosis of PMN is not induced by lipoarabinomannan or by a whole-cell lysate of M. tuberculosis and that neither tumor necrosis factor alpha nor CD11b, CD14, and Fcγ receptors are involved. Apoptosis of PMN from patients with active TB (TB-PMN) is induced by the interaction with the whole M. tuberculosis via Toll-like receptor 2 (TLR2), and, in contrast to spontaneous apoptosis, it involves the p38 mitogen-activated protein kinase (MAPK) pathway. These results correlate with a high expression of phosphorylated p38 (p-p38) in circulating TB-PMN and with the ability of M. tuberculosis to induce in vitro the expression of p-p38 in PMN. Therefore, when the bacterial burden is low, TB-PMN could be detecting nonopsonized M. tuberculosis via TLR2, leading to the activation of the p38 MAPK pathway, which in turn would induce PMN activation and apoptosis. This mechanism needs further confirmation at the site of infection.

Increased Susceptibility to Apoptosis of CD56dimCD16+ NK Cells Induces the Enrichment of IFN-γ-Producing CD56bright Cells in Tuberculous Pleurisy

Tuberculous pleuritis is a good model for the study of specific cells at the site of active Mycobacterium tuberculosis (Mtb) infection. We investigated the frequency and phenotype of NK cells in paired samples of peripheral blood and pleural fluid (PF) from patients with tuberculosis (TB) or parapneumonic infection. We demonstrated for the first time a reduction of NK cells in PF from TB with an enrichment in the CD56brightCD16− subset. In agreement, in PF NK cells we observed an increased expression of CD94, NKG2A, CD62L, and CCR7 molecules and lower expression of Bcl-2 and perforin. The activation markers CD69 and HLA-DR were also increased. The enrichment in the CD56bright subset was due to an increased susceptibility to apoptosis of CD56+CD16+ NK cells mediated by heat-labile and stable soluble factors present in tuberculous effusions and not in PF from other etiologies. Furthermore, in TB patients, Mtb-induced IFN-γ production by PF NK cells was not dependent on the presence of CD3+, CD19+, and CD14+ cells, suggesting a direct interaction of CD56bright cells with Mtb and/or the involvement of other accessory cells present at the site of Mtb infection.

Outbreaks of mycobacterium tuberculosis MDR strains induce high IL-17 T-cell response in patients with MDR tuberculosis that is closely associated with high antigen load.

BACKGROUND The proinflammatory cytokine interleukin 17 (IL-17) plays an important role in immune responses but it is also associated with tissue-damaging inflammation. So, we evaluated the ability of Mycobacterium tuberculosis clinical isolates to induce IL-17 in tuberculosis (TB) patients and in healthy human tuberculin reactors (PPD(+)HD). METHODS IL-17, interferon γ (IFN-γ), and interleukin 23 (IL-23) receptor expression were evaluated ex vivo and cultured peripheral blood mononuclear cells from TB and PPD(+)HD stimulated with irradiated clinical isolates from multidrug resistant (MDR) outbreaks M (Haarlem family) and Ra (Latin American-Mediterranean family), as well as drug-susceptible isolates belonging to the same families and laboratory strain H37Rv for 48 hours in T-cell subsets by flow cytometry. RESULTS We observed that: (1) MDR strains M and Ra are stronger IL-17 inducers than drug-susceptible Mtb strains of the Haarlem and Latin American-Mediterranean families, (2) MDR-TB patients show the highest IL-17 expression that is independent on the strain, (3) IL-17 expression is dependent on CD4(+) and CD8(+) T cells associates with persistently high antigen load. CONCLUSIONS IL-17--producing T cells could play an immunopathological role in MDR-TB promoting severe tissue damage, which may be associated with the low effectiveness of the second-line drugs employed in the treatment.

Mycobacterium tuberculosis-Induced Gamma Interferon Production by Natural Killer Cells Requires Cross Talk with Antigen-Presenting Cells Involving Toll-Like Receptors 2 and 4 and the Mannose Receptor in Tuberculous Pleurisy

ABSTRACT Tuberculous pleurisy allows the study of human cells at the site of active Mycobacterium tuberculosis infection. In this study, we found that among pleural fluid (PF) lymphocytes, natural killer (NK) cells are a major source of early gamma interferon (IFN-γ) upon M. tuberculosis stimulation, leading us to investigate the mechanisms and molecules involved in this process. We show that the whole bacterium is the best inducer of IFN-γ, although a high-molecular-weight fraction of culture filtrate proteins from M. tuberculosis H37Rv and the whole-cell lysate also induce its expression. The mannose receptor seems to mediate the inhibitory effect of mannosylated lipoarabinomannan, and Toll-like receptor 2 and 4 agonists activate NK cells but do not induce IFN-γ like M. tuberculosis does. Antigen-presenting cells (APC) and NK cells bind M. tuberculosis, and although interleukin-12 is required, it is not sufficient to induce IFN-γ expression, indicating that NK cell-APC contact takes place. Indeed, major histocompatibility complex class I, adhesion, and costimulatory molecules as well as NK receptors regulate IFN-γ induction. The signaling pathway is partially inhibited by dexamethasone and sensitive to Ca2+ flux and cyclosporine. Inhibition of p38 and extracellular-regulated kinase mitogen-activated protein kinase pathways reduces the number of IFN-γ+ NK cells. Phosphorylated p38 (p-p38) is detected in ex vivo PF-NK cells, and M. tuberculosis triggers p-p38 in PF-NK cells at the same time that binding between NK and M. tuberculosis reaches its maximum value. Thus, interplay between M. tuberculosis and NK cells/APC triggering IFN-γ would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a type 1 profile.

Patients with Multidrug-Resistant Tuberculosis Display Impaired Th1 Responses and Enhanced Regulatory T-Cell Levels in Response to an Outbreak of Multidrug-Resistant Mycobacterium tuberculosis M and Ra Strains

ABSTRACT In Argentina, multidrug-resistant tuberculosis (MDR-TB) outbreaks emerged among hospitalized patients with AIDS in the early 1990s and thereafter disseminated to the immunocompetent community. Epidemiological, bacteriological, and genotyping data allowed the identification of certain MDR Mycobacterium tuberculosis outbreak strains, such as the so-called strain M of the Haarlem lineage and strain Ra of the Latin America and Mediterranean lineage. In the current study, we evaluated the immune responses induced by strains M and Ra in peripheral blood mononuclear cells from patients with active MDR-TB or fully drug-susceptible tuberculosis (S-TB) and in purified protein derivative-positive healthy controls (group N). Our results demonstrated that strain M was a weaker gamma interferon (IFN-γ) inducer than H37Rv for group N. Strain M induced the highest interleukin-4 expression in CD4+ and CD8+ T cells from MDR- and S-TB patients, along with the lowest cytotoxic T-lymphocyte (CTL) activity in patients and controls. Hence, impairment of CTL activity is a hallmark of strain M and could be an evasion mechanism employed by this strain to avoid the killing of macrophages by M-specific CTL effectors. In addition, MDR-TB patients had an increased proportion of circulating regulatory T cells (Treg cells), and these cells were further expanded upon in vitro M. tuberculosis stimulation. Experimental Treg cell depletion increased IFN-γ expression and CTL activity in TB patients, with M- and Ra-induced CTL responses remaining low in MDR-TB patients. Altogether, these results suggest that immunity to MDR strains might depend upon a balance between the individual host response and the ability of different M. tuberculosis genotypes to drive Th1 or Th2 profiles.

In tuberculous pleural effusions, activated neutrophils undergo apoptosis and acquire a dendritic cell-like phenotype

Tuberculous pleuritis usually shows lymphocytic preponderance, but neutrophils are also present. Therefore, pleuritis is a good model for the study of neutrophil fate at sites of active Mycobacterium tuberculosis infection. We have previously demonstrated in vitro that M. tuberculosis-induced neutrophil apoptosis involves p38 mitogen protein kinase activation through Toll-like receptor 2. Herein, we demonstrate that, in tuberculous pleuritis, neutrophil apoptosis increases together with the expression of Toll-like receptor 2 and phosphorylated p38 (p-p38) kinase. In addition, receptors associated with activation/apoptotis (CD11b, CD64, tumor necrosis factor receptor, and Fas ligand) are up-regulated, together with a loss of CD16 expression. However, neutrophils express CD86, CD83, and major histocompatibility complex class II antigens, acquiring dendritic cell (DC) characteristics. Therefore, the cytokine milieu in the pleural space may influence signaling pathways on activated neutrophils, thereby inducing apoptosis and inhibiting their proinflammatory capacity, as well as allowing them acquire DC characteristics that influence the immune response.

Spontaneous or Mycobacterium tuberculosis‐induced apoptotic neutrophils exert opposite effects on the dendritic cell‐mediated immune response

Polymorphonuclear neutrophils (PMN) modulate the adaptive immune response through interactions with immature dendritic cells (iDC) while spontaneous apoptotic neutrophils PMNapo (PMNapo) may have an inhibitory effect on DC functions. We investigate the effect exerted by PMNapo in DC maturation and the role of Mycobacterium tuberculosis (Mtb)‐induced PMNapo in the cross‐presentation of mycobacterial antigens. We demonstrate that Mtb triggers the maturation of iDC while it is impaired by the presence of PMNapo, which abrogate Mtb‐induced expression of costimulatory and HLA class II molecules, reducing IL‐12 and IFN‐γ release by DC and partially inhibiting Mtb‐driven lymphocyte proliferation. This inhibitory effect is not observed in already Mtb‐matured DC, and it involves a direct interaction between DC and PMNapo, as supernatants from PMNapo cultures do not reveal this effect. Although PMNapo do not alter Mtb/DC‐SIGN interaction, they affect the intracellular signals leading to DC maturation without requiring their entry into DC. Phagocytosis of Mtb‐induced PMNapo by iDC leads to lymphoproliferation, which is significantly reduced by blocking CD36 and not DC‐SIGN on iDC. Therefore, cross‐presentation of Mtb antigens is taking place. Our findings suggest that the inflammatory milieu is subjected to a fine balance between non‐infected and Mtb‐induced PMNapo: non‐infected PMNapo limiting inflammation and Mtb‐induced PMNapo generating a specific immune activity.