Eduardo Abbate

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.

IL‐10 down‐regulates costimulatory molecules on Mycobacterium tuberculosis‐pulsed macrophages and impairs the lytic activity of CD4 and CD8 CTL in tuberculosis patients

Activation of T cells requires both TCR‐specific ligation and costimulation through accessory molecules during T cell priming. IFNγ is a key cytokine responsible for macrophage activation during Mycobacterium tuberculosis (Mtb) infection while IL‐10 is associated with suppression of cell mediated immunity in intracellular infection. In this paper we evaluated the role of IFNγ and IL‐10 on the function of cytotoxic T cells (CTL) and on the modulation of costimulatory molecules in healthy controls and patients with active tuberculosis (TB). γ‐irradiated‐Mtb (i‐Mtb) induced IL‐10 production from CD14+ cells from TB patients. Moreover, CD3+ T cells of patients with advanced disease also produced IL‐10 after i‐Mtb stimulation. In healthy donors, IL‐10 decreased the lytic activity of CD4+ and CD8+ T cells whereas it increased γδ‐mediated cytotoxicity. Furthermore, we found that the presence of IL‐10 induced a loss of the alternative processing pathways of antigen presentation along with a down‐regulation of the expression of costimulatory molecule expression on monocytes and macrophages from healthy individuals. Conversely, neutralization of endogenous IL‐10 or addition of IFNγ to either effector or target cells from TB patients induced a strong lytic activity mediated by CD8+ CTL together with an up‐regulation of CD54 and CD86 expression on target cells. Moreover, we observed that macrophages from TB patients could use alternative pathways for i‐Mtb presentation. Taken together, our results demonstrate that the presence of IL‐10 during Mtb infection might contribute to mycobacteria persistence inside host macrophages through a mechanism that involved inhibition of MHC‐restricted cytotoxicity against infected macrophages.

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.

NK cell activity in tuberculosis is associated with impaired CD11a and ICAM-1 expression: a regulatory role of monocytes in NK activation

Although the role of natural killer (NK) cells in mycobacterial infections is unclear, it has been postulated that they contribute to protective immunity through the production of interferon (IFN)‐γ. In this study, we evaluate the effect of interleukin (IL)‐10, IL‐15 and IL‐18 on NK lytic activity through the expression of CD16, CD11a and CD69 molecules and the induction of IFN‐γ production in patients with tuberculosis (TB) and healthy individuals (N). Our results showed an impairment of NK lytic activity and a gradual down‐regulation of costimulatory and adhesion molecules on NK cells which were dependent on the severity of the disease. NK lytic activity was increased by exogenous IL‐15 and IL‐18 in both TB and N, and by neutralization of endogenous IL‐10 only in TB; IL‐15 and IL‐18 increased CD69 receptor expression, while anti‐IL‐10 up‐regulated CD16 and CD11a expression in TB. Mycobacterium tuberculosis reduced the number of intracellular adhesion molecule (ICAM)‐1+ CD14+ cells, but in the presence of IL‐15, IL‐18 and anti‐IL‐10 its expression was up‐regulated. In cells from TB patients, the observed effects of IL‐15 and IL‐18 on NK function were not dependent on IL‐10 modulation of the surface expression of activator/adhesion molecules. In the absence of monocytes, IL‐10 activated NK cells, suggesting an indirect effect on their function. Furthermore, in TB patients the depletion of monocytes increased the production of IFN‐γ by NK cells. Therefore, monocytes from TB patients regulated the NK function involving IL‐10 which, through an indirect mechanism, led to the down‐regulation of costimulatory/adhesion molecules and/or IFN‐γ production.

Specific lytic activity against mycobacterial antigens is inversely correlated with the severity of tuberculosis

The ability of peripheral blood mononuclear cells (PBMC) from patients with active tuberculosis to display cytotoxic responses against autologous Mycobacterium tuberculosis (Mtb)‐pulsed macrophages was evaluated. Non‐MHC restricted cell‐dependent lytic activity was observed in ex vivo effector cells from tuberculosis patients and was mediated mainly by CD3+γδ TCR+ T (γδ T) cells bearing CD56 and/or CD16 molecules. MHC‐restricted and non‐MHC restricted cytotoxic T cells (CTL) were differentially expanded upon stimulation with Mtb in tuberculosis patients and normal controls (N). Class‐I restricted CD8+ CTL and class‐II restricted CD4+ CTL were generated in PPD+N and to a lesser extent in PPD–N. Mtb‐stimulated effector cells from tuberculosis patients became progressively non‐MHC restricted CD4–CD8–γδ T cells, while lytic activity of CD4+ and CD8+CTL decreased gradually as the disease became more severe. On the other hand, target cells were lysed by ex vivo cells from tuberculosis patients through the Fas‐FasL and perforin pathways. Mtb‐induced CD4+ CTL from tuberculosis patients and N controls preferentially employed the Fas‐FasL mechanism. Mtb‐induced CD8+ CTL effector cells from patients used the perforin‐based mechanism while cells from N controls also used the Fas‐FasL pathway. While Mtb‐induced γδ CTL from patients and PPD–N employed the latter mechanism cells from PPD+N individuals also used the perforin pathway. It can be concluded that shifts in the CTL response and the cytolytic mechanisms take place as the pulmonary involvement becomes more severe.

Inducible Costimulator: A Modulator of IFN-γ Production in Human Tuberculosis

Effective host defense against Mycobacterium tuberculosis requires the induction of Th1 cytokine responses. We investigated the regulated expression and functional role of the inducible costimulator (ICOS), a receptor known to regulate Th cytokine production, in the context of human tuberculosis. Patients with active disease, classified as high responder (HR) or low responder (LR) patients according to their in vitro T cell responses against the Ag, were evaluated for T cell expression of ICOS after M. tuberculosis-stimulation. We found that ICOS expression significantly correlated with IFN-γ production by tuberculosis patients. ICOS expression levels were regulated in HR patients by Th cytokines: Th1 cytokines increased ICOS levels, whereas Th2-polarizing conditions down-regulated ICOS in these individuals. Besides, in human polarized Th cells, engagement of ICOS increased M. tuberculosis IFN-γ production with a magnitude proportional to ICOS levels on those cells. Moreover, ICOS ligation augmented Ag-specific secretion of the Th1 cytokine IFN-γ from responsive individuals. In contrast, neither Th1 nor Th2 cytokines dramatically affected ICOS levels on Ag-stimulated T cells from LR patients, and ICOS activation did not enhance IFN-γ production. However, simultaneous activation of ICOS and CD3 slightly augmented IFN-γ secretion by LR patients. Together, our data suggest that the regulation of ICOS expression depends primarily on the response of T cells from tuberculosis patients to the specific Ag. IFN-γ released by M. tuberculosis-specific T cells modulates ICOS levels, and accordingly, ICOS ligation induces IFN-γ secretion. Thus, ICOS activation may promote the induction of protective Th1 cytokine responses to intracellular bacterial pathogens.

CD3 expression distinguishes two γδT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from γδT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating γδT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of γδT cells were differentiated by the CD3/γδT cell receptor (γδTCR) complex. The γδTCRlow subset had a higher CD3 to TCR ratio and was enriched in Vδ2+ cells, whereas most Vδ1+ cells belonged to the γδTCRhigh subset. In PB from TB, most γδTCRhigh were CD45RA+CCR7‐ and γδTCRlow were CD45RA+/−CCR7+CXCR3+. In the pleural space the proportion of CD45RA‐CCR7+CXCR3+ cells was higher. Neither spontaneous nor Mtb‐induced interferon (IFN)‐γ production was observed in PB‐γδT cells from TB; however, PE‐γδT cells showed a strong response. Both PB‐ and PE‐γδ T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE‐γδTCRlow cells were the most potent effector cells. Thus, γδT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As γδT cells produce IFN‐γ within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.

Monocytes and neutrophils from tuberculosis patients are insensitive to anti-inflammatory effects triggered by the prototypic formyl peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP)

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis where formyl peptides, which are cleavage products of bacterial and mitochondrial proteins, are present. In this study, we demonstrated that interferon gamma (IFN)‐γ and interleukin (IL)‐10 induced the overexpression of the receptor for the Fc portion of IgG I (FcγRI) in monocytes from tuberculosis (TB) patients, showing that these cells respond to IFN‐γ and IL‐10 signals. We also demonstrated that lower doses of IL‐10 render monocytes from TB patients less responsive to higher doses of the cytokine. Although the prototypic formyl peptide N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) is a well‐known proinflammatory agonist, we have demonstrated previously that preincubation of monocytes with FMLP inhibited the up‐regulation of FcγRI induced by IFN‐γ or IL‐10. This effect was not observed in monocytes from TB patientes. FMLP also induced the down‐regulation of the expression of FcγRI in monocytes that had been activated already with IFN‐γ. However, this effect of FMLP was not observed in monocytes from TB patients and supernatants from monocytes obtained from these patients were incapable of inducing the down‐regulation of FcγRI. In contrast to normal donors, supernatants from FMLP‐treated neutrophils from TB patients did not modify the basal level of expression of FcγRI in monocytes from normal donors. In conclusion, in this study we demonstrated the existence of two novel mechanisms that may contribute to the pathological effects generated by M. tuberculosis: the enhancement of FcγRI in response to IFN‐γ and IL‐10, and the unresponsiveness to the anti‐inflammatory effects induced by formyl peptides.