Variants of human DECTIN-1 rs16910526 are linked to differential reactive oxygen species production and susceptibility to tuberculosis.

BACKGROUND: Dectin-1 is a transmembrane receptor that plays a pivotal role in recognising fungi and Mycobacterium tuberculosis (Mtb). A specific variant, DECTIN-1 rs16910526, results in a truncated receptor that disrupts membrane expression and ligand binding and is clinically associated with recurrent cutaneous mycoses. Previous research has clarified the role of Dectin-1 in boosting immune defenses against mycobacteria by enhancing reactive oxygen species (ROS) production in neutrophils (PMNs). Here, we investigated the association between the rs16910526 variant and Dectin-1 expression in PMNs, as well as intracellular ROS production in response to Mtb. Furthermore, we explored the potential link between the rs16910526 gene variant and TB outcomes in Argentina. METHODS: DNA was extracted from blood samples obtained from a cohort of 178 TB patients and healthy subjects (HS) in Argentina. PCR amplification and sequencing were performed to identify the rs16910526 variant. Flow cytometry was utilised to assess Dectin-1 expression on the PMN plasma membrane and to measure intracellular ROS levels, as indicated by the oxidation of DHR123 in response to the Mtb antigen. RESULTS: PMNs carrying the rs16910526 variant exhibited diminished Dectin-1 expression and ROS production in response to Mtb (p < 0.0001). In a case‒control study, the rs16910526 variant had an allelic frequency of 0.112 in TB patients and 0.051 in HS. Notably, 10 out of 88 HS and 18 out of 62 TB patients harboured the variant (odds ratio [OR]: 2.55 [95% CI 1.1-5.9, p = 0.03]), indicating a potential association with TB disease. Furthermore, TB patients with the rs16910526 variant exhibited a delayed sputum smear conversion time (p < 0.004) and 100% positivity for acid-fast bacilli smears (p < 0.00001). CONCLUSION: Our study identified a significant association between the SNP variant rs16910526 in the DECTIN-1 gene and Dectin-1 expression in the PMN, leading to altered ROS production. The higher frequency of this variant in TB patients compared to HS suggests a possible link with susceptibility to TB disease in Argentina.

Apoptosis-mediated inhibition of human T-cell acute lymphoblastic leukemia upon treatment with Staphylococus Aureus enterotoxin-superantigen.

Patients with relapsed T cell acute lymphoblastic leukemia (T-ALL) have limited therapeutic options and poor prognosis. The finding of efficient strategies against this refractory neoplasm is a medical priority. Superantigens (SAgs) are viral and bacterial proteins that bind to major histocompatibility complex class II molecules as unprocessed proteins and subsequently interact with a high number of T cells expressing particular T cell receptor Vß chains. Although on mature T cells, SAgs usually trigger massive cell proliferation producing deleterious effects on the organism, in contrast, on immature T cells, they may trigger their death by apoptosis. On this basis, it was hypothesized that SAgs could also induce apoptosis in neoplastic T cells that are usually immature cells that probably conserve their particular Vß chains. In this work, we investigated the effect of the SAg Staphylococcus aureus enterotoxin E (SEE) (that specifically interacts with cells that express Vß8 chain), on human Jurkat T- leukemia line, that expresses Vß8 in its T receptor and it is a model of the highly aggressive recurrent T-ALL. Our results demonstrated that SEE could induce apoptosis in Jurkat cells in vitro. The induction of apoptosis was specific, correlated to the down regulation of surface Vß8 TCR expression and was triggered, at least in part, through the Fas/FasL extrinsic pathway. The apoptotic effect induced by SEE on Jurkat cells was therapeutically relevant. In effect, upon transplantation of Jurkat cells in the highly immunodeficient NSG mice, SEE treatment reduced dramatically tumor growth, decreased the infiltration of neoplastic cells in the bloodstream, spleen and lymph nodes and, most importantly, increased significantly the survival of mice. Taken together, these results raise the possibility that this strategy can be, in the future, a useful option for the treatment of recurrent T-ALL.

No Te Tratan Bien Porque Eres Mexicana: Intersectional Systemic Violence and Precarity in Latina Adolescent Life in the U.S. South.

Young Latina women (YLW) in Alabama are disproportionately affected by sexual health disparities. However, to access needed reproductive services, YLW must navigate a healthcare landscape that restricts access for youth. YLW also face racialized immigration enforcement in their communities which is designed to attrition the region's emergent Latina/o/x immigrant population. This paper describes the intersectional, structural forces that contribute to experienced systemic violence for YLW as they try to access sexual healthcare services. In 2017, we conducted semi-structured qualitative interviews with 20 YLW and 24 key stakeholders (parents, providers, Latino/a/x community leaders etc.) in West Alabama to examine attitudes and perceptions about sexual health and healthcare access (HCA) among YLW in the region. We used purposeful convenience sampling and snowballing to recruit a community-based sample. That is, we purposefully recruited YLW, adjusting through the recruitment period for a diverse sample, who represented the various voices that we were trying to capture in the study (i.e., younger and older adolescents, adolescents born in the U.S. and those born in other countries etc.). Through a focus on YLW's access to sexual/reproductive healthcare, we conclude that YLW experience systemic violence and resulting precarity because laws and health policies restrict access to evidence-based sexual health education and reproductive healthcare services. We discuss implications for future research and policy recommendations.

Reactive oxygen species production by human dendritic cells involves TLR2 and dectin-1 and is essential for efficient immune response against Mycobacteria.

Tuberculosis remains the single largest infectious disease with 10 million new cases and two million deaths that are estimated to occur yearly, more than any time in history. The intracellular replication of Mycobacterium tuberculosis (Mtb) and its spread from the lungs to other sites occur before the development of adaptive immune responses. Dendritic cells (DC) are professional antigen-presenting cells whose maturation is critical for the onset of the protective immune response against tuberculosis disease and may vary depending on the nature of the cell wall of Mtb strain. Here, we describe the role of the endogenous production of reactive oxygen species (ROS) on DC maturation and expansion of Mtb-specific lymphocytes. Here, we show that Mtb induces DC maturation through TLR2/dectin-1 by generating of ROS and through Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) in a ROS independently manner. Based on the differences observed in the ability to induce DC maturation, ROS production and lymphocyte proliferation by those Mtb families widespread in South America, i.e., Haarlem and Latin American Mediterranean and the reference strain H37Rv, we propose that variance in ROS production might contribute to immune evasion affecting DC maturation and antigen presentation.

Impaired dendritic cell differentiation of CD16-positive monocytes in tuberculosis: role of p38 MAPK.

Tuberculosis (TB) is one of the world's most pernicious diseases mainly due to immune evasion strategies displayed by its causative agent Mycobacterium tuberculosis (Mtb). Blood monocytes (Mos) represent an important source of DCs during chronic infections; consequently, the alteration of their differentiation constitutes an escape mechanism leading to mycobacterial persistence. We evaluated whether the CD16(+)/CD16(-) Mo ratio could be associated with the impaired Mo differentiation into DCs found in TB patients. The phenotype and ability to stimulate Mtb-specific memory clones DCs from isolated Mo subsets were assessed. We found that CD16(-) Mos differentiated into CD1a(+) DC-SIGN(high) cells achieving an efficient recall response, while CD16(+) Mos differentiated into a CD1a(-) DC-SIGN(low) population characterized by a poor mycobacterial Ag-presenting capacity. The high and sustained phosphorylated p38 expression observed in CD16(+) Mos was involved in the altered DC profile given that its blockage restored DC phenotype and its activation impaired CD16(-) Mo differentiation. Furthermore, depletion of CD16(+) Mos indeed improved the differentiation of Mos from TB patients toward CD1a(+) DC-SIGN(high) DCs. Therefore, Mos from TB patients are less prone to differentiate into DCs due to their increased proportion of CD16(+) Mos, suggesting that during Mtb infection Mo subsets may have different fates after entering the lungs.

Clinical isolates of Mycobacterium tuberculosis differ in their ability to induce respiratory burst and apoptosis in neutrophils as a possible mechanism of immune escape.

Tuberculosis pathogenesis was earlier thought to be mainly related to the host but now it appears to be clear that bacterial factors are also involved. Genetic variability of Mycobacterium tuberculosis (Mtb) could be slight but it may lead to sharp phenotypic differences. We have previously reported that nonopsonized Mtb H37Rv induce apoptosis of polymorphonuclear neutrophils (PMNs) by a mechanism that involves the p38 pathway. Here we evaluated the capability to induce PMN apoptosis of two prevalent Mtb lineages in Argentina, the Latin America and Mediterranean (LAM), and Haarlem, using the H37Rv as a reference strain. Results showed that LAM strains strongly induced apoptosis of PMN which correlated with the induction of reactive oxygen species (ROS) production and p38 activation. Interestingly, the highly prosperous multidrug-resistant M strain, belonging to the Haarlem lineage, lacked the ability to activate and to induce PMN apoptosis as a consequence of (1) a weak ROS production and (2) the contribution of antiapoptotic mechanisms mediated at least by ERK. Although with less skill, M is able to enter the PMN so that phenotypic differences could lead PMN to be a reservoir allowing some pathogens to prevail and persist over other strains in the community.

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.

Paradoxical role of CD16+CCR2+CCR5+ monocytes in tuberculosis: efficient APC in pleural effusion but also mark disease severity in blood.

The role of CD16(-) and CD16(+) Mo subsets in human TB remains unknown. Our aim was to characterize Mo subsets from TB patients and to assess whether the inflammatory milieu from TB pleurisy modulate their phenotype and recruitment. We found an expansion of peripheral CD16(+) Mo that correlated with disease severity and with TNF-α plasma levels. Circulating Mo from TB patients are activated, showing a higher CD14, CD16, and CD11b expression and Mtb binding than HS. Both subsets coexpressed CCR2/CCR5, showing a potential ability to migrate to the inflammatory site. In tuberculous PF, the CD16(+) subset was the main Mo/MΦ population, accumulation that can be favored by the induction of CD16 expression in CD16(-) Mo triggered by soluble factors found in this inflammatory milieu. CD16(+) Mo in PF were characterized by a high density of receptors for Mtb recognition (DC-SIGN, MR, CD11b) and for lipid-antigens presentation (CD1b), allowing them to induce a successful, specific T cell proliferation response. Hence, in tuberculous PF, CD16(+) Mo constitute the main APC population; whereas in PB, their predominance is associated with the severity of pulmonary TB, suggesting a paradoxical role of the CD16(+) Mo subset that depends on the cellular localization.

Mycobacterium tuberculosis impairs dendritic cell response by altering CD1b, DC-SIGN and MR profile.

During a chronic infection such as tuberculosis, the pool of tissue dendritic cells (DC) must be renewed by recruitment of both circulating DC progenitors and monocytes (Mo). However, the microenvironment of the inflammatory site affects Mo differentiation. As DC are critical for initiating a Mycobacterium tuberculosis-specific T-cell response, we argue that interference of M. tuberculosis with a correct DC generation would signify a mechanism of immune evasion. In this study, we showed that early interaction of γ-irradiated M. tuberculosis with Mo subverts DC differentiation in vitro. We found that irradiated M. tuberculosis effect involves (1) the loss of a significant fraction of monocyte population and (2) an altered differentiation process of the surviving monocyte subpopulation. Moreover, in the absence of irradiated M. tuberculosis, DC consist in a major DC-specific intercellular adhesion molecule 3-grabbing non-integrin receptor (DC-SIGN(high))/CD86(low) and minor DC-SIGN(low)/CD86(high) subpopulations, whereas in the presence of bacteria, there is an enrichment of DC-SIGN(low)/CD86(high) population. Besides, this population enlarged by irradiated M. tuberculosis, which is characterized by a reduced CD1b expression, correlates with a reduced induction of specific T-lymphocyte proliferation. The loss of CD1molecules partially involves toll-like receptors (TLR-2)/p38 MAPK activation. Finally, several features of Mo, which have been differentiated into DC in the presence of irradiated M. tuberculosis, resemble the features of DC obtained from patients with active tuberculosis. In conclusion, we suggest that M. tuberculosis escapes from acquired immune response in tuberculosis may be caused by an altered differentiation into DC leading to a poor M. tuberculosis-specific T-cell response.

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.

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-gamma) 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-gamma 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.

NK cells from tuberculous pleurisy express high ICAM-1 levels and exert stimulatory effect on local T cells.

Tuberculous pleurisy, one of the most common manifestations of extrapulmonary tuberculosis, is characterized by a T-cell-mediated hypersensitivity reaction along with a Th1 immune profile. In this study, we investigated functional cross-talk among T and NK cells in human tuberculous pleurisy. We found that endogenously activated pleural fluid-derived NK cells express high ICAM-1 levels and induce T-cell activation ex vivo through ICAM-1. Besides, upon in vitro stimulation with monokines and PAMP, resting peripheral blood NK cells increased ICAM-1 expression leading to cellular activation and Th1 polarization of autologous T cells. Furthermore, these effects were abolished by anti-ICAM-1 Ab. Hence, NK cells may contribute to the adaptive immune response by a direct cell-contact-dependent mechanism in the context of Mycobacterium tuberculosis infection.

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.

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-gamma) 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-gamma, 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-gamma 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-gamma 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-gamma 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-gamma+ 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-gamma would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a type 1 profile.

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-gamma 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.

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)-gamma. 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-gamma 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-gamma 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-gamma production.

Increased susceptibility to apoptosis of CD56dimCD16+ NK cells induces the enrichment of IFN-gamma-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-gamma 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.

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.

Mycobacterium tuberculosis triggers apoptosis in peripheral neutrophils involving toll-like receptor 2 and p38 mitogen protein kinase in tuberculosis patients.

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 Fcgamma 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.

Mycobacterium tuberculosis-induced activation accelerates apoptosis in peripheral blood neutrophils from patients with active tuberculosis.

The activation of circulating polymorphonuclear neutrophils (PMN) from patients with active tuberculosis (TB-PMN) may be associated with induction of apoptosis. Spontaneous or Mycobacterium tuberculosis (MTB)-induced apoptosis of PMN were evaluated by microscopy, DNA content, and their binding to Annexin V at 0, 3, and 18 h. In addition, the expression of CD11b and of CD16 were evaluated as parameters of activation and apoptosis, respectively. Recently isolated TB-PMN showed a higher CD11b expression than normal PMN (N-PMN), but there were no features of apoptosis, even though an enhancement of Fas expression was observed. Spontaneous apoptosis was accelerated in TB-PMN at 3 h, but no differences were observed in TB- and N-PMN at 18 h of culture. When stimulated with MTB, both TB- and N-PMN steadily increased CD11b expression along the culture period. MTB induced apoptosis of N-PMN at 3 h with loss of CD16 expression. By contrast, MTB delayed the apoptotic rate of TB-PMN, preserving the CD16 receptor at 3 h, whereas it accelerated apoptosis at 18 h, increasing at the same time the expression of CD11b. Taken together, these data suggest that the acceleration of apoptosis observed in TB-PMN could be associated with the MTB-induced activation.

Papel de la activación de los polimorfonucleares neutrófilos al proceso inflamatorio crónico en tuberculosis humana / Role of the neutrophil polymorphonuclears activation to the chronic inflammatory process in human tuberculosis

Los polimorfonucleares neutrófilos (PMN) participan en la formación del granuloma y preceden a los monocitos en las áreas de inflamación granulomatosa, liberando quimioquinas que atraen a los monocitos. El tumor necrosis factor alpha (TNFalfa), juega un papel importante en el desarrollo de la respuesta protectiva en tuberculosis (TB), participando en la formación del granuloma. Por otra parte está descrito que el TNFalfa promueve funciones del PMN y afecta la expresión de los receptores para el fragmento Fc de inmunoglobulina G (FcgammaRs), que a su vez median dichas funciones. Basados en estos antecedentes, decidimos evaluar el papel de los PMN en el proceso inflamatorio crónico de la tuberculosis, como regulador de la respuesta a través de la liberación de citoquinas (CK) y no por su contribución fagocítica, ya que es sabido que el Mycobacterium tuberculosis (M.tuberculosis), evade los mecanismos fagocíticos del neutrófilo. A fin de evaluar la contribución de los PMN en el proceso inflamatorio durante la TB activa, se investigó el estado de activación de los PMN circulantes de pacientes con TB (TB-PMN) comparándose con normales (N-PMN). Se evaluó la expresión de receptores para la porción Fc de IgG (FcgammaRI, FcgammaRII y FcgammaRIIIB), CD66b (marcador de degranulación) así como receptores para TNFalfa de 55 y 75 kDa(TNF-r55 y TNF-R75 respectivamente). Por otra parte se evaluaron parámetros funcionales como citotoxicidad, estallido respiratorio inducido por FMLP, y la producción de TNFalfa e IL-1beta... (TRUNCADO)