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.

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 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.

Mycobacterium tuberculosis multidrug resistant strain M induces an altered activation of cytotoxic CD8+ T cells.

In human tuberculosis (TB), CD8+ T cells contribute to host defense by the release of Th1 cytokines and the direct killing of Mycobacterium tuberculosis (Mtb)-infected macrophages via granule exocytosis pathway or the engagement of receptors on target cells. Previously we demonstrated that strain M, the most prevalent multidrug-resistant (MDR) Mtb strain in Argentine, is a weak inducer of IFN-γ and elicits a remarkably low CD8-dependent cytotoxic T cell activity (CTL). In contrast, the closely related strain 410, which caused a unique case of MDR-TB, elicits a CTL response similar to H37Rv. In this work we extend our previous study investigating some parameters that can account for this discrepancy. We evaluated the expressions of the lytic molecules perforin, granzyme B and granulysin and the chemokine CCL5 in CD8+ T cells as well as activation markers CD69 and CD25 and IL-2 expression in CD4+ and CD8+ T cells stimulated with strains H37Rv, M and 410. Our results demonstrate that M-stimulated CD8+ T cells from purified protein derivative positive healthy donors show low intracellular expression of perforin, granzyme B, granulysin and CCL5 together with an impaired ability to form conjugates with autologous M-pulsed macrophages. Besides, M induces low CD69 and IL-2 expression in CD4+ and CD8+ T cells, being CD69 and IL-2 expression closely associated. Furthermore, IL-2 addition enhanced perforin and granulysin expression as well as the degranulation marker CD107 in M-stimulated CD8+ T cells, making no differences with cells stimulated with strains H37Rv or 410. Thus, our results highlight the role of IL-2 in M-induced CTL activity that drives the proper activation of CD8+ T cells as well as CD4+ T cells collaboration.

Two genetically-related multidrug-resistant Mycobacterium tuberculosis strains induce divergent outcomes of infection in two human macrophage models.

Mycobacterium tuberculosis has a considerable degree of genetic variability resulting in different epidemiology and disease outcomes. We evaluated the pathogen-host cell interaction of two genetically closely-related multidrug-resistant M. tuberculosis strains of the Haarlem family, namely the strain M, responsible for an extensive multidrug-resistant tuberculosis outbreak, and its kin strain 410 which caused a single case in two decades. Intracellular growth and cytokine responses were evaluated in human monocyte-derived macrophages and dU937 macrophage-like cells. In monocyte-derived macrophages, strain M grew more slowly and induced lower levels of TNF-α and IL-10 than 410, contrasting with previous studies with other strains, where a direct correlation was observed between increased intracellular growth and epidemiological success. On the other hand, in dU937 cells, no difference in growth was observed between both strains, and strain M induced significantly higher TNF-α levels than strain 410. We found that both cell models differed critically in the expression of receptors for M. tuberculosis entry, which might explain the different infection outcomes. Our results in monocyte-derived macrophages suggest that strain M relies on a modest replication rate and cytokine induction, keeping a state of quiescence and remaining rather unnoticed by the host. Collectively, our results underscore the impact of M. tuberculosis intra-species variations on the outcome of host cell infection and show that results can differ depending on the in vitro infection model.

Social and environmental health determinants and their relationship with parasitic diseases in asymptomatic children from a shantytown in Buenos Aires, Argentina.

Health inequities are a common problem for all countries and are the result of not only adverse social conditions but also poor public policies. Today chronic diseases represent the most relevant threats and are a current challenge. Parasitic infections, a leading cause of child morbidity affecting low-income populations, can be transmitted because of an unhealthy environment. Notwithstanding, scarce data have been published on the epidemiological profile of intestinal parasitoses in asymptomatic children living in shantytowns. Vulnerable populations settled in slums are growing in Argentina, particularly in Buenos Aires city. Consequently, this work intended to screen healthy carriers of enteric parasites and determine the epidemiologic profile in asymptomatic children residing in one of those communities, to explore risk factors associated with the transmission of parasites, and to initiate a basic health education campaign to promote healthy behavior in the community. Fecal samples (n = 138) were analyzed by conventional parasitological methods and a survey gathered data on symptoms, family composition, and environmental and hygiene-related variables. High prevalence of feco-orally-transmitted parasitoses (83·3%) and polyparasitism were remarkable findings. The main environmental health determinants were those related to excreta disposal and water provision. Health promotion actions were performed through the diffusion of a set of posters with iconic images and brief messages for health education. Results suggest the need for an environmental sanitation policy to complement health promotion actions. It is essential to spread the results of investigations that address inequities and social determinants of health in order to integrate data with local political processes and alert on acceptable actions for developing appropriate interventions.

Differential induction of macrophage cell death by antigens of a clustered and a non-clustered multidrug-resistant Mycobacterium tuberculosis strain from Haarlem family.

Some multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) genotypes are the cause of large outbreaks, including strain M identified in Argentina. In contrast, its kin strain 410 has only caused a single case to date. Cell wall antigens from Mtb were associated with the modulation of macrophage (MΦ) cell death, and the ability to inhibit of MΦ apoptosis is considered a virulence mechanism. In this study, the ability these two clinical isolates with divergent epidemiology to induce MΦ cell death was evaluated using whole inactivated bacteria. We showed that gamma-irradiated (I-) strains induced MΦ necrosis, the strongest inducer being I-410. Cell death biased towards apoptosis with the heat-killed (hk) strains, both hk-MDR strains being poorer inducers of MΦ apoptosis than was H37Rv. These effects were partly due to their ability to induce anti-apoptotic mechanisms which were not related to the lack of tumor necrosis factor alpha induction or a compensatory effect of interleukin-10. The most noticeable difference between strain M and strain 410 was the ability shown by hk-M to interfere with apoptosis induced by hk-H37Rv. Thus, heat-stable and heat-labile antigens from these epidemiologically divergent Mtb strains differ in their ability to manipulate MΦ death.

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-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.