Juan Ignacio Basile

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.

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.

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‐SIGNhigh)/CD86low and minor DC‐SIGNlow/CD86high subpopulations, whereas in the presence of bacteria, there is an enrichment of DC‐SIGNlow/CD86high 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.

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.

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

Tuberculosis (TB) is one of the worlds 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‐SIGNhigh cells achieving an efficient recall response, while CD16+ Mos differentiated into a CD1a−DC‐SIGNlow 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‐SIGNhigh 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.

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (Treg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of Treg (CD4+CD25highforkhead box protein 3+), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4+CD25+, CD4+CD25highFoxP3+ and CD8+CD25+ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4+CD25low/negIL‐10+ in PB and CD4+CD25low/negIFN‐γ+ in PF; meanwhile, CD25high mainly expressed IL‐10 in both compartments. A high proportion of CD4+CD107+ and CD8+CD107+ cells was observed in PF. Treg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4+ and CD8+ degranulation responses and decreased CD4+IL‐10+ cells in PF. Our results demonstrated that in TB pleurisy Treg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4+ and CD8+ cells to degranulate in response to M. tuberculosis.

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.

Outbreaks of Mycobacterium tuberculosis MDR strains differentially induce neutrophil respiratory burst involving lipid rafts, p38 MAPK and Syk

BackgroundNeutrophils (PMN) are the first cells to infiltrate the lung after infection, and they play a significant protective role in the elimination of pathogen, by releasing preformed oxidants and proteolytic enzymes from granules and generating ROS, thus limiting inflammation by succumbing to apoptosis. In a previous study, we found marked differences in ROS-induced apoptosis between two Mycobacterium tuberculosis (Mtb) strains, M and Ra, representative of widespread Mtb families in South America, i.e. Haarlem and Latin-American Mediterranean (LAM), being strain M able to generate further drug resistance and to disseminate aggressively.MethodsIn this study we evaluate the nature of bacteria-PMN interaction by assessing ROS production, apoptosis, lipid raft coalescence, and phagocytosis induced by Mtb strains.ResultsDectin-1 and TLR2 participate in Mtb-induced ROS generation and apoptosis in PMN involving p38 MAPK and Syk activation with the participation of a TLR2-dependent coalescence of lipid rafts. Further, ROS production occurs during the phagocytosis of non-opsonized bacteria and involves α-glucans on the capsule. In contrast, strain M lacks the ability to induce ROS because of: 1) a reduced phagocytosis and 2) a failure in coalescence of lipid raft.ConclusionsThe differences in wall composition could explain the success of some strains which stay unnoticed by the host through inhibition of apoptosis and ROS but making possible its replication inside PMN as a potential evasion mechanism. Innate immune responses elicited by Mtb strain-to-strain variations need to be considered in TB vaccine development.

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.