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.

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.

Rethinking the term "glyphosate effect" through the evaluation of different glyphosate-based herbicide effects over aquatic microbial communities.

Glyphosate-based herbicides (GBH) -the most widely used herbicides in agriculture worldwide-are frequently generalized by the name of "glyphosate". However, GBH encompass a variety of glyphosate salts as active ingredient and different adjuvants, which differ between products. These herbicides reach water bodies and produce diverse impacts over aquatic communities. Yet, the risk assessment assays required for the approval focus mostly on active ingredients. Herein, we compared the effect of five different GBH as well as of monoisopropylamine salt of glyphosate (GIPA) on aquatic microbial communities from natural shallow lakes that were mixed and allowed to evolve in an outdoor pond. We performed an 8-day long assay under indoor control conditions to evaluate the effects of exposure on the structure of nano-plus microphytoplankton (net phytoplankton, with sizes between 2 and 20 µm and >20 µm, respectively) and picoplankton (size ranging between 0.2 and 2 µm) communities through microscopy and flow cytometry, respectively. Significantly different effects were evident on the structure of microbial communities dependent on the GBH, even with herbicides sharing similar active ingredients. Each GBH evoked increases of different magnitude in bacterioplankton abundance. Furthermore, GIPA and a formulation decreased the abundance of a phycocyanin-rich (PC-rich) picocyanobacteria (Pcy) cytometric population and GIPA further altered Pcy composition. Also, two GBH increased net phytoplankton total abundance and, unlike the tested GBH, no apparent effect of GIPA was detected on this community structure. These results demonstrate that GBH effects on aquatic microbial communities should not be summarized as "glyphosate" effects considering that the formulations have effects beyond those exerted by the active ingredients alone. This work intends to alert on the lack of real knowledge regarding the consequences of the variety of GBH on natural aquatic ecosystems. Indeed, the wide use of the term "glyphosate effect" should be thoroughly rethought.

A georeferenced rRNA amplicon database of aquatic microbiomes from South America.

The biogeography of bacterial communities is a key topic in Microbial Ecology. Regarding continental water, most studies are carried out in the northern hemisphere, leaving a gap on microorganism's diversity patterns on a global scale. South America harbours approximately one third of the world's total freshwater resources, and is one of these understudied regions. To fill this gap, we compiled 16S rRNA amplicon sequencing data of microbial communities across South America continental water ecosystems, presenting the first database µSudAqua[db]. The database contains over 866 georeferenced samples from 9 different ecoregions with contextual environmental information. For its integration and validation we constructed a curated database (µSudAqua[db.sp]) using samples sequenced by Illumina MiSeq platform with commonly used prokaryote universal primers. This comprised ~60% of the total georeferenced samples of the µSudAqua[db]. This compilation was carried out in the scope of the µSudAqua collaborative network and represents one of the most complete databases of continental water microbial communities from South America.

Unraveling the ecological processes modulating the population structure of Escherichia coli in a highly polluted urban stream network.

Escherichia coli dynamics in urban watersheds are affected by a complex balance among external inputs, niche modulation and genetic variability. To explore the ecological processes influencing E. coli spatial patterns, we analyzed its abundance and phylogenetic structure in water samples from a stream network with heterogeneous urban infrastructure and environmental conditions. Our results showed that environmental and infrastructure variables, such as macrophyte coverage, DIN and sewerage density, mostly explained E. coli abundance. Moreover, main generalist phylogroups A and B1 were found in high proportion, which, together with an observed negative relationship between E. coli abundance and phylogroup diversity, suggests that their dominance might be due to competitive exclusion. Lower frequency phylogroups were associated with sites of higher ecological disturbance, mainly involving simplified habitats, higher drainage infrastructure and septic tank density. In addition to the strong negative relationship between phylogroup diversity and dominance, the occurrence of these phylogroups would be associated with increased facilitated dispersal. Nutrients also contributed to explaining phylogroup distribution. Our study proposes the differential contribution of distinct ecological processes to the patterns of E. coli in an urban watershed, which is useful for the monitoring and management of fecal pollution.

Aerobic methanotrophs in an urban water cycle system: Community structure and network interaction pattern.

Aerobic methane-oxidizing bacteria (MOB) play an important role in reducing methane emissions in nature. Most current researches focus on the natural habitats (e.g., lakes, reservoirs, wetlands, paddy fields, etc.). However, methanotrophs and the methane-oxidizing process remain essentially unclear in artificial habitat, such as the urban water cycle systems. Here, high-throughput sequencing and qPCR were used to analyze the community structure and abundance of MOB. Six different systems were selected from Yunyang City, Chongqing, China, including the raw water system (RW), the water supply pipe network system (SP), the wastewater pipe network system (WP), the hospital wastewater treatment system (HP), the municipal wastewater treatment plant system (WT) and the downstream river system (ST) of a wastewater treatment plant. Results clearly showed that the MOB community structure and network interaction patterns of the urban water cycle system were different from those of natural water bodies. Type I MOB was the dominant clade in HP. Methylocysis in Type II was the most abundant genus among the whole urban water cycle system, indicating that this genus had a high adaptability to the environment. Temperature, dissolved oxygen, pH and concentration significantly affected the MOB communities in the urban water cycle system. The network of MOB in WT was the most complicated, and there were competitive relationships among species in WP. The structure of the network in HP was unstable, and therefore, it was vulnerable to environmental disturbances. Methylocystis (Type II) and Methylomonas (Type I) were the most important keystone species in the entire urban water cycle system. Overall, these findings broaden the understanding of the distribution and interaction patterns of MOB communities in an urban water cycle system and provide valuable clues for ecosystem restoration and environmental management.

New findings on the effect of glyphosate on autotrophic and heterotrophic picoplankton structure: A microcosm approach.

Massive use of glyphosate-based herbicides in agricultural activities has led to the appearance of this herbicide in freshwater systems, which represents a potential threat to these systems and their communities. These herbicides can affect autotrophic and heterotrophic picoplankton abundance. However, little is known about glyphosate impact on the whole structure of these assemblages. Herein, we used an 8-day long microcosm approach under indoor controlled conditions to analyze changes in the structure of picoplankton exposed to a single pulse of glyphosate. The analyzed picoplankton correspond to two outdoor ponds with contrasting states: "clear" (chlorophyll-a = 3.48 µg L-1± 1.15; nephelometric turbidity, NTU = 1) and "turbid" (chlorophyll-a = 105.96 µg L-1 ± 15.3; NTU = 48). We evaluated herbicide impact on different picoplankton cytometric populations and further explored changes in bacterial dominant operational taxonomic units (OTUs) fingerprinting. We observed that glyphosate induced a drastic decrease in the abundance of phycocyanin-rich picocyanobacteria. Particularly, in the turbid system this effect resulted in an 85 % decrease in the abundance of the whole autotrophic picoplankton. Glyphosate also changed the structure of the heterotrophic fraction by means of changing bacterial dominant OTUs fingerprinting patterns in both systems and by shifting the relative abundances of cytometric groups in the clear scenario. These results demonstrate that upon glyphosate exposure picoplanktonic fractions face not only the already reported changes in abundance, but also alterations in the composition of cytometric groups and of bacterial dominant operational taxonomic units. This research provides suitable and still little explored tools to analyze agrochemical effects on picoplanktonic communities.

C5aR contributes to the weak Th1 profile induced by an outbreak strain of Mycobacterium tuberculosis.

C5a anaphylatoxin is a component of the complement system involved in the modulation of T-cell polarization. Herein we investigated whether C5a receptors, C5aR and C5L2, modulate the cytokine profiles induced by Mycobacterium tuberculosis (Mtb). We analyzed the impact of both receptors on T helper cell polarization induced by the multidrug resistant outbreak strain named M, which is a poor IFN-γ inducer compared with the laboratory strain H37Rv. To this aim, we first blocked C5aR or C5L2 of peripheral blood monocytes (Mo) from patients with tuberculosis and healthy donors, then we stimulated the Mo either with H37Rv or the M strain, and finally we analyzed cytokine profiles of Mo/macrophages (MΦ) and CD4+ T-cells. We found that: (i) Mtb modulated the expression of both C5a receptors, (ii) C5aR inhibited the expansion of CD4+IFN-γ+ lymphocytes stimulated by the M strain but not by H37Rv, (iii) both receptors modulated the Mo/MΦ cytokine expression induced by Mtb. We conclude that C5aR, but not C5L2, plays a role in T helper cell polarization induced by Mtb and that this effect is strain- and donor-dependent. We speculate that the epidemiologically successful M strain takes advantage of this C5aR-mediated inhibition of Th1 polarization to survive within the host.

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.

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.

A functional misexpression screen uncovers a role for enabled in progressive neurodegeneration.

Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism.