Genome-Wide Gene Expression Analysis of Mtb-Infected DC Highlights the Rapamycin-Driven Modulation of Regulatory Cytokines via the mTOR/GSK-3ß Axis.

In human primary dendritic cells (DC) rapamycin-an autophagy inducer and protein synthesis inhibitor-overcomes the autophagy block induced by Mycobacterium tuberculosis (Mtb) and promotes a Th1 response via IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was further investigated by analyzing both transcriptome and translatome gene profiles. Hundreds of differentially expressed genes (DEGs) were identified by transcriptome and translatome analyses of Mtb-infected DC, and some of these genes were found further modulated by rapamycin. The majority of transcriptome-associated DEGs overlapped with those present in the translatome, suggesting that transcriptionally stimulated mRNAs are also actively translated. In silico analysis of DEGs revealed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused an enrichment of IFN-ß, IFN-λ and IFN-stimulated gene transcripts in the polysome-associated RNA fraction. In addition, rapamycin led to an increase of IL-12, IL-23, IL-1ß, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3ß, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3ß acts as molecular switch for the regulation of the cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy induction contributes to DC activation during Mtb infection and points to rapamycin and GSK-3ß modulators as promising compounds for host-directed therapy in the control of Mtb infection.

Moxifloxacin Activates the SOS Response in Mycobacterium tuberculosis in a Dose- and Time-Dependent Manner.

Previous studies on Escherichia coli demonstrated that sub-minimum inhibitory concentration (MIC) of fluoroquinolones induced the SOS response, increasing drug tolerance. We characterized the transcriptional response to moxifloxacin in Mycobacterium tuberculosis. Reference strain H37Rv was treated with moxifloxacin and gene expression studied by qRT-PCR. Five SOS regulon genes, recA, lexA, dnaE2, Rv3074 and Rv3776, were induced in a dose- and time-dependent manner. A range of moxifloxacin concentrations induced recA, with a peak observed at 2 × MIC (0.25 µg/mL) after 16 h. Another seven SOS responses and three DNA repair genes were significantly induced by moxifloxacin. Induction of recA by moxifloxacin was higher in log-phase than in early- and stationary-phase cells, and absent in dormant bacilli. Furthermore, in an H37Rv fluoroquinolone-resistant mutant carrying the D94G mutation in the gyrA gene, the SOS response was induced at drug concentrations higher than the mutant MIC value. The 2 × MIC of moxifloxacin determined no significant changes in gene expression in a panel of 32 genes, except for up-regulation of the relK toxin and of Rv3290c and Rv2517c, two persistence-related genes. Overall, our data show that activation of the SOS response by moxifloxacin, a likely link to increased mutation rate and persister formation, is time, dose, physiological state and, possibly, MIC dependent.

The Combination Rifampin-Nitazoxanide, but Not Rifampin-Isoniazid-Pyrazinamide-Ethambutol, Kills Dormant Mycobacterium tuberculosis in Hypoxia at Neutral pH.

The activities of rifampin, nitazoxanide, PA-824, and sutezolid were tested against dormant Mycobacterium tuberculosis under conditions mimicking caseous granulomas (hypoxia at pH 7.3) in comparison with those of the combination rifampin-isoniazid-pyrazinamide-ethambutol (R-I-Z-E), which is used for human therapy. Mycobacterial viability was monitored by CFU and regrowth in MGIT 960. As shown by lack of regrowth in MGIT, rifampin-nitazoxanide-containing combinations, but not R-I-Z-E, killed dormant cells in 28 to 35 days. These observations might be important in designing new tuberculosis therapies.

Mycobacterium tuberculosis-induced miR-155 subverts autophagy by targeting ATG3 in human dendritic cells.

Autophagy is a primordial eukaryotic pathway, which provides the immune system with multiple mechanisms for the elimination of invading pathogens including Mycobacterium tuberculosis (Mtb). As a consequence, Mtb has evolved different strategies to hijack the autophagy process. Given the crucial role of human primary dendritic cells (DC) in host immunity control, we characterized Mtb-DC interplay by studying the contribution of cellular microRNAs (miRNAs) in the post-transcriptional regulation of autophagy related genes. From the expression profile of de-regulated miRNAs obtained in Mtb-infected human DC, we identified 7 miRNAs whose expression was previously found to be altered in specimens of TB patients. Among them, gene ontology analysis showed that miR-155, miR-155* and miR-146a target mRNAs with a significant enrichment in biological processes linked to autophagy. Interestingly, miR-155 was significantly stimulated by live and virulent Mtb and enriched in polysome-associated RNA fraction, where actively translated mRNAs reside. The putative pair interaction among the E2 conjugating enzyme involved in LC3-lipidation and autophagosome formation-ATG3-and miR-155 arose by target prediction analysis, was confirmed by both luciferase reporter assay and Atg3 immunoblotting analysis of miR-155-transfected DC, which showed also a consistent Atg3 protein and LC3 lipidated form reduction. Late in infection, when miR-155 expression peaked, both the level of Atg3 and the number of LC3 puncta per cell (autophagosomes) decreased dramatically. In accordance, miR-155 silencing rescued autophagosome number in Mtb infected DC and enhanced autolysosome fusion, thereby supporting a previously unidentified role of the miR-155 as inhibitor of ATG3 expression. Taken together, our findings suggest how Mtb can manipulate cellular miRNA expression to regulate Atg3 for its own survival, and highlight the importance to develop novel therapeutic strategies against tuberculosis that would boost autophagy.

A method permissive to fixation and permeabilization for the multiparametric analysis of apoptotic and necrotic cell phenotype by flow cytometry.

Annexin-V/propidium iodide method (A-V/PI) is a common flow cytometric method for the multiparametric analysis of cells in apoptosis. However, A-V/PI does not permit fixation and/or permeabilization of cells making impossible evaluation of intracellular markers, restricting the analysis in a narrow time frame after staining and excluding the possibility to study pathogen-infected cells. We developed a method permitting fixation and permeabilization of stained cells: Fixed Apoptotic/Necrotic (FAN) cells test. FAN relies on the same principle of A-V/PI, but uses reagents that maintain their binding and fluorescence characteristics after fixation/permeabilization: a fluorochrome-labeled anti-phosphatidylserine antibody and fluorescent amine-binding dyes. FAN was tested to discriminate apoptotic and necrotic cells using different stimuli on several cell types and results were always comparable to those obtained using A-V/PI. FAN, unlike A-V/PI, permitted to correlate cell death with intracellular and surface markers expression and to perform cytometry even two weeks after sample preparation. As fixation of stained cells inactivates infective pathogens, we used FAN in an in vitro model of Mycobacterium tuberculosis (Mtb) infection of macrophages to monitor cellular infection and cell death induction. Using a red-fluorescent Mtb, fluorochrome labeled anti-TNF-α and anti-MHC class II monoclonal antibodies, FAN permitted to establish that the extent of macrophage death correlates with intracellular Mtb content and that dying cells accumulate TNF-α and down-modulate MHC class II molecules. Results suggest that FAN may represent an additional tool to study programmed cell death particularly useful when fixation procedures are required for a safe infected sample analysis or to comparatively analyze multiple samples. © 2017 International Society for Advancement of Cytometry.

Mycobacterium tuberculosis gene expression at different stages of hypoxia-induced dormancy and upon resuscitation.

The physiology of dormant Mycobacterium tuberculosis was studied in detail by examining the gene expression of 51 genes using quantitative Reverse-Transcription Polymerase Chain Reaction. A forty-day period of dormancy in the Wayne culture model depicted four major transcription patterns. Some sigma factors and many metabolic genes were constant, whereas genes belonging to the dormancy regulon were activated on day 9. In particular, alpha-crystallin mRNA showed more than a 1,000-fold increase compared to replicating bacilli. Genes belonging to the enduring hypoxic response were up-regulated at day 16, notably, transcription factors sigma B and E. Early genes typical of log-phase bacilli, esat-6 and fbpB, were uniformly down-regulated during dormancy. Late stages of dormancy showed a drop in gene expression likely due to a lack of substrates in anaerobic respiration as demonstrated by the transcriptional activation observed following nitrates addition. Among genes involved in nitrate metabolism, narG was strongly up-regulated by nitrates addition. Dormant bacilli responded very rapidly when exposed to oxygen and fresh medium, showing a transcriptional activation of many genes, including resuscitation-promoting factors, within one hour. Our observations extend the current knowledge on dormant M. tuberculosis gene expression and its response to nutrients and to aerobic and anaerobic respiration.

Impact of Mycobacterium tuberculosis RD1-locus on human primary dendritic cell immune functions.

Modern strategies to develop vaccines against Mycobacterium tuberculosis (Mtb) aim to improve the current Bacillus Calmette-Guerin (BCG) vaccine or to attenuate the virulence of Mtb vaccine candidates. In the present study, the impact of wild type or mutated region of difference 1 (RD1) variants on the immunogenicity of Mtb and BCG recombinants was investigated in human primary dendritic cells (DC). A comparative analysis of transcriptome, signalling pathway activation, maturation, apoptosis, cytokine production and capacity to promote Th1 responses demonstrated that DC sense quantitative and qualitative differences in the expression of RD1-encoded factors--ESAT6 and CFP10--within BCG or Mtb backgrounds. Expansion of IFN-γ producing T cells was promoted by BCG::RD1-challenged DC, as compared to their BCG-infected counterparts. Although Mtb recombinants acted as a strong Th-1 promoting stimulus, even with RD1 deletion, the attenuated Mtb strain carrying a C-terminus truncated ESAT-6 elicited a robust Th1 promoting phenotype in DC. Collectively, these studies indicate a necessary but not sufficient role for the RD1 locus in promoting DC immune-regulatory functions. Additional mycobacterial factors are likely required to endow DC with a high Th1 polarizing capacity, a desirable attribute for a successful control of Mtb infection.

Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses.

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-ß-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated ß-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

Dual effect of Thymosin α 1 on human monocyte-derived dendritic cell in vitro stimulated with viral and bacterial toll-like receptor agonists.

OBJECTIVES: Thymosin α 1 (Tα1) recently gained interest as immune adjuvant for vaccines because of its ability to modulate the T-cell/dendritic cell (DC) axis and to improve antibody production. The objective of this study was to determine whether Tα1 would address in vitro the response of human primary monocyte-derived DC, crucial regulators of vaccine-induced immunity, upon exposure to different toll-like receptor (TLR) agonists or infection with viruses or bacteria. METHODS: DC maturation and production of pro-inflammatory cytokines were analyzed. RESULTS: Our data revealed a dual effect of Tα1 on DC biology upon viral or bacterial stimulation. Interestingly, Tα1 enhanced human leukocyte antigen (HLA)-I and II surface expression and secretion of IL-6, TNF-α and IL-8 when DCs were treated with viral TLR3 and TLR7/8 agonists. Similarly, in pandemic H1N1 influenza A-infected DCs, Tα1 raised the expression of maturation markers and type I and III Interferon (IFN). In contrast, following bacterial TLR2 and 4 stimulation, as well as upon Bacillus Calmette-Guerin infection, the presence of Tα1 in DC cultures drastically lowered the analyzed cellular parameters. CONCLUSION: The knowledge that Tα1 pleiotropic effect might ameliorate anti-viral immune responses and, at the same time, dampen inflammation caused by bacterial infections could lay the groundwork for a more appropriate therapeutic application of this molecule.

A human dendritic cell-based in vitro model to assess Mycobacterium tuberculosis SO2 vaccine immunogenicity.

Among the tuberculosis (TB) vaccine candidates, SO2 is the prototype of the first live-attenuated vaccine that recently entered into clinical trials. To investigate the capacity of SO2 to stimulate an appropriate immune response in vitro within a human immunological context, a comparative analysis of the effects promoted by SO2, the current Bacille Calmette-Guerin (BCG) vaccine and Mycobacterium tuberculosis (Mtb) was conducted in human primary dendritic cells (DC), which are critical modulators of vaccine-induced immunity. In particular, we found that SO2 promotes the expression of maturation markers similarly to BCG but at a lower extent than Mtb. Moreover, SO2-infected DC released higher levels of interleukin (IL)-23 than BCG-infected cells, which account for the expansion of interferon (IFN)-γ-producing T cells in an IL-12-independent manner. In the autologous mixed leukocyte reaction setting, the expansion of IL-17-producing T cells was also observed in response to SO2 infection. Interestingly, apoptosis and autophagic flux, events required for the antigen presentation within MHC class II complex, were not affected in DC infected with SO2, conversely to what observed upon Mtb stimulation. Collectively, our results indicate that SO2 represents a promising TB vaccine candidate, which displays an attenuated phenotype and promotes in DC a stronger capacity to stimulate the Th response than BCG vaccine. Interestingly, the data obtained by using the human DC-based experimental setting mirrored the results derived from studies in animal models, suggesting that this system could be used for an efficient and rapid down-selection of new TB vaccine candidates, contributing to achieve the "3Rs" objective.

Dormant Mycobacterium tuberculosis fails to block phagosome maturation and shows unexpected capacity to stimulate specific human T lymphocytes.

Dormancy is defined as a stable but reversible nonreplicating state of Mycobacterium tuberculosis. It is currently thought that dormant M. tuberculosis (D-Mtb) is responsible for latent tuberculosis (TB) infection. Recently, D-Mtb was also shown in sputa of patients with active TB, but the capacity of D-Mtb to stimulate specific immune responses was not investigated. We observed that purified protein derivative-specific human CD4(+) T lymphocytes recognize mycobacterial Ags more efficiently when macrophages are infected with D-Mtb instead of replicating M. tuberculosis (R-Mtb). The different Ag recognition occurs even when the two forms of mycobacteria equally infect and stimulate macrophages, which secrete the same cytokine pattern and express MHC class I and II molecules at the same levels. However, D-Mtb but not R-Mtb colocalizes with mature phagolysosome marker LAMP-1 and with vacuolar proton ATPase in macrophages. D-Mtb, unlike R-Mtb, is unable to interfere with phagosome pH and does not inhibit the proteolytic efficiency of macrophages. We show that D-Mtb downmodulates the gene Rv3875 encoding for ESAT-6, which is required by R-Mtb to block phagosome maturation together with Rv3310 gene product SapM, previously shown to be downregulated in D-Mtb. Thus, our results indicate that D-Mtb cannot escape MHC class II Ag-processing pathway because it lacks the expression of genes required to block the phagosome maturation. Data suggest that switching to dormancy not only represents a mechanism of survival in latent TB infection, but also a M. tuberculosis strategy to modulate the immune response in different stages of TB.

Identification of Haemophilus influenzae clones associated with invasive disease a decade after introduction of H. influenzae serotype b vaccination in Italy.

The introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines has changed the epidemiology of invasive H. influenzae disease, with a shift in the predominant serotype from Hib to nonencapsulated H. influenzae (ncHi). The objective of this study was to identify the genotypes/clones associated with invasive H. influenzae disease in Italy. Eighty-seven H. influenzae strains isolated in the years 2009 to 2011 within the National Surveillance of Invasive Bacterial Disease program were analyzed. Strains were characterized by serotyping, antimicrobial susceptibility testing, and multilocus sequence typing (MLST). Genetic polymorphisms in the bla(TEM) gene promoter region as well as the occurrence of both adhesin genes (hmwA and hia) and the IgA1 protease-encoding gene (igaB) were also investigated. Of 87 strains, 67 were ncHi and 20 were encapsulated. Eleven strains were ß-lactamase positive, harboring the bla(TEM) gene. Most bla(TEM) genes (10/11) were associated with a Pdel promoter region exhibiting a 135-bp deletion; the remaining strain possessed the Pa/Pb overlapping promoter. MLST analysis showed that encapsulated isolates were clonal, with each serotype sharing a few related sequence types (STs). Forty-six different STs were identified among the 67 ncHi strains. Despite this heterogeneity, a group of closely related STs (ST103, ST139, and ST145) encompassed almost 25% of all ncHi strains and 45.5% of the ß-lactamase producers carrying the Pdel promoter. These major ST clones were found to be associated with the hmwA gene but not with the igaB gene. To conclude, although the heterogeneity of the ncHi population was confirmed, diffusion of major successful ST clones was documented.

Mycobacterium tuberculosis may escape helper T cell recognition by infecting human fibroblasts.

The host immune response can limit Mycobacterium tuberculosis (Mtb) spreading in primary tuberculosis (TB) without eradicating all bacilli, which can persist causing latent TB infection and are responsible for reactivation TB. Persistent Mtb is confined to granulomas within phagocytes, but it is also found in other non-immune cells. We focused on fibroblasts since these cells participate to the granuloma formation and were shown to be infected in latent TB infections. We show that in vitro both Mtb and Bacille Calmette-Guérin actively replicate in human fibroblasts. Mycobacterial infection of fibroblasts causes a significant inhibition of interferon (IFN)-γ induced membrane expression of major histocompatibility complex class II molecules in these cells. The functional consequence of in vitro infection is a significant reduction of the fibroblast capacity to present peptides and soluble proteins to autologous specific CD4(+) T cell clones. Moreover, fibroblasts are capable of presenting antigen derived from the processing of heat-killed Mtb, but not from viable Mtb. Data indicate that IFN-γ treated fibroblasts are capable of presenting antigens derived from the processing of whole bacteria in addition to the capacity to present peptides and isolated proteins. Interestingly, Mtb infected fibroblasts lose this capacity, suggesting that Mtb may evade T helper immune surveillance by infecting fibroblasts.

ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells.

Emerging evidence points to an important role of autophagy in the immune response mediated by dendritic cells (DC) against Mycobacterium tuberculosis (Mtb). Since current vaccination based on Bacillus Calmette-Guerin (BCG) is unable to stop the tuberculosis epidemic, a deeper comprehension of the alterations induced by Mtb in DC is essential for setting new vaccine strategies. Here, we compared the capacity of virulent (H37Rv) and avirulent (H37Ra) Mtb strains as well as BCG to modulate autophagy in human primary DC. We found that Mtb H37Rv impairs autophagy at the step of autophagosome-lysosome fusion. In contrast, neither Mtb H37Ra nor BCG strains were able to hamper autophagosome maturation. Both these attenuated strains have a functional inhibition of the 6kD early secreted antigenic target ESAT-6, an effector protein of the ESAT-6 Secretion System-1(ESX-1)/type VII secretion system. Notably, the ability to inhibit autophagy was fully restored in recombinant BCG and Mtb H37Ra strains in which ESAT-6 secretion was re-established by genetic complementation using either the ESX-1 region from Mtb (BCG::ESX-1) or the PhoP gene (Mtb H37Ra::PhoP), a regulator of ESAT-6 secretion. Importantly, the autophagic block induced by Mtb was overcome by rapamycin treatment leading to an increased interleukin-12 expression and, in turn, to an enhanced capacity to expand a Th1-oriented response. Collectively, our study demonstrated that Mtb alters the autophagic machinery through the ESX-1 system, and thereby opens new exciting perspectives to better understand the relationship between Mtb virulence and its ability to escape the DC-mediated immune response.

Infection of human THP-1 cells with dormant Mycobacterium tuberculosis.

Dormant, non-replicating Mycobacterium tuberculosis H37Rv strain cultured in hypoxic conditions was used to infect THP-1 cells. CFUs counting, Kinyoun staining and electron microscopy showed that dormant bacilli infected THP-1 cells at a rate similar to replicating M. tuberculosis, but failed to grow during the first 6 days of infection. The absence of growth was specific to the intracellular compartment, as demonstrated by efficient growth in liquid medium. Quantification of ß-actin mRNA recovered from infected cells showed that, in contrast with log-phase bacteria, infection with dormant bacilli determined a reduced THP-1 cell death. Gene expression of intracellular non-replicating bacteria showed a pattern typical of a dormant state. Intracellular dormant bacteria induced the activation of genes associated to a proinflammatory response in THP-1 cells. Though, higher levels of TNFα, IL-1ß and IL-8 mRNAs compared to aerobic H37Rv infected cells were not paralleled by increased cytokine accumulation in the supernatants. Moreover, dormant bacilli induced a higher expression of inducible cox-2 gene, accompanied by increased PGE2 secretion. Overall, our data describe a new model of in vitro infection using dormant M. tuberculosis that could provide the basis for understanding how non-replicating bacilli survive intracellularly and influence the maintenance of the hypoxic granuloma.

Treatment of tuberculosis in a region with high drug resistance: outcomes, drug resistance amplification and re-infection.

INTRODUCTION: Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries. METHODS: We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment. RESULTS: At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome. CONCLUSION: In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.

The M. tuberculosis phosphate-binding lipoproteins PstS1 and PstS3 induce Th1 and Th17 responses that are not associated with protection against M. tuberculosis infection.

The M. tuberculosis phosphate-binding transporter lipoproteins PstS1 and PstS3 were good immunogens inducing CD8(+) T-cell activation and both Th1 and Th17 immunity in mice. However, this antigen-specific immunity, even when amplified by administration of the protein with the adjuvant LTK63 or by the DNA priming/protein boosting regimen, was not able to contain M. tuberculosis replication in the lungs of infected mice. The lack of protection might be ascribed with the scarce/absent capacity of PstS1/PstS3 antigens to modulate the IFN-γ response elicited by M. tuberculosis infection during which, however, PstS1-specific IL-17 secreting cells were generated in both unvaccinated and BCG-vaccinated mice. In spite of a lack of protection by PstS1/PstS3 immunizations, our results do show that PstS1 is able to induce IL-17 response upon M. tuberculosis infection which is of interest in the study of anti-M. tuberculosis immunity and as potential immunomodulator in combined vaccines.

Expression of proinflammatory and regulatory cytokines via NF-κB and MAPK-dependent and IFN regulatory factor-3-independent mechanisms in human primary monocytes infected by Mycobacterium tuberculosis.

Knowledge of the molecular events regulating the innate response to Mycobacterium tuberculosis (Mtb) is critical for understanding immunological pathogenesis and protection from tuberculosis. To this aim, the regulation and the expression of regulatory and proinflammatory cytokines were investigated in human primary monocytes upon Mtb infection. We found that Mtb-infected monocytes preferentially express a proinflammatory cytokine profile, including IL-6, TNF-α, and IL-1ß. Conversely, among the regulatory cytokines, Mtb elicited IL-10 and IL-23 release while no expression of IL-12p70, IL-27, and IFN-ß was observed. The analysis of the signalling pathways leading to this selective cytokine expression showed that in monocytes Mtb activates MAPK and NF-κB but is unable to stimulate IRF-3 phosphorylation, a transcription factor required for IL-12p35 and IFN-ß gene expression. Thus, by inducing a specific cytokine profile, Mtb can influence the immunoregulatory properties of monocytes, which represent important target of novel vaccinal strategies against Mtb infection.