Antimicrobial Effect of Cannabidiol on Intracellular Mycobacterium tuberculosis.

Introduction: Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), has killed nearly one billion people during the last two centuries. Nowadays, TB remains a major global health problem ranked among the top 10 causes of death worldwide. One of the main challenges in developing new strategies to fight TB is focused on reducing the duration and complexity of drug regimens. Cannabidiol (CBD) is the main nonpsychoactive ingredient extracted from the Cannabis sativa L. plant, which has been shown to be biologically active against bacteria. The purpose of this work was to investigate the antimicrobial effect of CBD on M. tuberculosis intracellular infection. Materials and Methods: To assess the minimum inhibitory concentration (MIC) of CBD on mycobacterial strains, the MTT assay was performed on Mycobacterium smegmatis, and the Colony-Forming Unit (CFU) assay was conducted on MtbH37Rv. Additionally, the cytotoxic effect of CBD on THP-1 cells was assessed by MTT assay. Moreover, macrophages derived from the THP-1 cell were infected with MtbH37Rv (multiplicity of infection 1:10) to evaluate the intracellular activity of CBD by determining the CFU/mL. Results: Antimicrobial activity against M. smegmatis (MIC=100 µM) and MtbH37Rv (MIC=25 µM) cultures was exhibited by CBD. Furthermore, the effect of CBD was also evaluated on MtbH37Rv infected macrophage cells. Interestingly, a reduction in viable intracellular MtbH37Rv bacteria was observed after 24 h of treatment. Moreover, CBD exhibited a safe profile toward human THP-1 cells, since it showed no toxicity (CC50=1075 µM) at a concentration of antibacterial effect (selectivity index 43). Conclusion: These results extend the knowledge regarding the antimicrobial activity of CBD and demonstrate its ability to kill the human intracellular pathogen M. tuberculosis.

Beyond the Clinic: The Activation of Diverse Cellular and Humoral Factors Shapes the Immunological Status of Patients with Active Tuberculosis.

Mycobacterium tuberculosis (Mtb), the etiologic agent of tuberculosis (TB), has killed nearly one billion people in the last two centuries. Nowadays, TB remains a major global health problem, ranking among the thirteen leading causes of death worldwide. Human TB infection spans different levels of stages: incipient, subclinical, latent and active TB, all of them with varying symptoms, microbiological characteristics, immune responses and pathologies profiles. After infection, Mtb interacts with diverse cells of both innate and adaptive immune compartments, playing a crucial role in the modulation and development of the pathology. Underlying TB clinical manifestations, individual immunological profiles can be identified in patients with active TB according to the strength of their immune responses to Mtb infection, defining diverse endotypes. Those different endotypes are regulated by a complex interaction of the patient's cellular metabolism, genetic background, epigenetics, and gene transcriptional regulation. Here, we review immunological categorizations of TB patients based on the activation of different cellular populations (both myeloid and lymphocytic subsets) and humoral mediators (such as cytokines and lipid mediators). The analysis of the participating factors that operate during active Mtb infection shaping the immunological status or immune endotypes of TB patients could contribute to the development of Host Directed Therapy.

Association of IFN-γ +874 A/T SNP and hypermethylation of the -53 CpG site with tuberculosis susceptibility.

Introduction: Tuberculosis (TB) is now the 2nd leading infectious killer after COVID-19 and the 13th leading cause of death worldwide. Moreover, TB is a lethal combination for HIV-patients. Th1 responses and particularly IFN-γ are crucial for immune protection against Mycobacterium tuberculosis infection. Many gene variants for IFNG that confer susceptibility to TB have been described in multiple ethnic populations. Likewise, some epigenetic modifications have been evaluated, being CpG methylation the major epigenetic mark that makes chromatin inaccessible to transcription factors, thus avoiding the initiation of IFNG transcription. Methods: We evaluated both genetic and epigenetic changes involved in IFN-γ production and TB susceptibility in Argentine population. Amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed for the IFN-γ +874 A/T polymorphism (rs2430561) genotyping in 199 healthy donors (HD) and 173 tuberculosis (TB) patients. IFN-γ levels from M. tuberculosis-stimulated PBMCs were measured by ELISA. The methylation status at the -53 CpG site of the IFNG promoter in individuals with latent infection (LTBI), TB and HD was determine by pyrosequencing. Results: Using a case-control study, we found that A allele and, consequently, AA genotype were overrepresented in patients with active disease. Moreover, HD carrying T allele (AT or TT genotype) evidenced an augmented IFN-γ secretion compared to TB patients. Codominance was the genetic model that best fits our results according to the Akaike information criterion (AIC). In addition, increased methylation levels at the -53 CpG site in the IFN-γ promoter were observed in whole blood of patients with active TB compared to LTBI individuals. Discussion: IFN-γ is regulated by genetic variants and epigenetic modifications during TB. Besides, AA genotype of the rs2430561 single nucleotide polymorphism could be considered as a potential TB susceptibility genetic biomarker in Argentina and the methylation of the -53 CpG site could result in a useful predictor of TB reactivation.

PGE2 displays immunosuppressive effects during human active tuberculosis.

Prostaglandin E2 (PGE2), an active lipid compound derived from arachidonic acid, regulates different stages of the immune response of the host during several pathologies such as chronic infections or cancer. In fact, manipulation of PGE2 levels was proposed as an approach for countering the Type I IFN signature of tuberculosis (TB). However, very limited information regarding the PGE2 pathway in patients with active TB is currently available. In the present work, we demonstrated that PGE2 exerts a potent immunosuppressive action during the immune response of the human host against Mycobacterium tuberculosis (Mtb) infection. Actually, we showed that PGE2 significantly reduced the surface expression of several immunological receptors, the lymphoproliferation and the production of proinflammatory cytokines. In addition, PGE2 promoted autophagy in monocytes and neutrophils cultured with Mtb antigens. These results suggest that PGE2 might be attenuating the excessive inflammatory immune response caused by Mtb, emerging as an attractive therapeutic target. Taken together, our findings contribute to the knowledge of the role of PGE2 in the human host resistance to Mtb and highlight the potential of this lipid mediator as a tool to improve anti-TB treatment.

Shedding Light on Autophagy During Human Tuberculosis. A Long Way to Go.

Immunity against Mycobacterium tuberculosis (Mtb) is highly complex, and the outcome of the infection depends on the role of several immune mediators with particular temporal dynamics on the host microenvironment. Autophagy is a central homeostatic mechanism that plays a role on immunity against intracellular pathogens, including Mtb. Enhanced autophagy in macrophages mediates elimination of intracellular Mtb through lytic and antimicrobial properties only found in autolysosomes. Additionally, it has been demonstrated that standard anti-tuberculosis chemotherapy depends on host autophagy to coordinate successful antimicrobial responses to mycobacteria. Notably, autophagy constitutes an anti-inflammatory mechanism that protects against endomembrane damage triggered by several endogenous components or infectious agents and precludes excessive inflammation. It has also been reported that autophagy can be modulated by cytokines and other immunological signals. Most of the studies on autophagy as a defense mechanism against Mycobacterium have been performed using murine models or human cell lines. However, very limited information exists about the autophagic response in cells from tuberculosis patients. Herein, we review studies that face the autophagy process in tuberculosis patients as a component of the immune response of the human host against an intracellular microorganism such as Mtb. Interestingly, these findings might contribute to recognize new targets for the development of novel therapeutic tools to combat Mtb. Actually, either as a potential successful vaccine or a complementary immunotherapy, efforts are needed to further elucidate the role of autophagy during the immune response of the human host, which will allow to achieve protective and therapeutic benefits in human tuberculosis.

Neutrophil autophagy during human active tuberculosis is modulated by SLAMF1.

Neutrophils infected with Mycobacterium tuberculosis (Mtb) predominate in tuberculosis patients' lungs. Neutrophils phagocytose the pathogen, but the mechanism of pathogen elimination is controversial. Macroautophagy/autophagy, a crucial mechanism for several neutrophil functions, can be modulated by immunological mediators. The costimulatory molecule SLAMF1 can act as a microbial sensor in macrophages being also able to interact with autophagy-related proteins. Here, we demonstrate for the first time that human neutrophils express SLAMF1 upon Mtb-stimulation. Furthermore, SLAMF1 was found colocalizing with LC3B+ vesicles, and activation of SLAMF1 increased neutrophil autophagy induced by Mtb. Finally, tuberculosis patients' neutrophils displayed reduced levels of SLAMF1 and lower levels of autophagy against Mtb as compared to healthy controls. Altogether, these results indicate that SLAMF1 participates in neutrophil autophagy during active tuberculosis.Abbreviations: AFB: acid-fast bacilli; BafA1: bafilomycin A1; CLL: chronic lymphocytic leukemia; DPI: diphenyleneiodonium; EVs: extracellular vesicles; FBS: fetal bovine serum; HD: healthy donors; HR: high responder (tuberculosis patient); IFNG: interferon gamma; IL1B: interleukin 1 beta; IL17A: interleukin 17A; IL8: interleukin 8; LR: low responder (tuberculosis patient); mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK14/p38: mitogen-activated protein kinase 14; Mtb: Mycobacterium tuberculosis; Mtb-Ag: Mycobacterium tuberculosis, Strain H37Rv, whole cell lysate; NETs: neutrophils extracellular traps; PPD: purified protein derivative; ROS: reactive oxygen species; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; SLAMF1: signaling lymphocytic activation molecule family member 1; TB: tuberculosis; TLR: toll like receptor.

SLAMF1 signaling induces Mycobacterium tuberculosis uptake leading to endolysosomal maturation in human macrophages.

Tuberculosis dates back to ancient times but it is not a problem of the past. Each year, millions of people die from tuberculosis. After inhalation of infectious droplet nuclei, Mycobacterium tuberculosis reaches the lungs where it can manipulate the immune system and survive within host macrophages, establishing a persistent infection. The signaling lymphocytic activation molecule family member 1 (SLAMF1) is a self-ligand receptor that can internalize gram-negative bacteria and regulate macrophages' phagosomal functions. In tuberculosis, SLAMF1 promotes Th1-protective responses. In this work, we studied the role of SLAMF1 on macrophages' functions during M. tuberculosis infection. Our results showed that both M. tuberculosis and IFN-γ stimulation induce SLAMF1 expression in macrophages from healthy donor and Tohoku Hospital Pediatrcs-1 cells. Costimulation through SLAMF1 with an agonistic antibody resulted in an enhanced internalization of M. tuberculosis by macrophages. Interestingly, we found that SLAMF1 interacts with M. tuberculosis and colocalizes with the bacteria and with early and late endosomes/lysosomes markers (EEA1 and LAMP2), suggesting that SLAMF1 recognize M. tuberculosis and participate in the endolysosomal maturation process. Notably, increased levels of SLAMF1 were detected in CD14 cells from pleural effusions of tuberculosis patients, indicating that SLAMF1 might have an active function at the site of infection. Taken together, our results provide evidence that SLAMF1 improves the uptake of M. tuberculosis by human monocyte-derived macrophages.

IL-12 DNA Displays Efficient Adjuvant Effects Improving Immunogenicity of Ag85A in DNA Prime/MVA Boost Immunizations.

Mycobacterium tuberculosis (Mtb) infection is one of the leading causes of death worldwide. The Modified Vaccinia Ankara (MVA) vaccine vector expressing the mycobacterial antigen 85A (MVA85A) was demonstrated to be safe, although it did not improve BCG efficacy, denoting the need to search for improved tuberculosis vaccines. In this work, we investigated the effect of IL-12 DNA -as an adjuvant- on an Ag85A DNA prime/MVA85A boost vaccination regimen. We evaluated the immune response profile elicited in mice and the protection conferred against intratracheal Mtb H37Rv challenge. We observed that the immunization scheme including DNA-A85A+DNA-IL-12/MVA85A induced a strong IFN-γ production to Ag85A in vitro, with a significant expansion of IFN-γ+CD4+ and IFN-γ+CD8+ anti-Ag85A lymphocytes. Furthermore, we also detected a significant increase in the proportion of specific CD8+CD107+ T cells against Ag85A. Additionally, inclusion of IL-12 DNA in the DNA-A85A/MVA85A vaccine scheme induced a marked augment in anti-Ag85A IgG levels. Interestingly, after 30 days of infection with Mtb H37Rv, DNA-A85A+DNA-IL-12/MVA85A vaccinated mice displayed a significant reduction in lung bacterial burden. Together, our findings suggest that IL-12 DNA might be useful as a molecular adjuvant in an Ag85A DNA/MVA prime-boost vaccine against Mtb infection.

Glucocorticoids uncover a critical role for ASH2L on BCL-X expression regulation in leukemia cells.

Targeting the apoptosis machinery is a promising therapeutic approach in myeloid malignancies. BCL2L1 is a well-known glucocorticoid-responsive gene and a key apoptosis regulator that, when over-expressed, can contribute to tumor development, progression and therapeutic resistance. Moreover, synthetic glucocorticoids, like dexamethasone, are frequently used in the treatment of hematopoietic diseases due to its pro-apoptotic properties. We report here that the trithorax protein ASH2L, considered one of the core subunits of H3K4-specific MLL/SET methyltransferase complexes, contributes to anti-apoptotic BCL-XL over-expression and cell survival in patient-derived myeloid leukemia cells. We find that the unliganded glucocorticoid receptor (uGR) and ASH2L interact in a common protein complex through a chromatin looping determined by uGR and ASH2L binding to BCL2L1 specific +58 HRE and promoter region, respectively. Upon addition of dexamethasone, GR and ASH2L recruitment is reduced, BCL-XL expression diminishes and apoptosis is induced consequently. Overall, our findings indicate that uGR and ASH2L may act as key regulatory players of BCL- XL upregulation in AML cells.

The Non-synonymous rs763780 Single-Nucleotide Polymorphism in IL17F Gene Is Associated With Susceptibility to Tuberculosis and Advanced Disease Severity in Argentina.

Th17 lymphocytes, that produce IL17A, IL17F, and IL22, play a crucial role during the immune response against Mycobacterium tuberculosis (Mtb) infection. Whereas, the contribution of IL17A in immunity to tuberculosis is usually accepted, the role of IL17F has been scarcely studied so far. The aim of this work was to evaluate the existence of a potential association of the non-synonymous variant rs763780 SNP of the IL17F gene with human tuberculosis. Accordingly, by comparing healthy donors (HD) and tuberculosis patients (TB) populations we demonstrated an association between the C allele of the SNP and the susceptibility to tuberculosis disease in Argentina. Furthermore, we found that peripheral blood mononuclear cells (PBMCs) from individuals with a more effective immune response against Mtb secreted the highest levels of IL17F when stimulated with a lysate of Mtb (Mtb-Ag). Besides, we evidenced that Mtb-Ag-stimulated PBMCs from HD carrying the C variant of the SNP displayed the lowest IFNG secretion, proliferation index, and SLAM expression as compared to TT carriers. Moreover, Mtb-Ag-stimulated PBMCs from TB carrying the C allele produced the lowest levels of IFNG, the highest level of IL17A, and the minimum proliferation indexes as compared to TT TB, suggesting a relationship between the C allele and tuberculosis severity. In fact, TB carrying the C allele presented a more severe disease, with the highest bacilli burden in sputum. Together, our findings identify the IL17F rs763780 SNP as a biomarker of tuberculosis susceptibility and advanced disease severity in Argentina, suggesting that IL17F could be a critical cytokine in tuberculosis immunity.

IL17A augments autophagy in Mycobacterium tuberculosis-infected monocytes from patients with active tuberculosis in association with the severity of the disease.

During mycobacterial infection, macroautophagy/autophagy, a process modulated by cytokines, is essential for mounting successful host responses. Autophagy collaborates with human immune responses against Mycobacterium tuberculosis (Mt) in association with specific IFNG secreted against the pathogen. However, IFNG alone is not sufficient to the complete bacterial eradication, and other cytokines might be required. Actually, induction of Th1 and Th17 immune responses are required for protection against Mt. Accordingly, we showed that IL17A and IFNG expression in lymphocytes from tuberculosis patients correlates with disease severity. Here we investigate the role of IFNG and IL17A during autophagy in monocytes infected with Mt H37Rv or the mutant MtΔRD1. Patients with active disease were classified as high responder (HR) or low responder (LR) according to their T cell responses against Mt. IL17A augmented autophagy in infected monocytes from HR patients through a mechanism that activated MAPK1/ERK2-MAPK3/ERK1 but, during infection of monocytes from LR patients, IL17A had no effect on the autophagic response. In contrast, addition of IFNG to infected monocytes, increased autophagy by activating MAPK14/p38 α both in HR and LR patients. Interestingly, proteins codified in the RD1 region did not interfere with IFNG and IL17A autophagy induction. Therefore, in severe tuberculosis patients' monocytes, IL17A was unable to augment autophagy because of a defect in the MAPK1/3 signaling pathway. In contrast, both IFNG and IL17A increased autophagy levels in patients with strong immunity to Mt, promoting mycobacterial killing. Our findings might contribute to recognize new targets for the development of novel therapeutic tools to fight the pathogen.

Cross-talk between CD31 and the signaling lymphocytic activation molecule-associated protein during interferon- gamma production against Mycobacterium tuberculosis.

Effective host defense against tuberculosis requires Th1 cytokine responses. We studied the regulation of interferon (IFN)- gamma production during tuberculosis by investigating the role of CD31, a receptor that attenuates T cell receptor signals. After antigen stimulation, CD3(+)CD31(+) blood lymphocytes decreased in healthy donors and in tuberculosis patients with robust Th1 responses to Mycobacterium tuberculosis and IFN- gamma was secreted only by CD31(-) T cells. In contrast, in patients with weak Th1 cytokine responses to M. tuberculosis, the level of CD3(+)CD31(+) lymphocytes was increased and IFN- gamma production was low. Furthermore, the inverse relationship between CD31 expression and IFN- gamma production was in contrast to signaling lymphocytic activation molecule (SLAM) expression, an IFN- gamma inducer in tuberculosis. Interestingly, CD31 bound to SLAM-associated protein (SAP), an IFN- gamma inhibitor in tuberculosis, and when CD31 and SAP were coexpressed in lymphocytes, their association inhibited the IFN- gamma response to M. tuberculosis. Thus, CD31, when binding to SAP, interferes with Th1 responses, suggesting that CD31 has a key regulatory role in the signaling pathway(s) leading to the IFN- gamma response to M. tuberculosis.