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.

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.

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.

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.

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.