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      Extracellular Vesicles Released from Mycobacterium tuberculosis-Infected Neutrophils Promote Macrophage Autophagy and Decrease Intracellular Mycobacterial Survival


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          Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the lungs, macrophages and neutrophils are the first immune cells that have contact with the infecting mycobacteria. Neutrophils are phagocytic cells that kill microorganisms through several mechanisms, which include the lytic enzymes and antimicrobial peptides that are found in their lysosomes, and the production of reactive oxygen species. Neutrophils also release extracellular vesicles (EVs) (100–1,000 nm in diameter) to the extracellular milieu; these EVs consist of a lipid bilayer surrounding a hydrophilic core and participate in intercellular communication. We previously demonstrated that human neutrophils infected in vitro with Mtb H37Rv release EVs (EV-TB), but the effect of these EVs on other cells relevant for the control of Mtb infection, such as macrophages, has not been completely analyzed. In this study, we characterized the EVs produced by non-stimulated human neutrophils (EV-NS), and the EVs produced by neutrophils stimulated with an activator (PMA), a peptide derived from bacterial proteins (fMLF) or Mtb, and observed that the four EVs differed in their size. Ligands for toll-like receptor (TLR) 2/6 were detected in EV-TB, and these EVs favored a modest increase in the expression of the co-stimulatory molecules CD80, a higher expression of CD86, and the production of higher amounts of TNF-α and IL-6, and of lower amounts of TGF-β, in autologous human macrophages, compared with the other EVs. EV-TB reduced the amount of intracellular Mtb in macrophages, and increased superoxide anion production in these cells. TLR2/6 ligation and superoxide anion production are known inducers of autophagy; accordingly, we found that EV-TB induced higher expression of the autophagy-related marker LC3-II in macrophages, and the co-localization of LC3-II with Mtb inside infected macrophages. The intracellular mycobacterial load increased when autophagy was inhibited with wortmannin in these cells. In conclusion, our results demonstrate that neutrophils produce different EVs in response to diverse activators, and that EV-TB activate macrophages and promote the clearance of intracellular Mtb through early superoxide anion production and autophagy induction, which is a novel role for neutrophil-derived EVs in the immune response to Mtb.

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          Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo.

          Intracellular pathogens and the molecules they express have limited contact with the immune system. Here, we show that macrophages infected with intracellular pathogens Mycobacterium tuberculosis, M bovis BCG, Salmonella typhimurium, or Toxoplasma gondii release from cells small vesicles known as exosomes which contain pathogen-associated molecular patterns (PAMPs). These exosomes, when exposed to uninfected macrophages, stimulate a proinflammatory response in a Toll-like receptor- and myeloid differentiation factor 88-dependent manner. Further, exosomes isolated from the bronchoalveolar lavage fluid (BALF) of M bovis BCG-infected mice contain the mycobacteria components lipoarabinomannan and the 19-kDa lipoprotein and can stimulate TNF-alpha production in naive macrophages. Moreover, exosomes isolated from M bovis BCG- and M tuberculosis-infected macrophages, when injected intranasally into mice, stimulate TNF-alpha and IL-12 production as well as neutrophil and macrophage recruitment in the lung. These studies identify a previously unknown function for exosomes in promoting intercellular communication during an immune response to intracellular pathogens, and we hypothesize that extracellular release of exosomes containing PAMPs is an important mechanism of immune surveillance.
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            Identification of nitric oxide synthase as a protective locus against tuberculosis.

            Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2(-/-) mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-L-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.
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              Neutrophils are the predominant infected phagocytic cells in the airways of patients with active pulmonary TB.

              The exact role of neutrophils in the pathogenesis of TB is poorly understood. Recent evidence suggests that neutrophils are not simply scavenging phagocytes in Mycobacterium tuberculosis (Mtb) infection. Three different types of clinical specimens from patients with active pulmonary TB who underwent lung surgery were examined: sputum, BAL fluid, and cavity contents. Differential cell separation and quantification were performed for intracellular and extracellular bacteria, and bacterial length was measured using microscopy. Neutrophils were more abundant than macrophages in sputum (86.6% +/- 2.2% vs 8.4% +/- 1.3%) and in BAL fluid (78.8% +/- 5.8% vs 11.8% +/- 4.1%). Inside the cavity, lymphocytes (41.3% +/- 11.2%) were the most abundant cell type, followed by neutrophils (38.8% +/- 9.4%) and macrophages (19.5% +/- 7.5%). More intracellular bacilli were found in neutrophils than macrophages in sputum (67.6% +/- 5.6% vs 25.2% +/- 6.5%), in BAL fluid (65.1% +/- 14.4% vs 28.3% +/- 11.6%), and in cavities (61.8% +/- 13.3% vs 23.9% +/- 9.3%). The lengths of Mtb were shortest in cavities (1.9+/- 0.1 microm), followed by in sputum (2.9 +/- 0.1 microm) and in BAL fluid (3.6 +/- 0.2 microm). Our results show that neutrophils are the predominant cell types infected with Mtb in patients with TB and that these intracellular bacteria appear to replicate rapidly. These results are consistent with a role for neutrophils in providing a permissive site for a final burst of active replication of the bacilli prior to transmission.

                Author and article information

                URI : http://frontiersin.org/people/u/261004
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                Front Immunol
                Front Immunol
                Front. Immunol.
                Frontiers in Immunology
                Frontiers Media S.A.
                19 February 2018
                : 9
                : 272
                [1] 1Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN) , Mexico City, Mexico
                [2] 2Departamento de Fisiología y Farmacología, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México (UNAM) , Mexico City, Mexico
                [3] 3Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN) , Mexico City, Mexico
                [4] 4Laboratorio de Citometría de Flujo de Diagnóstico Molecular de Leucemias y Terapia Celular SA. De CV. (DILETEC) , Mexico City, Mexico
                [5] 5Laboratorios Nacionales de Servicios Experimentales (LANSE), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN) , Mexico City, Mexico
                [6] 6Departamento de Bioquímica, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN) , Mexico City, Mexico
                [7] 7Departamento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN) , Mexico City, Mexico
                [8] 8Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI), Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN) , Mexico City, Mexico
                Author notes

                Edited by: Christopher Gregory, University of Edinburgh, United Kingdom

                Reviewed by: Maria Cecilia G. Marcondes, San Diego Biomedical Research Institute, United States; Bruce Milne Hall, University of New South Wales, Australia

                *Correspondence: Rommel Chacón-Salinas, rommelchacons@ 123456yahoo.com.mx ; Iris Estrada-García, iestrada5@ 123456hotmail.com

                Specialty section: This article was submitted to Immunological Tolerance and Regulation, a section of the journal Frontiers in Immunology

                Copyright © 2018 Alvarez-Jiménez, Leyva-Paredes, García-Martínez, Vázquez-Flores, García-Paredes, Campillo-Navarro, Romo-Cruz, Rosales-García, Castañeda-Casimiro, González-Pozos, Hernández, Wong-Baeza, García-Pérez, Ortiz-Navarrete, Estrada-Parra, Serafín-López, Wong-Baeza, Chacón-Salinas and Estrada-García.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                : 03 November 2017
                : 30 January 2018
                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 44, Pages: 12, Words: 8402
                Funded by: Consejo Nacional de Ciencia y Tecnología 10.13039/501100003141
                Award ID: 105278, 221002, 157100
                Funded by: Secretaría de Investigación y Posgrado, Instituto Politécnico Nacional 10.13039/501100007161
                Award ID: SIP-IPN, 20180537
                Original Research

                extracellular vesicles,neutrophils,tuberculosis,macrophage,autophagy
                extracellular vesicles, neutrophils, tuberculosis, macrophage, autophagy


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