Pulmonary immune responses to Mycobacterium tuberculosis in exposed individuals

RESEARCH ARTICLE Pulmonary immune responses to Mycobacterium tuberculosis in exposed individuals Christian Herzmann1*, Martin Ernst2, Christoph Lange3,4,5, Steffen Stenger6, Stefan H. E. Kaufmann7, Norbert Reiling8, Tom Schaberg9, Lize van der Merwe1,10, Jeroen Maertzdorf7, for the Tb or not Tb consortium¶ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Center for Clinical Studies, Research Center Borstel, Borstel, Germany, 2 Division of Clinical Infectious Diseases, Research Center Borstel, Borstel, Germany, 3 German Center for Infection Research (DZIF), Clinical Tuberculosis Unit, Borstel, Germany, 4 International Health / Infectious Diseases, University of Lübeck, Lübeck, Germany, 5 Department of Medicine, Karolinska Institute, Stockholm, Sweden, 6 Institute for Medical Microbiology and Hygiene, University Hospital Ulm, Ulm, Germany, 7 Department of Immunology, Max Planck Institute for Infection Biology, Berlin, Germany, 8 Division of Microbial Interface Biology, Research Center Borstel, Borstel, Germany, 9 Center of Pneumology, Agaplesion Deaconess Hospital Rotenburg, Rotenburg, Germany, 10 LizeStats Consulting, Frankraal, Overstrand, Western Cape, South Africa ¶ Membership of the Tb or not Tb consortium is provided in the Acknowledgments. * OPEN ACCESS Citation: Herzmann C, Ernst M, Lange C, Stenger S, Kaufmann SHE, Reiling N, et al. (2017) Pulmonary immune responses to Mycobacterium tuberculosis in exposed individuals. PLoS ONE 12(11): e0187882. https://doi.org/10.1371/journal. pone.0187882 Editor: Olivier Neyrolles, Institut de Pharmacologie et de Biologie Structurale, FRANCE Abstract Background Blood based Interferon-(IFN)-γ release assays (IGRAs) have a poor predictive value for the development of tuberculosis. This study aimed to investigate the correlation between IGRAs and pulmonary immune responses in tuberculosis contacts in Germany. Received: July 26, 2017 Accepted: October 28, 2017 Published: November 10, 2017 Copyright: © 2017 Herzmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This observational, multicentre, prospective study was carried out by the research consortium on “Pulmonary Tuberculosis – Host and Pathogen Determinants of Resistance and Disease Progression- (TB or not TB)”, funded by the German Ministry of Education and Research (BMBF, reference 01KI0784). The funder provided support in the form of salaries for one author [CH], Methods IGRAs were performed on bronchoalveolar lavage (BAL) cells and peripheral blood from close healthy contacts of patients with culturally confirmed tuberculosis. Cellular BAL composition was determined by flow cytometry. BAL cells were co-cultured with three strains of Mycobacterium tuberculosis (Mtb) and Mtb derived antigens including Purified Protein Derivative (PPD), 6 kD Early Secretory Antigenic Target (ESAT-6) and 10 kD Culture Filtrate Protein (CFP-10). Levels of 29 cytokines and chemokines were analyzed in the supernatants by multiplex assay. Associations and effects were examined using linear mixedeffects models. Results There were wide variations of inter-individual cytokine levels in BAL cell culture supernatants. Mycobacterial infection and stimulation with PPD showed a clear induction of several macrophage and lymphocyte associated cytokines, reflecting activation of these cell types. No robust correlation between cytokine patterns and blood IGRA status of the donor was observed, except for slightly higher Interleukin-2 (IL-2) responses in BAL cells from IGRApositive donors upon mycobacterial infection compared to cells from IGRA-negative donors. PLOS ONE | https://doi.org/10.1371/journal.pone.0187882 November 10, 2017 1 / 18 Pulmonary immune responses to Mycobacterium tuberculosis in exposed individuals but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. LvdM is the sole employee and owner of the statistics consulting company LizeStats Consulting. LizeStats Consulting provides a salary to LvdM, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Stronger correlations were observed when cytokine patterns were stratified according to BAL IGRA status. BAL cells from donors with BAL IGRA-positive responses produced significantly more IFN-γ and IL-2 upon PPD stimulation and mycobacterial infection than cells from BAL IGRA-negative individuals. Correlations between BAL composition and basal cytokine release from unstimulated cells were suggestive of pre-activated lymphocytes but impaired macrophage activity in BAL IGRA-positive donors, in contrast to BAL IGRAnegative donors. Competing interests: Lize van der Merwe is the sole employee and owner of the statistics consulting company LizeStats Consulting. This does not alter our adherence to PLOS ONE policies on sharing data and materials. In vitro BAL cell cytokine responses to M. tuberculosis antigens or infection do not reflect blood IGRA status but do correlate with stronger cellular responses in BAL IGRA-positive donors. The cytokine patterns observed suggest a pre-activated state of lymphocytes and suppressed macrophage responsiveness in BAL cells from BAL IGRA-positive individuals. Conclusions Background About one quarter of the world’s population is estimated to be infected with Mycobacterium tuberculosis [1]. Although the bacterium is transmitted via aerosol inhalation, the estimates for infection rates are based on non-respiratory assays. Both the tuberculin skin test (TST) and the blood based Interferon-γ release assay (IGRA) measure a systemic host immune response that is driven by T lymphocytes primed to M. tuberculosis antigens. The value of these two assays is controversial for several reasons. First, even after documented exposure to patients with contagious pulmonary tuberculosis, less than half of the contact persons develop a positive systemic immune response [2,3]. Second, individuals that are latently infected—as defined by a positive test in the absence of radiological or clinical signs suggestive of tuberculosis—rarely develop tuberculosis after exposure [4,5]. Third, TST and IGRAs may produce conflicting results in approximately 20% of the tested persons [6]. Fourth, it remains unknown whether a positive test is driven by persisting viable bacteria within the host or a lasting immune response to dead bacteria and mycobacterial antigens or based on memory responses in absence of nominal antigen. 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