Networked T Cell Death following Macrophage Infection by Mycobacterium tuberculosis

et al. (2012) Networked T Cell Death following Macrophage Infection by Mycobacterium tuberculosis. PLoS ONE 7(6): e38488. doi:10.1371/journal.pone.0038488 Networked T Cell Death following Macrophage Infection by Mycobacterium tuberculosis Stephen H.-F. Macdonald 0 Elliott Woodward 0 Michelle M. Coleman 0 Emma R. Dorris 0 Parthiban Nadarajan 0 Wui-Mei Chew 0 Anne-Marie McLaughlin 0 Joseph Keane 0 Pere-Joan Cardona, Fundacio Institut d'Investigacio en Cie`ncies de la Salut Germans Trias i Pujol. Universitat Auto` noma de Barcelona. Ciberes, Spain 0 Department of Clinical Medicine, Trinity Institute of Molecular Medicine, St. James's Hospital , Dublin , Ireland Background: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb) impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. Methodology/Principal Findings: We found that lymphopenia (,1.56109 cells/l) was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtbinfected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb) or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cellfree supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-a or Fas. The supernatant cytotoxic signal(s) were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtbsecreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG)- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. Conclusions: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as interfere with microbial eradication in the granuloma. - Funding: This work was funded by the Science Foundation Ireland (SFI; www.sfi.ie) and the Health Research Board (HRB; www.hrb.ie). JK, SHFM and MMC were supported by SFI as part of the Immunology Research Centre, (SFI) Strategic Research Cluster grant (07/SRC/B1144 Cluster Grant E02209420). ERD was funded by the HRBs PhD Scholars Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Tuberculosis (TB) in an immunosuppressive illness and lymphopenia often occurs in TB patients [1,2]. Rather than being an epiphenomenon, it is more likely that this T cell deficiency contributes to pathogen persistence in the host, and the lack of a meaningful immune response during chronic TB infection. Indeed, intracellular pathogens such as Mycobacterium tuberculosis (Mtb) must suppress immunity to survive within an infected host [3,4], and the status of host T lymphocytes is a critical factor in determining the resolution of chronic infections, like tuberculosis. The mechanism of lymphopenia in tuberculosis patients is poorly understood, but may involve activation-induced apoptosis or sequestration of lymphocytes to inflamed organs such as the lung. T cells enhance the activity of phagocytes against Mtb and other intracellular microbes by delivering activating signals, including interferon-c (IFN-c) [5], subsequently upregulating key processes such as nitric oxide generation and apoptosis [6,7]. Accordingly, the significant T cell depletion which accompanies active TB disease is associated with poor prognosis [1,8] as well as diminished cytokine responses which may persist even following successful antitubercular therapy [9]. T cell apoptosis is also an abundant feature of the granuloma, which is a highly organised structure comprising a necrotic centre containing bacteria, dead and infected macrophages, as well as multinucleate giant cells, surrounded by a peripheral cuff of lymphocytes [10,11]. Host phagocyte death can be inhibited by Mtb [12], and it has been shown, by Mustafa et al., that infected cells at the granuloma centre express reduced levels of apoptotic markers such as caspase 3 than those that are uninfected [13]. However significant levels of proapoptotic surface molecules such as Fas ligand are expressed at the periphery of the granuloma, where T cells accumulate [14]. This may represent an infection-induced keep out signal that prevents T cell penetration into the granuloma centre. Additionally, Hirsch et al. have described an increased susceptibility to apoptosis in T cells taken from tuberculosis patients [15], and research by Sharma et al. indicated that infected macrophages can signal T cell apoptosis [16]. The mechanism of this T cell death response is unclear, and it is not known if Mtb-infected human alveolar macrophages (AMs) can supply this immunosuppressive death signal. We found that lymphopenia was prevalent in our culture positive tuberculosis patients, and there was a statistically significant recovery of lymphocyte counts after anti-TB treatment. Because animal model work had associated low lymphocyte counts with death following mycobacterial infection [17], we sought to investigate the toxic effect of infected macrophages on T cells. We have previously reported that human alveolar macrophage infection by Mtb can elicit signals that kill infected cells and nearby uninfected bystander macrophages, in what is likely a hostprotective response. The macrophage death response deprives the bacilli of their replicative niche cell and facilitates delivery of mycobacterial antigens to APCs, enabling antigen presentation [18,19]. Even as the infected human alveolar macrophage death response may support TB immunity, it is likely that a death signal delivered to nearby T cells would diminish the host response [12,20]. We now report that Mtb-infected human alveolar macrophages do cause networked, caspase-dependent lymphocyte apoptosis. Like alveolar macrophages, cell-free culture supernatants from Mtb-infected THP-1 macrophages reliably induced this T cell killing phenotype. We found that this human macrophage cell line recapitulates this host resp (...truncated)