Increased Frequency of Myeloid-derived Suppressor Cells during Active Tuberculosis and after Recent Mycobacterium tuberculosis Infection Suppresses T-Cell Function Nelita du Plessis1, Laurianne Loebenberg1, Magdalena Kriel1, Florian von Groote-Bidlingmaier2, Eliana Ribechini3, Andre G. Loxton1, Paul D. van Helden1, Manfred B. Lutz3, and Gerhard Walzl1 1 Division of Molecular Biology and Human Genetics, MRC Centre for Molecular and Cellular Biology, DST/NRF Centre of Excellence for Biomedical TB Research, and 2Division of Pulmonology, Department of Medicine, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; and 3Institute of Virology and Immunobiology, University of Wu ¨ rzburg, Wu ¨ rzburg, Germany
Rationale: Inadequacy of T-cell responses may result in the development of tuberculosis (TB). Myeloid-derived suppressor cells (MDSCs) have been described as suppressors of T-cell function in cancer biology and recently in several infectious diseases. Objectives: To explore the presence and role of MDSCs in TB. Methods: We analyzed surface markers of MDSCs in peripheral blood and at the site of disease in TB cases and in patients with lung cancer, and in peripheral blood of asymptomatic tuberculin skin test–positive individuals with recent (household) or remote exposure to Mycobacterium tuberculosis (M.tb) and in uninfected healthy control subjects. To evaluate the suppressive capacity of MDSCs, cells of household contacts infected with M.tb and TB cases were isolated and cocultured with CD31 T cells. Measurements and Main Results: Our results demonstrate an increased presence of MDSCs after recent M.tb infection and disease. We confirm their suppression of CD41 T-cell function, including reduced cytokine responses and inhibition of CD41 T-cell proliferation. Only MDSCs from TB cases reduced T-cell activation, altered T-cell trafficking, and suppressed CD81 T-cell functions. M.tb-expanded MDSCs were associated with significantly higher IL-1b, IL-6, IL-8, granulocyte colony–stimulating factor, and monocyte chemotactic protein-1, and reduced granulocyte-macrophage colony–stimulating factor and macrophage inflammatory protein-1 beta levels in coculture. Conclusions: These data reveal that innate MDSCs are induced not only during active TB at similar levels as found in cancer, but also in healthy individuals after recent exposure to M.tb. These cells diminish protective T-cell responses and may contribute to the inability of hosts to eradicate the infection and add to the subsequent development of TB disease.
(Received in original form February 8, 2013; accepted in final form July 5, 2013) Supported by the National Research Foundation South Africa, German Research Foundation (DFG LU 851/6-1), and International Research Training Group of the DFG (IRTG 1522). N.d.P., G.W., and M.B.L. were supported by common funding through the NRF/IRTG1522. The work of E.R. and M.B.L. was supported by DFG grant LU851/6-1. Author Contributions: Study concept and design, M.B.L., G.W., N.d.P., and E.R. Clinical characterization of participants, F.v.G.-B. and M.K. Acquisition of data, L.L. and N.d.P. Statistical analysis, L.L., N.d.P., and A.G.L. Analysis and interpretation of data, G.W., N.d.P., M.B.L., and E.R. Drafting of the manuscript, N.d.P. Critical revisions to the manuscript for important intellectual content, G.W., M.B.L., P.D.v.H., and M.K. Obtained funding, G.W., M.B.L., and N.d.P. Study supervision, G.W. Correspondence and requests for reprints should be addressed to Nelita du Plessis, Ph.D., Stellenbosch University, Department of Biomedical Sciences, DST/NRF Centre of Excellence in Biomedical TB Research, Division of Molecular Biology and Human Genetics, Cape Town, Western Cape, South Africa. E-mail:
[email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 188, Iss. 6, pp 724–732, Sep 15, 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1164/rccm.201302-0249OC on July 25, 2013 Internet address: www.atsjournals.org
AT A GLANCE COMMENTARY Scientific Knowledge on the Subject
The presence and role of myeloid-derived suppressor cells in tuberculosis (TB) have not been explored previously. What This Study Adds to the Field
We show that myeloid-derived suppressor cell frequencies are increased and suppress T-cell function in TB cases and recently exposed contacts of TB cases. Our results provide new insights into how the innate immune system contributes to the failure to control human Mycobacterium tuberculosis infection. Keywords: myeloid-derived suppressor cells; Mycobacterium tuberculosis; immunosuppression
Efficient host protection against Mycobacterium tuberculosis (M.tb) infection is associated with the induction, activation, and proliferation of TH1 and TH17 cells; their release of the cytokines IL-2, tumor necrosis factor (TNF)-a, IFN-g, and IL-17; and resultant activation of effector monocytes (1, 2). Numerous studies have also shown that CD8 T cell–mediated killing of infected host cells provides defense against M.tb infection (3, 4). However, effector and memory responses often fail to eradicate M.tb from the host, and suppression of cell-mediated immunity is associated with the development of tuberculosis (TB) disease (5). Such immune failure is observed in 5–10% of all individuals infected with M.tb (6). Antiinflammatory innate subsets (7–9), regulatory T cells (Tregs) (10), and TH2 (11) responses suppress and counteract protective T-cell immunity, leading to depressed T-cell activation and reduced IFN-g and IL-2 production. Recently, a heterologous population of undifferentiated immature innate cells with the ability to suppress T-cell responses in an antigen-specific and nonspecific manner has been described as myeloid-derived suppressor cells (MDSCs) (12, 13). MDSCs are early myeloid progenitor HLADR(2/lo)CD11b(1) cells consisting of granulocytic and monocytic fractions. Human monocytic MDSCs are described as CD14(1) or S100A9(1) cells, whereas granulocytic MDSCs are CD15(1) (14–16). Other markers include CD80, CD83, DC-SIGN, CD33, and IL-4Ra (17–20). Immune suppression by MDSCs seems to be dependent on the disease or microenvironmental context (21, 22), with effects ranging from inhibition of T-cell activation and proliferation by up-regulation of arginase and reactive oxygen species (23–25) to induction of Tregs (26, 27), reduction of TH1 responses (28), induction of T-cell apoptosis, and impairment of T-cell migration (29).
Du Plessis, Loebenberg, Kriel, et al.: Myeloid-derived Suppressor Cells in Tuberculosis
MDSCs accumulate in lymphoid tissues, the periphery and site of disease in several chronic inflammatory disorders, such as inflammatory bowel disease (30), diabetes (31), and encephalomyelitis (32). Among healthy hosts, however, they are present only at low frequencies (33). MDSC-mediated suppression of host immunity during chronic inflammation is often crucial to immune regulation and tolerance to limit immunopathology, but potentially detrimental during other pathologic disorders and infections. The unfavorable effects of MDSCs are evident in tumor biology where they accumulate and suppress TH1 responses, promoting tumor progression by angiogenesis and metastasis (34–36). MDSCs have also gained attention because of their adverse role in infectious diseases, such as HIV and simian immunodeficiency virus (37–39), influenza (40), and candidiasis (41). However, their accumulation in mycobacterial infections has only been reported in a mouse model of Mycobacterium bovis bacillus Calmette-Guérin vaccination (42) and complete Freund adjuvant injection (43). These MDSCs blocked T-cell proliferation, dampened T-cell priming, and were unable to kill mycobacteria (43). Nevertheless, no evidence of a functional role for MDSCs in human TB exists. We hypothesized that patients with active, untreated TB would have higher frequencies of MDSCs compared with healthy control subjects. Some of the results of this study have been previously reported in the form of an abstract (44).
METHODS Study Subjects Participants with active TB and household contacts (HHC) were recruited from two health care clinics in South Africa. Patients with pleural TB (PLTB) and pleural malignancy were recruited from the Pulmonology Division of Tygerberg Academic Hospital. All TB cases had untreated, first-episode pulmonary TB with clinical and chest radiographic characteristics compatible with active TB and at least one sputum culture positive for M.tb on mycobacteria growth indicator tube culture, with confirmation of acid-fast bacilli through Ziehl-Neelsen staining and polymerase chain reaction. All patients had negative sputum cultures at the end of treatment. PLTB was diagnosed with a combination of clinical, microbiologic, and histopathologic features including exudative pleural effusions, the presence of acid-fast bacilli by microscopy or M.tb by culture in pleural fluid, or a positive culture or granulomatous inflammation with caseous necrosis on histology of closed pleural needle biopsy specimens. HHC of patients with TB were living in the same household as an adult, sputum smear–positive individual whose diagnosis was no longer than 3 months before enrolment of the HHC into the study. HHC had no clinical, radiologic, or microbiologic evidence of active TB but were tuberculin skin test– positive (TST1) greater than or equal to 10 mm. The South African National TB program does not administer isoniazid preventative treatment for HIV-uninfected adults with latent M.tb infection. Healthy uninfected control subjects were recruited from TST-negative laboratory personnel with no known recent exposure to an active TB case. Healthy remotely exposed control subjects consisted of TST(1) individuals with no history of TB disease or known recent exposure to an active TB case within the past 12 months. All healthy control subjects had no clinical, radiologic, or microbiologic evidence of active TB; were from white and South African Coloured1 population groups; and had no known medical condition. Patients with pleural malignancy had exudative pleural effusions with cytologic and/or histopathologic confirmation of malignancy. All participants were HIV-uninfected adults and all M.tb strains were drug sensitive. Additional characteristics of patients and control subjects are described in Table E1 in the online supplement. Written informed consent was obtained from participants and study approval by the Stellenbosch University Ethics Review Committee (N10/02/055 and 1
In South Africa, the collective term “Coloured” is recognized and officially accepted to describe individuals of mixed ancestry. Although we acknowledge that in some cultures this term may have acquired a derogatory connotation, this is not intended here.
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N05/11/187). Heparinized blood (40 ml) and/or pleural fluid were collected at recruitment. All samples were processed within 2 hours of collection.
Flow Cytometry For intracellular staining, cells were cultured with 10 mg/ml Brefeldin A (Sigma-Aldrich, St. Louis, MO) for the last 6 hours of culture. Erythrocytes were lysed (FACS Lysing solution; BD Biosciences, San Jose, CA) and cells fixed and permeabilized (IC Fixation/Permeabilization buffer; eBioscience Inc., San Diego, CA). Cells (1 3 106), whole blood (100 ml), or pleural effusions (100 ml) were stained with antibodies against cytoplasmic (IFN-g, IL-2, IL-10, TNF-a, Ki67, 7AAD) or surface proteins (LIN1 [lineage marker 1 cocktail contains CD3, CD14, CD16, CD19, CD20, and CD56; used as exclusion marker], CD33, HLA-DR, CD14, CD80, CD11b, CD4, CD3, CD8, CD69, CD62L, Annexin-V) for 20 minutes and washed as per manufacturer’s instructions (BD Biosciences; eBioscience). Cell acquisitions (
