LAG3 Expression in Active Mycobacterium tuberculosis Infections
2014; Elsevier BV; Volume: 185; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2014.11.003
ISSN1525-2191
AutoresBonnie Phillips, Smriti Mehra, Muhammad Ahsan, Moisés Selman, Shabaana A. Khader, Deepak Kaushal,
Tópico(s)Immune Cell Function and Interaction
ResumoMycobacterium tuberculosis (MTB) is a highly successful pathogen because of its ability to persist in human lungs for long periods of time. MTB modulates several aspects of the host immune response. Lymphocyte-activation gene 3 (LAG3) is a protein with a high affinity for the CD4 receptor and is expressed mainly by regulatory T cells with immunomodulatory functions. To understand the function of LAG3 during MTB infection, a nonhuman primate model of tuberculosis, which recapitulates key aspects of natural human infection in rhesus macaques (Macaca mulatta), was used. We show that the expression of LAG3 is highly induced in the lungs and particularly in the granulomatous lesions of macaques experimentally infected with MTB. Furthermore, we show that LAG3 expression is not induced in the lungs and lung granulomas of animals exhibiting latent tuberculosis infection. However, simian immunodeficiency virus–induced reactivation of latent tuberculosis infection results in an increased expression of LAG3 in the lungs. This response is not observed in nonhuman primates infected with non-MTB bacterial pathogens, nor with simian immunodeficiency virus alone. Our data show that LAG3 was expressed primarily on CD4+ T cells, presumably by regulatory T cells but also by natural killer cells. The expression of LAG3 coincides with high bacterial burdens and changes in the host type 1 helper T-cell response. Mycobacterium tuberculosis (MTB) is a highly successful pathogen because of its ability to persist in human lungs for long periods of time. MTB modulates several aspects of the host immune response. Lymphocyte-activation gene 3 (LAG3) is a protein with a high affinity for the CD4 receptor and is expressed mainly by regulatory T cells with immunomodulatory functions. To understand the function of LAG3 during MTB infection, a nonhuman primate model of tuberculosis, which recapitulates key aspects of natural human infection in rhesus macaques (Macaca mulatta), was used. We show that the expression of LAG3 is highly induced in the lungs and particularly in the granulomatous lesions of macaques experimentally infected with MTB. Furthermore, we show that LAG3 expression is not induced in the lungs and lung granulomas of animals exhibiting latent tuberculosis infection. However, simian immunodeficiency virus–induced reactivation of latent tuberculosis infection results in an increased expression of LAG3 in the lungs. This response is not observed in nonhuman primates infected with non-MTB bacterial pathogens, nor with simian immunodeficiency virus alone. Our data show that LAG3 was expressed primarily on CD4+ T cells, presumably by regulatory T cells but also by natural killer cells. The expression of LAG3 coincides with high bacterial burdens and changes in the host type 1 helper T-cell response. Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), is thought to have infected more than one third of the world's current population.1WHO: Global Tuberculosis Report 2013. World Health Organization, Geneva, Switzerland2013Google Scholar MTB is responsible for approximately 1.3 million deaths a year, meaning that this pathogen results in greater mortality than any other infectious bacterium.2Raviglione M.C. The new Stop TB Strategy and the Global Plan to Stop TB, 2006-2015.Bull World Health Organ. 2007; 85: 327Crossref PubMed Scopus (48) Google Scholar, 3Philips J.A. Ernst J.D. Tuberculosis pathogenesis and immunity.Annu Rev Pathol. 2012; 7: 353-384Crossref PubMed Scopus (275) Google Scholar Each year, approximately 9 million people become newly infected with MTB.1WHO: Global Tuberculosis Report 2013. World Health Organization, Geneva, Switzerland2013Google Scholar Of those individuals infected with MTB, approximately 90% remain latently infected, with an asymptomatic infection that is not cleared by the immune response.4Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project.JAMA. 1999; 282: 677-686Crossref PubMed Scopus (2726) Google Scholar Only 5% to 10% of MTB-infected individuals progress to active disease, where a breakdown of MTB containment and clinical symptoms of TB take place.5Bloom B.R. Murray C.J. Tuberculosis: commentary on a reemergent killer.Science. 1992; 257: 1055-1064Crossref PubMed Scopus (1244) Google Scholar During active TB, the release of MTB bacilli occurs, resulting in individuals who are highly infectious, leading to the spread of MTB. The granuloma is crucial to determining the progression or control of MTB infection.6Russell D.G. Who puts the tubercle in tuberculosis?.Nat Rev Microbiol. 2007; 5: 39-47Crossref PubMed Scopus (480) Google Scholar In human pulmonary TB, the structure of the lung granuloma within the host is well organized and composed mainly of immune cells. A classic-type MTB-induced lung granuloma consists of a necrotic central region encircled by monocyte-derived cells, including infected macrophages, epithelioid macrophages, multinucleated giant cells, and foamy macrophages, which is then surrounded by an outer ring of mostly T and B lymphoid-type cells and fibroblasts.6Russell D.G. Who puts the tubercle in tuberculosis?.Nat Rev Microbiol. 2007; 5: 39-47Crossref PubMed Scopus (480) Google Scholar, 7Ramakrishnan L. Revisiting the role of the granuloma in tuberculosis.Nat Rev Immunol. 2012; 12: 352-366Crossref PubMed Scopus (522) Google Scholar The most accepted view is that the formation of this lung granuloma is the host's attempt to contain and control the growth of MTB bacilli, yet it has been suggested that granuloma formation might inadvertently assist in the persistence of infection.8Ehlers S. Schaible U.E. The granuloma in tuberculosis: dynamics of a host-pathogen collusion.Front Immunol. 2012; 3: 411PubMed Google Scholar, 9Guirado E. Schlesinger L.S. Modeling the Mycobacterium tuberculosis granuloma: the critical battlefield in host immunity and disease.Front Immunol. 2013; 4: 98Crossref PubMed Scopus (171) Google Scholar These two views hint at the constant struggle that occurs between host and pathogen to gain advantage during infection. Immunomodulation within the host is critical for successful containment of the bacilli within the lung granuloma. A suppressed immune response will prevent granuloma formation and maintenance, and an overactive response will result in excessive inflammation, causing immunopathogenesis and allowing for the proliferation and spread of MTB.10Lin P.L. Myers A. Smith L. Bigbee C. Bigbee M. Fuhrman C. Grieser H. Chiosea I. Voitenek N.N. Capuano S.V. Klein E. Flynn J.L. Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model.Arthritis Rheum. 2010; 62: 340-350Crossref PubMed Scopus (47) Google Scholar, 11Fenhalls G. Stevens L. Bezuidenhout J. Amphlett G.E. Duncan K. Bardin P. Lukey P.T. Distribution of IFN-gamma, IL-4 and TNF-alpha protein and CD8 T cells producing IL-12p40 mRNA in human lung tuberculous granulomas.Immunology. 2002; 105: 325-335Crossref PubMed Scopus (63) Google Scholar MTB has previously been shown to use certain immunomodulatory proteins of the host to regulate the immune response in its favor; this has been observed with the up-regulation of IL-10, as well as potentially with the increased presence of indoleamine 2,3-dioxygenase.12Redford P.S. Murray P.J. O'Garra A. The role of IL-10 in immune regulation during M. tuberculosis infection.Mucosal Immunol. 2011; 4: 261-270Crossref PubMed Scopus (334) Google Scholar, 13Cooper A.M. Cell-mediated immune responses in tuberculosis.Annu Rev Immunol. 2009; 27: 393-422Crossref PubMed Scopus (843) Google Scholar, 14Mehra S. Alvarez X. Didier P.J. Doyle L.A. Blanchard J.L. Lackner A.A. Kaushal D. Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.J Infect Dis. 2013; 207: 1115-1127Crossref PubMed Scopus (72) Google Scholar Moreover, previous studies have illustrated that infected macrophages produce the immunosuppressive cytokine IL-10 that can inhibit the production of IL-12, thus controlling T-cell differentiation.12Redford P.S. Murray P.J. O'Garra A. The role of IL-10 in immune regulation during M. tuberculosis infection.Mucosal Immunol. 2011; 4: 261-270Crossref PubMed Scopus (334) Google Scholar, 15Giacomini E. Iona E. Ferroni L. Miettinen M. Fattorini L. Orefici G. Julkunen I. Coccia E.M. Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response.J Immunol. 2001; 166: 7033-7041Crossref PubMed Scopus (355) Google Scholar Expression of IL-10 in regulatory T cells (Tregs) during early MTB infection has been observed in virulent MTB strains, resulting in the dampening of the type 1 helper T-cell (Th1) response.12Redford P.S. Murray P.J. O'Garra A. The role of IL-10 in immune regulation during M. tuberculosis infection.Mucosal Immunol. 2011; 4: 261-270Crossref PubMed Scopus (334) Google Scholar Another method through which MTB is able to control the host response to infection is through the suppression of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) within dendritic cells, which causes decreased dendritic cell function and maturation.16Geijtenbeek T.B. Van Vliet S.J. Koppel E.A. Sanchez-Hernandez M. Vandenbroucke-Grauls C.M. Appelmelk B. Van Kooyk Y. Mycobacteria target DC-SIGN to suppress dendritic cell function.J Exp Med. 2003; 197: 7-17Crossref PubMed Scopus (900) Google Scholar Lymphocyte-activation gene 3 (LAG3) protein is expressed on populations of activated T cells, such as Tregs and natural killer (NK) cells, and some monocyte-derived cell populations.17Poirier N. Haudebourg T. Brignone C. Dilek N. Hervouet J. Minault D. Coulon F. de Silly R.V. Triebel F. Blancho G. Vanhove B. Antibody-mediated depletion of lymphocyte-activation gene-3 (LAG-3(+) )-activated T lymphocytes prevents delayed-type hypersensitivity in non-human primates.Clin Exp Immunol. 2011; 164: 265-274Crossref PubMed Scopus (23) Google Scholar, 18Macon-Lemaitre L. Triebel F. The negative regulatory function of the lymphocyte-activation gene-3 co-receptor (CD223) on human T cells.Immunology. 2005; 115: 170-178Crossref PubMed Scopus (137) Google Scholar LAG3 is a negative costimulatory receptor that is homologous to CD4, yet has a 2-log higher affinity for major histocompatibility complex II.17Poirier N. Haudebourg T. Brignone C. Dilek N. Hervouet J. Minault D. Coulon F. de Silly R.V. Triebel F. Blancho G. Vanhove B. Antibody-mediated depletion of lymphocyte-activation gene-3 (LAG-3(+) )-activated T lymphocytes prevents delayed-type hypersensitivity in non-human primates.Clin Exp Immunol. 2011; 164: 265-274Crossref PubMed Scopus (23) Google Scholar This molecule dampens the immune response through the activation and resulting proliferation of Tregs, as well as via the inhibition of monocyte differentiation, both of which have deleterious downstream effects on Th1 effector T-cell proliferation and function, and are essential for an adequate host response to control MTB infection.17Poirier N. Haudebourg T. Brignone C. Dilek N. Hervouet J. Minault D. Coulon F. de Silly R.V. Triebel F. Blancho G. Vanhove B. Antibody-mediated depletion of lymphocyte-activation gene-3 (LAG-3(+) )-activated T lymphocytes prevents delayed-type hypersensitivity in non-human primates.Clin Exp Immunol. 2011; 164: 265-274Crossref PubMed Scopus (23) Google Scholar, 18Macon-Lemaitre L. Triebel F. The negative regulatory function of the lymphocyte-activation gene-3 co-receptor (CD223) on human T cells.Immunology. 2005; 115: 170-178Crossref PubMed Scopus (137) Google Scholar, 19Li N. Wang Y. Forbes K. Vignali K.M. Heale B.S. Saftig P. Hartmann D. Black R.A. Rossi J.J. Blobel C.P. Dempsey P.J. Workman C.J. Vignali D.A. Metalloproteases regulate T-cell proliferation and effector function via LAG-3.EMBO J. 2007; 26: 494-504Crossref PubMed Scopus (168) Google Scholar, 20Workman C.J. Wang Y. El Kasmi K.C. Pardoll D.M. Murray P.J. Drake C.G. Vignali D.A. LAG-3 regulates plasmacytoid dendritic cell homeostasis.J Immunol. 2009; 182: 1885-1891Crossref PubMed Scopus (163) Google Scholar LAG3 up-regulation has already been shown to be detrimental to the host response in certain chronic infections, such as hepatitis B virus and Plasmodium falciparum.21Li F.J. Zhang Y. Jin G.X. Yao L. Wu D.Q. Expression of LAG-3 is coincident with the impaired effector function of HBV-specific CD8(+) T cell in HCC patients.Immunol Lett. 2013; 150: 116-122Crossref PubMed Scopus (94) Google Scholar, 22Illingworth J. Butler N.S. Roetynck S. Mwacharo J. Pierce S.K. Bejon P. Crompton P.D. Marsh K. Ndungu F.M. Chronic exposure to Plasmodium falciparum is associated with phenotypic evidence of B and T cell exhaustion.J Immunol. 2013; 190: 1038-1047Crossref PubMed Google Scholar The blockade of LAG3 with monoclonal antibodies has resulted in an enhanced ability of antigen-presenting cells to generate a Th1 response, with increased levels of interferon (IFN)-γ being present.23Andreae S. Piras F. Burdin N. Triebel F. Maturation and activation of dendritic cells induced by lymphocyte activation gene-3 (CD223).J Immunol. 2002; 168: 3874-3880Crossref PubMed Scopus (132) Google Scholar Building on these facts, it appears as though LAG3 and IL-10 could play similar roles during an MTB infection in both the inhibition of the Th1 immune response, as well as with presentation of antigen. Our group has previously shown that the granuloma-rich lung tissue from actively MTB-infected rhesus macaques has a 25-fold higher expression of LAG3 RNA than that of naïve and latently infected animals.24Mehra S. Golden N.A. Dutta N.K. Midkiff C.C. Alvarez X. Doyle L.A. Asher M. Russell-Lodrigue K. Monjure C. Roy C.J. Blanchard J.L. Didier P.J. Veazey R.S. Lackner A.A. Kaushal D. Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.J Med Primatol. 2011; 40: 233-243Crossref PubMed Scopus (87) Google Scholar We propose that LAG3 plays a role in modulating the local lung immune response to MTB to attempt to contain infection through dampening the immune response to reduce host-mediated immunopathogenesis. We believe that LAG3 up-regulation correlates with active TB due to a diminished immune response and may be functionally linked to IL-10.12Redford P.S. Murray P.J. O'Garra A. The role of IL-10 in immune regulation during M. tuberculosis infection.Mucosal Immunol. 2011; 4: 261-270Crossref PubMed Scopus (334) Google Scholar As part of the current study, we have tested this hypothesis in the rhesus macaque (Macaca mulatta) model. This system was used because of its ability to reproducibly emulate the progression of MTB infection, where the clinical signs experienced by nonhuman primates (NHPs) during active infection and MTB-induced lung granuloma structure closely mirror what is experienced in humans.25Kaushal D. Mehra S. Didier P.J. Lackner A.A. The non-human primate model of tuberculosis.J Med Primatol. 2012; 41: 191-201Crossref PubMed Scopus (114) Google Scholar Furthermore, NHPs are able to recapitulate latency, as well as reactivation when infected with simian immunodeficiency virus (SIV).5Bloom B.R. Murray C.J. Tuberculosis: commentary on a reemergent killer.Science. 1992; 257: 1055-1064Crossref PubMed Scopus (1244) Google Scholar, 26Mehra S. Golden N.A. Stuckey K. Didier P.J. Doyle L.A. Russell-Lodrigue K.E. Sugimoto C. Hasegawa A. Sivasubramani S.K. Roy C.J. Alvarez X. Kuroda M.J. Blanchard J.L. Lackner A.A. Kaushal D. The Mycobacterium tuberculosis stress response factor SigH is required for bacterial burden as well as immunopathology in primate lungs.J Infect Dis. 2012; 205: 1203-1213Crossref PubMed Scopus (58) Google Scholar Our findings show that LAG3 expression is localized to the lungs of animals experiencing active TB. Furthermore, we show that LAG3 is expressed within the outer periphery of MTB-induced lung granulomas. The cells expressing LAG3 are believed to be regulatory T cells and NK cells. Archived paraffinized lung tissue biopsy samples from humans diagnosed with TB were collected in accordance with a protocol approved by the Ethics Committee of the National Institute of Respiratory Diseases (Mexico City, Mexico), as previously described.27Slight S.R. Rangel-Moreno J. Gopal R. Lin Y. Fallert Junecko B.A. Mehra S. Selman M. Becerril-Villanueva E. Baquera-Heredia J. Pavon L. Kaushal D. Reinhart T.A. Randall T.D. Khader S.A. CXCR5(+) T helper cells mediate protective immunity against tuberculosis.J Clin Invest. 2013; 123: 712-726PubMed Google Scholar Adult male Indian origin rhesus macaques acquired from the Tulane National Primate Research Center (Covington, LA) breeding colony were used for our studies. These animals were quarantined for 90 days and were tested for previous exposure to MTB with the tuberculin skin test (TST) and an NHP IFN-γ release assay (Primagam; Life Technologies-Thermo Fisher Scientific, Waltham, MA).28Dutta N.K. Mehra S. Didier P.J. Roy C.J. Doyle L.A. Alvarez X. Ratterree M. Be N.A. Lamichhane G. Jain S.K. Lacey M.R. Lackner A.A. Kaushal D. Genetic requirements for the survival of tubercle bacilli in primates.J Infect Dis. 2010; 201: 1743-1752Crossref PubMed Scopus (133) Google Scholar MTB-infected animals were housed in Biosafety Level 3 conditions. Blood draws, bronchoalveolar lavage (BAL), and all other physical data collection were performed as previously described.28Dutta N.K. Mehra S. Didier P.J. Roy C.J. Doyle L.A. Alvarez X. Ratterree M. Be N.A. Lamichhane G. Jain S.K. Lacey M.R. Lackner A.A. Kaushal D. Genetic requirements for the survival of tubercle bacilli in primates.J Infect Dis. 2010; 201: 1743-1752Crossref PubMed Scopus (133) Google Scholar, 29Gormus B.J. Blanchard J.L. Alvarez X.H. Didier P.J. Evidence for a rhesus monkey model of asymptomatic tuberculosis.J Med Primatol. 2004; 33: 134-145Crossref PubMed Scopus (53) Google Scholar Samples were collected for this study from animals with active TB as well as latent TB infection (LTBI) and reactivation, which had been described in detail earlier.14Mehra S. Alvarez X. Didier P.J. Doyle L.A. Blanchard J.L. Lackner A.A. Kaushal D. Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.J Infect Dis. 2013; 207: 1115-1127Crossref PubMed Scopus (72) Google Scholar, 24Mehra S. Golden N.A. Dutta N.K. Midkiff C.C. Alvarez X. Doyle L.A. Asher M. Russell-Lodrigue K. Monjure C. Roy C.J. Blanchard J.L. Didier P.J. Veazey R.S. Lackner A.A. Kaushal D. Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.J Med Primatol. 2011; 40: 233-243Crossref PubMed Scopus (87) Google Scholar The Tulane National Primate Research Center Institutional Animal Care and Use Committee and the Institutional Biosafety Committee approved all procedures. The experimental design for MTB infection via inhalation and SIV coinfection has been previously illustrated.24Mehra S. Golden N.A. Dutta N.K. Midkiff C.C. Alvarez X. Doyle L.A. Asher M. Russell-Lodrigue K. Monjure C. Roy C.J. Blanchard J.L. Didier P.J. Veazey R.S. Lackner A.A. Kaushal D. Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.J Med Primatol. 2011; 40: 233-243Crossref PubMed Scopus (87) Google Scholar Herein, we used 32 Indian rhesus macaques (Table 1). For the study of active TB, 1000 colony-forming units (CFUs) of MTB CDC1551 was deposited into the lungs of 10 animals via the head-only aerosol method, as previously described.24Mehra S. Golden N.A. Dutta N.K. Midkiff C.C. Alvarez X. Doyle L.A. Asher M. Russell-Lodrigue K. Monjure C. Roy C.J. Blanchard J.L. Didier P.J. Veazey R.S. Lackner A.A. Kaushal D. Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.J Med Primatol. 2011; 40: 233-243Crossref PubMed Scopus (87) Google Scholar For the study of LTBI, 22 remaining animals were infected through the same method to deposit 50 CFUs of MTB CDC1551.24Mehra S. Golden N.A. Dutta N.K. Midkiff C.C. Alvarez X. Doyle L.A. Asher M. Russell-Lodrigue K. Monjure C. Roy C.J. Blanchard J.L. Didier P.J. Veazey R.S. Lackner A.A. Kaushal D. Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.J Med Primatol. 2011; 40: 233-243Crossref PubMed Scopus (87) Google Scholar MTB infection was confirmed by conversion to positive TST and PRIMAGAM. Over a period of 9 weeks, the animals were bled weekly for complete blood cell count, serum chemistry analysis, and serum C-reactive protein (CRP) assays. BAL collection was also performed at weeks 3 and 7 to determine MTB levels. In a subset of animals infected with the lower dose of MTB and with no signs of disease, SIV infections were performed with 300 samples of 50% tissue culture–infective dose of SIVmac239 virus diluted in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) and injected i.v. at week 9 after MTB infection. The criteria to determine the onset of active TB, LTBI, and reactivation of LTBI included the following: TST and PRIMAGAM-IFN-γ release assay positivity, serum CRP levels, and bacterial CFUs in the BAL of these animals over the course of infection. All procedures had been described in detail in our prior publications.14Mehra S. Alvarez X. Didier P.J. Doyle L.A. Blanchard J.L. Lackner A.A. Kaushal D. Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.J Infect Dis. 2013; 207: 1115-1127Crossref PubMed Scopus (72) Google Scholar, 24Mehra S. Golden N.A. Dutta N.K. Midkiff C.C. Alvarez X. Doyle L.A. Asher M. Russell-Lodrigue K. Monjure C. Roy C.J. Blanchard J.L. Didier P.J. Veazey R.S. Lackner A.A. Kaushal D. Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus.J Med Primatol. 2011; 40: 233-243Crossref PubMed Scopus (87) Google Scholar, 25Kaushal D. Mehra S. Didier P.J. Lackner A.A. The non-human primate model of tuberculosis.J Med Primatol. 2012; 41: 191-201Crossref PubMed Scopus (114) Google Scholar, 28Dutta N.K. Mehra S. Didier P.J. Roy C.J. Doyle L.A. Alvarez X. Ratterree M. Be N.A. Lamichhane G. Jain S.K. Lacey M.R. Lackner A.A. Kaushal D. Genetic requirements for the survival of tubercle bacilli in primates.J Infect Dis. 2010; 201: 1743-1752Crossref PubMed Scopus (133) Google Scholar Plasma SIV levels were measured using an SIV assay developed by the Tulane National Primate Research Center Pathogen Quantification and Detection Core.15Giacomini E. Iona E. Ferroni L. Miettinen M. Fattorini L. Orefici G. Julkunen I. Coccia E.M. Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response.J Immunol. 2001; 166: 7033-7041Crossref PubMed Scopus (355) Google Scholar Animals with acute brucellosis were infected with Brucella melitensis via aerosol with between 8.5 × 105 and 1.3 × 106 CFUs, as previously described.30Lee K.M. Chiu K.B. Sansing H.A. Didier P.J. Ficht T.A. Arenas-Gamboa A.M. Roy C.J. Maclean A.G. Aerosol-induced brucellosis increases TLR-2 expression and increased complexity in the microanatomy of astroglia in rhesus macaques.Front Cell Infect Microbiol. 2013; 3: 86Crossref PubMed Scopus (30) Google Scholar Animals infected with SIV alone were administered the virus i.v. with 100 animal infectious dose.31Kincaid E.Z. Ernst J.D. Mycobacterium tuberculosis exerts gene-selective inhibition of transcriptional responses to IFN-gamma without inhibiting STAT1 function.J Immunol. 2003; 171: 2042-2049Crossref PubMed Scopus (99) Google Scholar Humane end points were predefined in this protocol and applied as a measure of reduction of discomfort.Table 1Classification of TB Status in MTB-Infected Rhesus MacaquesIdentification codeInfection/treatmentClassificationAge at infection (years)Mamu subtypeTST (−2 weeks)TST (3 weeks)TST (7 weeks)E or ENTime to necropsy (days)HD0001MTBActive TB9.5NANNNPPPNDE21HD0002MTBActive TB10.3NANNNPPPNDE25HD0003MTBActive TB2.4A-11 and DR2011NNNPPPNDE25HD0004MTBActive TB3.5A-02, A-11, and B-01NNNPPPNDE34HD0005MTBActive TB6.6A-11 and B-17NNNPPPNDE34HD0006MTBActive TB3.7A-08 and B-01NNNPPPNDE35HD0007MTBActive TB7.8A-02NNNPPPNDE38HD0008MTBActive TB13.8A-02 and B-01NNNPPPNDE51HD0009MTBActive TB13.4A-02 and B-01NNNPPPNDE52HD0010MTBActive TB9.4A-02, A-08, B01, and DR2011NNNPPPNDE61LD0001MTBActive TB9.3A-02 and DR2011NNNNDPPPE105LD0002MTBActive TB6.5NANNNNDPPPE133LD0003MTBActive TB3.5NANNNPPPNDE46LD0004MTBActive TB3.7A08NNNPPPNDE43LD0005MTBLTBI9.4A-02 and B-17NNNNDPPPEN126LD0006MTBLTBI9.4A-08NNNNDPPPEN136LD0007MTBLTBI11.6NANNNNDPPPEN53LD0008MTBLTBI12.5A-01 and DR2011NNNNDPPPEN52LD0009MTBLTBI4.5DR2011NNNNDPPPEN127LD0010MTBLTBI3.4A-01 and A-08NNNNDPPPEN120LD0011MTBLTBI6.4NANNNPPPPPPEN190LD0012MTB or SIVReactivation3.1A08NNNNDPPPE147LD0013MTB or SIVReactivation3.5A-01, A-02, and DR2011NNNNDPPPE114LD0014MTB or SIVReactivation3.5B-01NNNNDPPPE112LD0015MTB or SIVReactivation3.3A-01 and B-01NNNNDPPPE127LD0016MTB or SIVReactivation3.4A-01 and B-01NNNNDPPPE167LD0017MTB or SIVReactivation8.5A-11NNNNDPPPE153LD0018MTB or SIVReactivation3.4A-02 and B-01NNNNDPPPE104LD0019MTB or SIVReactivation2.2NANNNNDPPPE167LD0020MTB or SIVLTBI4.5A-01 and B-17NNNNDPPPEN154LD0021MTB or SIVLTBI8.2A-11NNNNDPPPEN126LD0022MTB or SIVLTBI3.1A-01 and A-08NNNNDPPPEN137Animals were divided into two subsets on the basis of high- and low-dose MTB infection. TST data were obtained 24, 48, and 72 hours after mammalian tuberculin injection. Negative results for the TST were represented with an N, and a P showed positive results. SIV coinfection was performed 63 days after MTB infection.E, euthanasia; EN, experimental necropsy; HD, animals that received high-dose MTB infection when included in animal identification code; LD, animals that received low-dose MTB infection when included in identification code; LTBI, latent tuberculosis infection; MTB, Mycobacterium tuberculosis; N, a single negative; NA, data not available; ND, not done; P, a single positive test result at each time of reading; SIV, simian immunodeficiency virus; TB, tuberculosis; TST, tuberculin skin test. Open table in a new tab Animals were divided into two subsets on the basis of high- and low-dose MTB infection. TST data were obtained 24, 48, and 72 hours after mammalian tuberculin injection. Negative results for the TST were represented with an N, and a P showed positive results. SIV coinfection was performed 63 days after MTB infection. E, euthanasia; EN, experimental necropsy; HD, animals that received high-dose MTB infection when included in animal identification code; LD, animals that received low-dose MTB infection when included in identification code; LTBI, latent tuberculosis infection; MTB, Mycobacterium tuberculosis; N, a single negative; NA, data not available; ND, not done; P, a single positive test result at each time of reading; SIV, simian immunodeficiency virus; TB, tuberculosis; TST, tuberculin skin test. RNA was extracted from lung tissue and BAL that had been stored at −80°C. Lung, spleen, bronchial lymph node, and BAL were placed in TRIzol (Life Technologies, Grand Island, NY) before RNA extraction. Tissue was then homogenized in M tubes (Miltenyi Biotec, Auburn, CA) with a GentleMACS Dissociator (Miltenyi Biotec) using the RNA_02 setting. All samples were run through QIAshredders (Qiagen, Valencia, CA) to ensure cell lysis. RNA was then extracted with a TRIzol/RNeasy Kit (Qiagen) hybrid protocol. DNA was removed from the RNA samples using a TURBO DNA-free Kit (Life Technologies). RNA was quantified with the NanoDrop 2000 (Thermo Scientific, Wilmington, DE). Because of low RNA yield, BAL samples were amplified with MessageAmp aRNA Amplification Kit (Life Technologies) and concentrated with a Vacufuge (Eppendorf, Hauppauge, NY). cDNA synthesis was performed with the High Capacity RNA-to-cDNA Kit (Life Technologies). Primers were as follows: LAG3, 5′-TCTTTCCTTACTGCCAAGTGGGCT-3′ (forward) and 5′-AATGTGACAGTGGCATTGAGCTGC-3′ (reverse); IL-10, 5′-TGAGAACCACGACCCAGACATCAA-3′ (forward) and 5′-AAAGGCATTCTTCACCTGCTCCAC-3′ (reverse); and β-actin, 5′-TCGTCCACCGCAAATGC-3′ (forward) and 5′-TCAAGAAAGGGTGTAACGCAACT-3′ (reverse). These primers were designed and optimized (Integrated DNA Technologies, Coralville, IA). The reactions were performed on an ABI 7900 RT-PCR machine (Applied Biosystems, Carlsbad, CA) with SYBR Green (Life Technologies) as a detector. Samples were performed in duplicate, and both positive and negative controls (nontemplate controls) were used. The expression levels were calculated using the 2E−ΔΔCT method, where samples were normalized against β-actin expression and uninfected controls. Lung tissue was homogenized in M tubes with a GentleMACS Dissociator using the Protien_01 setting with Tissue Extraction Reagent I (Invitrogen, Life Technologies, Grand Island, NY) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich). Supernatant was filtered with a 0.2-μm sterile polyethersulfone filter (VWR, Radnor, PA). Samples were then acid treated (the pH was <3.0 and then neutralized). Milliplex TGFβ Magnetic Bead 3 Plex Kit (Millipore, Billerica, MA) was used to detect presence of cytokine using the Bio-Plex 200 array reader (Bio-Rad, Hercules, CA) and analyzed with Bio-Plex Manager software version 6.1 (Bio-Rad). Flow cytometry staining was performed on bl
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