Microenvironments in Tuberculous Granulomas Are Delineated by Distinct Populations of Macrophage Subsets and Expression of Nitric Oxide Synthase and This information is current as Arginase Isoforms of September 26, 2021. Joshua T. Mattila, Olabisi O. Ojo, Diane Kepka-Lenhart, Simeone Marino, Jin Hee Kim, Seok Yong Eum, Laura E. Via, Clifton E. Barry III, Edwin Klein, Denise E. Kirschner, Sidney M. Morris, Jr., Philana Ling Lin and JoAnne L. Downloaded from Flynn J Immunol 2013; 191:773-784; Prepublished online 7 June 2013; doi: 10.4049/jimmunol.1300113 http://www.jimmunol.org/content/191/2/773 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/06/07/jimmunol.130011 Material 3.DC1 References This article cites 66 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/191/2/773.full#ref-list-1 by guest on September 26, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Microenvironments in Tuberculous Granulomas Are Delineated by Distinct Populations of Macrophage Subsets and Expression of Nitric Oxide Synthase and Arginase Isoforms Joshua T. Mattila,* Olabisi O. Ojo,* Diane Kepka-Lenhart,* Simeone Marino,† Jin Hee Kim,‡ Seok Yong Eum,x Laura E. Via,{ Clifton E. Barry, III,{ Edwin Klein,‖ Denise E. Kirschner,† Sidney M. Morris, Jr.,* Philana Ling Lin,# and JoAnne L. Flynn*,** Macrophages in granulomas are both antimycobacterial effector and host cell for Mycobacterium tuberculosis, yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we Downloaded from examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial NO synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared with nongranulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, whereas epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68, and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1, and Arg2 proteins were identified in macrophages and localized similarly in granulomas http://www.jimmunol.org/ across species, with greater eNOS expression and ratio of iNOS/Arg1 expression in epithelioid macrophages as compared with cells in the lymphocyte cuff. iNOS, Arg1, and Arg2 expression in neutrophils was also identified. The combination of phenotypic and functional markers support that macrophages with anti-inflammatory phenotypes localized to outer regions of granulomas, whereas the inner regions were more likely to contain macrophages with proinflammatory, presumably bactericidal, phenotypes. Together, these data support the concept that granulomas have organized microenvironments that balance antimicrobial anti- inflammatory responses to limit pathology in the lungs. The Journal of Immunology, 2013, 191: 773–784. he initial immunologic events following Mycobacterium sufficient to control intracellular replication in the majority of cases by guest on September 26, 2021 tuberculosis infection include cytokine- and chemokine- but must interact with activated T cells (2). The product of these T mediated recruitment of monocytes, neutrophils, and interactions is the granuloma, an organized structure rich in mac- tissue-resident macrophages (1). Macrophages alone are not rophages and lymphocytes that acts as a functional unit for con- trolling M. tuberculosis infection. The presence of granulomas is not necessarily indicative of controlled M. tuberculosis infection *Department of Microbiology and Molecular Genetics, University of Pittsburgh, because hosts with active tuberculosis (TB) have numerous gran- Pittsburgh, PA 15261; †Department of Microbiology and Immunology, University ulomas, but the fact that most M. tuberculosis–infected indi- of Michigan Medical School, Ann Arbor, MI 48109; ‡National Masan Tuberculosis Hospital, Changwon 631-710, Republic of Korea; xInternational Tuberculosis Re- viduals never experience active disease (1) suggests that search Institute, Changwon 631-710, Republic of Korea; {Tuberculosis Research granulomas in immunologically competent individuals can be highly Section, Laboratory of Clinical Infectious Disease, National Institute of Allergy and ‖ effective at restraining bacterial growth and dissemination. Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; Division of Laboratory Animal Resources, University of Pittsburgh, Pittsburgh PA 15261; #De- The necrotic (caseous) granuloma is the most common lesion partment of Pediatrics, Children’s Hospital of Pittsburgh of the University of Pittsburgh associated with active TB. Necrotic granulomas have an outer Medical Center, Pittsburgh PA 15224; and **Center for Vaccine Research, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261 lymphocyte cuff dominated by T and B cells and a macro- phage-rich middle region that surrounds an amorphous center Received for publication January 14, 2013. Accepted for publication May 7, 2013. of caseous necrosis. Other types of granulomas include completely This work was supported by National Institutes of Health (NIH) Grants HL074845 (to J.L.F.), HL106804 (to J.L.F. and D.E.K.), HL092883 (to J.L.F. and D.E.K.), cellular, nonnecrotic granulomas and suppurative granulomas EB012579 (to D.E.K. and J.L.F.), GM057384-11 (to S.M.M.); the Division of Intra- (heavily infiltrated by neutrophils) (3). In experimentally infected mural Research, National Institute of Allergy and Infectious Diseases, NIH and the Korean Ministry of Health, Welfare and Family Affairs (to C.E.B.); NIH Grant cynomolgus macaques, caseous granulomas form by 4–6 wk AI060525-05 (to J.L.F., supporting J.T.M.); NIH Grant AI077183 (to J.T.M.); the postinfection (4, 5), whereas nonnecrotic granulomas are not ob- Bill and Melinda Gates Foundation (to J.L.F., P.L.L., and C.E.B.); NIH Grant K08 served until later and primarily during active or reactivated disease AI063101 (to P.L.L.); the Otis Foundation (to P.L.L.); and the Heiser Program for Research in Tuberculosis and Leprosy (to J.T.M.). (5). Partially mineralized and highly fibrotic (fibrocalcific) granu- Address correspondence and reprint requests to Prof. JoAnne L. Flynn, Center for lomas are associated with clinically latent infection but can also be Vaccine Research, University of Pittsburgh School of Medicine, W1144 Biomedical found in active TB. Other granuloma types, including caseous Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261. E-mail address: joanne@ granulomas, can be present in latent infection as well, indicating pitt.edu that the types of granulomas that are present incompletely describe The online version of this article contains supplemental material. the infection status of the host. This being said, latent M. tuberculosis Abbreviations used in this article: Arg, arginase; eNOS, endothelial NO synthase; iNOS, inducible NO synthase; L-NIL, N6-(1-iminoethyl)-L-lysine; nNOS, neural NO infection is associated with lower numbers of granulomas than active synthase; NOS, NO synthase; qRT-PCR, quantitative RT-PCR; TB, tuberculosis. TB (5). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300113 774 MYELOID CELL NOS AND ARGINASE ISOFORMS IN PRIMATE GRANULOMAS The high level of stratification in human and nonhuman pri- Materials and Methods mate granulomas (3, 6) suggests that protection depends on Tissue processing and sectioning microenvironments (7, 8) that promote bacterial clearance while All animal procedures and husbandry practices were included in protocols minimizing damage to uninvolved tissues adjacent to the granuloma. approved by the University of Pittsburgh’s Institutional Animal Use and Macrophage subsets engaging in anti-inflammatory or proin- Care Committee. Cynomolgus macaques were infected with low-dose flammatory processes are likely to be important, yet poorly under- (∼25 CFU) Erdman-strain M. tuberculosis as previously described (4). stood, mediators determining the characteristics of these micro- Macaques with active TB were humanely euthanized and necropsied as environments. Activated macrophages are often classified as either previously described (4, 5). All samples obtained were from animals un- dergoing necropsy as part of other studies. For immunohistochemistry, classically activated M1 (proinflammatory) macrophages that engage granuloma-containing tissues were excised and fixed in 10% neutral in bactericidal activity or alternatively activated M2 (anti- buffered formalin prior to placement in histology cassettes and paraffin inflammatory) macrophages that mediate