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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/

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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 controlling 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 macrophage-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 undergoing 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 prohealing responses. embedding. Tissues were cut into 5-mm–thick sections by the University Expression of inducible NO synthase (iNOS) is the hallmark of of Pittsburgh Medical Center’s in situ histology laboratory and mounted on SuperFrost Plus slides (Thermo Fisher Scientific, Waltham, MA). proinflammatory macrophages and, in murine systems, is necessary Formalin-fixed paraffin-embedded human lung tissue samples containing for improved resistance to TB (9–11). iNOS-expressing macro- granulomas were dissected from tissue removed during therapeutic lung phages have been identified in the lungs of humans with TB (12–14), resection surgery at the National Masan Tuberculosis Hospital from although a correlation between human TB and deficient iNOS ex- patients refractory to second line drug therapy. Tissue collection (2003– pression has proven difficult (15). The other NOS isoforms, endo- 2007) was approved by the National Masan Tuberculosis Hospital institutional review board, an exemption from National Institutes of Health, thelial NOS (eNOS) and neural NOS (nNOS), can also be present in and with written consent of the subjects; samples were deidentified when granulomas (12), but it is not known whether they have homeostatic provided for analysis. Downloaded from or bactericidal functions. Prohealing anti-inflammatory macrophages are characterized Immunofluorescence, immunohistochemistry, imaging, and by arginase 1 (Arg1) expression (16, 17), although this is best image analysis defined in murine systems. Arginases can compete with NO Formalin-fixed paraffin-embedded tissue sections from M. tuberculosis– synthases for L-arginine and generate urea and L-ornithine, which infected macaques m907, m9209 m1307, m1707, m3809, m9905, m10708, m21802, m15304, and m13207 were selected for study. All animals except can be subsequently converted to L-proline (18), an amino acid http://www.jimmunol.org/ m10708 had active TB at the time of necropsy. Tissue sections were used in collagen synthesis and wound healing (19, 20), or to deparaffinized in xylene, 100% ethanol, and 95% ethanol. Tissue sections polyamines (18), which can play a variety of roles in cell phys- were then placed into an Ag retrieval buffer (20 mM Tris/820 mM EDTA/ iology and pathophysiology (21). Although anti-inflammatory 0.00005% Tween 20 [pH 9]) containing a pressure cooker (Manttra, Pis- macrophages have important functions in healing and antihel- cataway, NJ), incubated under pressure for 7 min before removal from the minth responses (22), arginase expression can diminish protec- hotplate, and allowed to cool slowly over 30 min. Sections were incubated in blocking buffer (2.5% BSA in PBS) for 30 min at 37˚C, prior to addition tion against intracellular pathogens, including M. tuberculosis of primary Ab diluted in blocking buffer. Abs for immunohistochemistry (23, 24). Macrophage arginase expression has been identified in were against human CD3ε (ready-to-use format, 1:2 dilution; DakoCyto- tuberculous lung from humans (25) and also in PBMCs of mation, Carpintera, CA), CD11c (clone 5D11, 1:30 dilution; Leica patients infected with M. tuberculosis (26), but the contribution Microsystems, Buffalo Grove, IL), CD68 (clone KP1, 1:50 dilution; Lab by guest on September 26, 2021 Vision, San Diego, CA), CD163 (clone 10D6, 1:30 dilution; Lab Vision), of arginase to protective or pathologic responses in M. tuberculosis calprotectin (clone MAC387; 1:100 dilution; Lab Vision), HAM56 (ready- infection in humans remains undetermined. The NOS/arginase to-use format, 1:2 dilution; Enzo Life Sciences, Farmingdale, NY), iNOS paradigm of macrophage activation is best defined for mice, (rabbit polyclonal; Lab Vision), eNOS (rabbit polyclonal; Lab Vision), however, and the signals in primates that drive macrophage acti- Arg1 (clone 19/arginase1, 1:100 dilution; BD Biosciences), arginase 2 vation remain largely undefined. Considering this, it is likely that (rabbit polyclonal, 1:40 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and nitrotyrosine (rabbit polyclonal, 1:100 dilution; Millipore, Billerica, macrophage polarization occurs on a spectrum (27) with classical MA). The specificity of iNOS and eNOS Abs were confirmed by Western and alternative activation on opposing ends of a spectrum but with blotting of iNOS and eNOS (0.1 mg/lane) in conjunction with 1 mg most macrophages having characteristics somewhere along that M. tuberculosis lysate to confirm that the Abs were isoform specific and continuum. not reactive with bacterial proteins (data not shown). We were unable to identify an anti-nNOS Ab that worked for immunohistochemistry in ma- We examined granulomas from cynomolgus macaques with active caque tissues and was not cross-reactive with other NOS isoforms (data not or clinically latent M. tuberculosis infection to determine whether shown). Tissue sections were incubated at room temperature in cocktails specific populations of macrophage localize to specific microenvi- of primary Abs for 1 h. Secondary Abs, purchased from either Jackson ronments in different granuloma types. Moreover, because NOS ImmunoResearch Laboratories (West Grove, PA) or Life Technologies, and arginase expression may mediate antimycobacterial activity and were diluted in blocking buffer and applied to tissue sections that had been washed three to five times with IHC wash buffer (0.2% Tween-20 in PBS) immunopathology, we sought to identify how NOS and arginase and incubated for 1 h at room temp in the dark. HAM56 was stained with expression relates to the distribution of microenvironment-specific an anti-mouse m-chain–specific secondary Ab (Jackson ImmunoResearch macrophage populations. Studies using mathematical modeling Laboratories) that was made in donkey and not crossreactive with mouse have suggested that granulomas are organized in a nonrandom IgG Abs. The specificity of secondary Abs was confirmed either by isotype or no-primary controls using the same staining and imaging protocol as fashion, and chemokine and cytokine gradients also exist, helping to sections containing stained with primary Abs. The slides were then washed establish cell patterns (28). We present biochemical, molecular, and three to five times with IHC buffer and directly labeled conjugates applied. immunohistochemical evidence demonstrating that macrophages Abs for direct labeling were chosen based on their ability to work well and neutrophils in macaque granulomas can express functional NOS following significant dilution when used with a secondary Ab. The unla- and arginase enzymes and are organized into different microen- beled Abs were labeled with either Alexa Fluor 488 or Alexa Fluor 647 using the Zenon direct labeling kit (Life Technologies). Tissue sections vironments. Furthermore, we identified similarities in macrophage were incubated with direct conjugates for 2 h at room temperature or distribution and NOS and arginase expression between macaque and overnight at 4˚C. Slides were washed four times with IHC wash buffer, human granulomas, suggesting these functions are conserved across once with PBS, and then coverslips were applied using Prolong Gold primate species. These results provide new data on the diversity of mounting medium containing DAPI (Life Technologies). Slides were cured for 24 h at room temperature before imaging. Granulomas were imaged macrophages and neutrophils in granulomas and their effector ca- with either an Olympus Fluoview 500 or Fluoview 1000 laser scanning pacity and thus may lead to an improved understanding of the confocal microscope (Olympus, Center Valley, PA) maintained by the mechanisms underlying antimycobacterial responses. University of Pittsburgh’s Center for Biologic Imaging and a Fluoview 1000 The Journal of Immunology 775 laser scanning confocal microscope maintained by the University of Initial attempts to quantify NOS activity in lung homogenates was done 3 3 Pittsburgh’s Microbiology and Molecular Genetics Department. Individual using the [ H]L-arginine to [ H]citrulline conversion-based assay but tissue sections from animals with active TB frequently contained multiple yielded unacceptable levels of background signal that was not inhibited by granulomas of various sizes and type; we chose to image granulomas with selective NOS or arginase inhibitors. Consequently, NOS activity was 2 features that were representative of that particular granuloma type. Three- determined using a cell lysate–based assay that measured nitrite (NO2 ) color images (red, green, and far red [pseudocolored as blue]) were acquired via the Griess reaction. Lung tissue was homogenized and lysed in 1–3 ml sequentially, followed by a DAPI image (gray) showing nuclei. Images 25 mM Tris-HCl (pH 7.4) with 1 mM EDTA and 1 mM EGTA and filter (either single sections or serial Z sections acquired at 1-mm intervals) were sterilized by passing through a 0.22-mm syringe filter. Protein concen- acquired and saved as TIFF-format images. Z series images were opened trations in tissue homogenates were measured by BCA protein assay with MacBiophotonics ImageJ (available at www.macbiophotonics.ca/ (Thermo Fisher Scientific). NOS assays were performed by adding 10 ml software.htm) or FIJI (available at http://pacific.mpi-cbg.de/wiki/index.php/ tissue homogenate to 40 ml assay buffer (75 mM L-arginine, 1 mM Downloads) and saved as maximum-intensity projections. At least three NADPH, 0.25 mM calmodulin, 2.5 mM FAD, 2.5 mM FMN, 10 mM tet- fields in the macrophage–lymphocyte region were imaged at 3400–600 rahydrobiopterin, 440 mM nor-NOHA [Cayman Chemical Company, Ann original magnification for counting cells in tissues. Images were opened in Arbor, MI], 120 mM CaCl2, and 120 mM MgCl2 in 100 mM HEPES) Photoshop CS4 (Adobe Systems, San Jose CA), overlaid with a grid to followed by an overnight incubation at 37˚C to ensure the reaction went to facilitate analysis, and counted manually by examining each channel sep- completion. A replicate control sample was prepared in the same buffer but arately or in combination for positively stained cells. The number of nuclei included N6-(1-iminoethyl)-L-lysine (L-NIL; Cayman Chemical), a selec- per image, which was used to determine the number of cells within an tive iNOS inhibitor. Recombinant nitrate reductase was added to the re- image, was assessed with CellProfiler v2.0 (available at http://www. action to convert nitrate to nitrite and then the reaction assayed with cellprofiler.org/). Granulomas were too large to be imaged by one 3200 freshly prepared Griess reagent (0.05% naphthylethylenediamine dihy- original magnification field; consequently, multiple overlapping fields drochloride and 0.5% sulfanilamide in 2.5% H3PO4) on a plate reader were acquired and the image of the entire granuloma assembled into (Molecular Devices, Sunnyvale, CA) with a standard curve prepared from a single montage using Photoshop (Adobe Systems). Preliminary work nitrite-containing homogenization buffer. Data were analyzed by measur- Downloaded from indicated that counting individual cells in granulomas for phenotypic ing the concentration of nitrite (based on the standard curve) in the un- analysis by automated or manual means was not going to be feasible due inhibited and L-NIL–inhibited samples. Two factors were taken into to the complexity of the environment; consequently, we used a region-based consideration in the data analysis: the intrinsic coloration of the homog- approach to analyze staining (signal) intensity in lymphocyte cuff or epi- enate imparted by hemoglobin and significantly different protein concen- thelioid macrophage regions. For analysis of region-based characteristics trations related to differences in the size of tissues that were homogenized. (macrophage markers, iNOS/Arg1 expression), nonoverlapping image fields To account for these factors, we divided the measured nitrite concentration (3200 original magnification) of granulomas containing both epithelioid in the uninhibited sample by the measured nitrite concentration in the L- macrophage and lymphocyte cuff regions were acquired as previously NIL–inhibited control to determine the percentage of control, and then this http://www.jimmunol.org/ indicated. From these images, regions of interest were drawn around epi- value was divided by the mass of protein per assay. Normalized data are thelioid macrophage or lymphocyte cuff regions, and the mean pixel inten- expressed as percentage of control per microgram protein. Unless other- sity of the red, green, and blue channels was determined with Photoshop’s wise noted, reagents were purchased from Sigma-Aldrich. histogram tool (Adobe Systems). The iNOS/Arg1 ratio was calculated by dividing the mean iNOS signal by the mean Arg1 signal. Pairwise com- Quantitative real-time PCR parisons were made between macrophage surface marker signal or iNOS/Arg1 signal ratio were made between the epithelioid macrophage and lymphocyte RNA was obtained from eight M. tuberculosis culture–positive (involved) cuff regions. and four culture-negative (uninvolved) tissues from m907, m1307, m1707, m9803, m16705, m13207, m10403, and m16705 stored in RNAlater (Life Technologies, Grand Island, NY) at 280˚C at necropsy. Tissues were Auramine-rhodamine staining by guest on September 26, 2021 disrupted in TRIzol (Life Technologies) under BSL3 conditions, and RNA Granulomas were stained with auramine-rhodamine reagents to visualize the was isolated using a standard phenol-chloroform isolation followed by mycobacterial cell wall component mycolic acid. Tissue sections were de- cleanup with RNeasy columns (Qiagen, Valencia, CA). RNA was quanti- paraffinized as previously indicated and equilibrated in distilled H2Ofor5 fied on a DU-800 spectrophotometer (Beckman Coulter, Brea, CA) and min before being stained with auramine-rhodamine for 30 min. Excess stain used for quantitative RT-PCR (qRT-PCR). Arg1 (HP200032) and Arg2 was washed off with distilled H2O and decolorized in multiple washes of (HP205107) primers were purchased from Origene (Rockville, MD), isopropanol with 5% HCl (v/v) before being counterstained with potassium whereas eNOS primers were purchased from Realtimeprimers.com (Elkins permanganate for 50 s. Slides were washed and air dried before imaging. All Park, PA). iNOS (forward: 59-TCTTGGTCAAAGCTGTGCTC-39;re- auramine-rhodamine staining reagents were purchased from BD Bio- verse: 59-CATTGCCAAACGTACTGGTC-39) and nNOS (forward: 59- sciences. Auramine-rhodamine–stained slides were imaged on an Olympus TAGCTTCCAGAGTGACAAAGTGACC-39; reverse: 59-TGTTCCAGG- Provis epifluorescence microscope (Olympus) at 3200 original magnifica- GATCAGGCTGGTATTC-39) primers were based on the respective human tion. Following this, the immersion oil was removed in a xylene bath, and the RNA sequences and the identity of the product confirmed by sequencing. slides were then stained with H&E using standard protocols. Coverslips were Hypoxanthine phosphoribosyltransferase mRNA (forward: 59-TTACCT- mounted on H&E-stained slides with Permount (Thermo Fisher Scientific), CACTGCTTTCCGGAG-39; reverse: 59-AGTCTGGCTTATATCCAACAT- and the same tissue section used for the auramine-rhodamine staining was 39) was quantified as an internal control. Unless otherwise indicated, pri- used to acquire the H&E image on the same microscope previously used to mers were purchased from Sigma-Aldrich. Amplification was performed ensure consistent magnification and perspective. To determine the localiza- on an iCycler (Bio-Rad, Hercules, CA) using AMV reverse transcriptase tion of M. tuberculosis or M. tuberculosis Ags in granulomas and colocalize (Promega, Madison, WI) and GoTaq qPCR Master Mix reagents (Promega). them with cells, the red auramine-rhodamine image was overlaid on a gray- Samples were assayed in duplicate for each tissue. Relative quantification scale image of the H&E-stained granuloma with Photoshop (Adobe Systems) based on the DD threshold cycle method (30) with hypoxanthine phos- and this position located on the color version of the H&E. phoribosyltransferase (also performed in duplicate) was used as a house- keeping control. NOS and arginase activity assays Flow cytometry to identify calprotectin as a neutrophil marker Granuloma-containing tissues for arginase assays that had been flash frozen in liquid nitrogen at necropsy and stored at 280˚C were homogenized with Whole blood from macaques was isolated as previously described (31). a Medimachine tissue homogenizer (BD Biosciences) under BSL3 con- Preliminary experiments demonstrated that Percoll gradient isolation of ditions. Tissues were homogenized in PBS containing 0.1% Triton X-100 PBMCs from whole blood separates cells into two fractions, a lymphocyte and protease inhibitor mixture (Thermo Fisher Scientific) without EDTA and monocyte-rich fraction and a neutrophil and RBC-rich fraction. and filtered through a 0.22-mm syringe filter. Protein concentrations were Neutrophils in the RBC fraction had physical characteristics that did not measured using a BCA Protein Assay Kit (Thermo Fisher Scientific) with differ from neutrophils in RBC-lysed whole blood. Neutrophils from the BSA as a protein standard. Arginase or NOS activity were assayed under RBC pellet were stained after lysing the RBCs with PharmLyse (BD BSL2 conditions using filter-sterilized samples. Arginase activity was Biosciences) and the cells fixed and permeablized with the Cytofix/ determined as previously described (29) by following the conversion of Cytoperm and Perm/Wash buffers (BD Biosciences) for intracellular Ag 14 14 L-[guanidino- C]arginine to [ C]urea, which was quantified by scintilla- staining. Cells were stained for CD163 (clone eBioGHI/61; eBioscience, 14 14 tion counting after its conversion to CO2 and trapping as Na2 CO3. San Diego, CA) and calprotectin (clone MAC387; Lab Vision) that had Tissues for NOS assays were from recently necropsied animals that had been labeled with the Zenon labeling kit (Life Technologies). Cells phe- been homogenized (see below) and stored at 280˚C until the time of assay. notypes were read using an LSRII flow cytometer (BD Biosciences). 776 MYELOID CELL NOS AND ARGINASE ISOFORMS IN PRIMATE GRANULOMAS

Data analysis and statistics granulomas. qRT-PCR indicated Arg1, Arg2, eNOS, and iNOS ex- Data were analyzed with GraphPad Prism 5.0 (GraphPad, La Jolla, CA). pression was upregulated in granuloma-containing tissues relative to Comparisons between treatments were made using the nonparametric uninfected control tissues (Fig. 2A), with Arg1 and iNOS upregu- Mann–Whitney U test, and pairwise comparisons between granuloma lated more than Arg2 and eNOS, respectively. Although nNOS ex- regions were made using the nonparametric Wilcoxon matched-pairs pression was identified, its expression in granulomas was not , signed-rank test with p 0.05 considered statistically significant. Flow strongly upregulated above uninfected lung. Biochemical assays cytometric data were analyzed using the FlowJo software package (Tree Star, Ashland, OR). for enzyme activity in tissue lysates demonstrated that granuloma- containing tissues had significantly more arginase and NOS activity Results than uninvolved tissues (Fig. 2B–D). The NOS activity associated Arg1, Arg2, iNOS, and eNOS are expressed and functional in with individual granulomas within a monkey was highly variable the granuloma but generally higher than the NOS activity from uninvolved lung from that same monkey (Fig. 2C). When these data were aggre- Factors initiating macrophage polarization and phenotypes of gated and compared, granulomas showed significantly more NOS classically or alternatively activated macrophages have not been activity than uninvolved tissue from infected animals (Fig. 2D). fully resolved in primates. In mice, NOS expression defines Although the iNOS activity of many granulomas was inhibited by classically activated macrophages, whereas arginase expression L-NIL, it was not possible to inhibit the NOS activity in some delineates alternatively activated macrophage populations; con- tissues, suggesting other NOS isoforms contribute to NO production sequently, we used Arg1, Arg2, iNOS, or eNOS (Fig. 1) as markers (data not shown). Finally, we identified nitrotyrosine, the nitro- for macrophage function with the caveat that these markers may sylated tyrosine residues produced after NO–protein interactions, Downloaded from not fully describe macrophage polarization. Moreover, because of in granuloma macrophages and neutrophils (Fig. 2E), supporting the difficulty assigning an activation state to primate macrophages, that the NOS species present in granulomas are functional. we will use the term proinflammatory to indicate macrophages with dominant NOS expression and anti-inflammatory to charac- Identification of macrophage- and neutrophil-specific Abs for terize macrophages with dominant arginase expression. Arg1 was cynomolgus macaques abundant in macrophages, whereas the most intense Arg2 signal Despite the abundance of macrophages in granulomas, little is http://www.jimmunol.org/ occurred as discrete granules in cells with segmented nuclei and known about their molecular identities. Two categories of macro- at much lower levels in macrophages (Fig. 1A). iNOS and eNOS phages found in human granulomas are described morphologically were identified in macrophages, each with a characteristic staining (33) as epithelioid macrophages and foamy macrophages. Epithe- pattern. Macrophage iNOS had a punctate appearance and ap- lioid macrophages are defined by their high cytoplasm/nucleus peared to be scattered throughout the cytoplasm (Fig. 1A), ratios and diffusely eosinophilic cytoplasm (34, 35) and are par- whereas eNOS staining in epithelioid macrophages was more in- ticularly abundant in granuloma regions adjacent to caseous ne- tense and associated with the cell membrane (Fig. 1A). We also crosis. Foamy macrophages are also associated with tuberculous found substantial iNOS and Arg1 coexpression, particularly in granulomas and are identifiable by their foamy, lipid-rich cyto- macrophages (Fig. 1B) but also in neutrophils (Fig. 1B). These by guest on September 26, 2021 plasm (2). To better characterize the presence and location of these results demonstrate that macrophage activation is not binary but cells in granulomas, we used Abs against human macrophage Ags occurs along a spectrum. that we validated for use in macaque tissues. These Ags, including qRT-PCR and biochemical assays for arginase and NOS activity CD11c, CD68, CD163, HAM56, and calprotectin (Mac387), reli- confirmed that arginase and NOS are expressed and functional in ably stained cells in macaque tissues, whereas we excluded other commonly used macrophage markers, including CD11b and CD14 because of their broad myeloid cell expression. Dendritic cell– specific ICAM-3–grabbing nonintegrin (CD209), a marker associated with dendritic cells, was expressed by macrophages in throughout granulomas (data not shown) and, consequently, was not included in this study. We also excluded F4/80, a commonly used macrophage marker in murine systems because the human homolog (EMR1) is an eosinophil-associated protein (36) and CD15, a marker for human neutrophils because it expression on a subset of lymphocytes and lung epithelial cells (data not shown). These Abs can react with other structures (e.g., HAM56 and CD163 with endothelium and CD68 with fibroblasts), but endothelium is not abundant in granulomas and fibroblasts associated with granulomas did not stain positively for CD68; data not shown). Staining patterns for CD11c+,CD68+,CD163+,andHAM56+ cells were morphologically consistent with macrophage-like cells (Supple- mental Fig. 1). The Ab Mac387 is often described as a macrophage FIGURE 1. Immunohistochemical identification of arginase and NOS marker, yet in cynomolgus macaques, the morphology of calprotectin- isoforms in cells from cynomolgus macaque granulomas. (A) Epithelioid expressing cells was more neutrophil-like. Subsequent experiments macrophages, as depicted by H&E staining (H&E) were stained for Arg1, identified calprotectin-bright cells in cynomolgus macaques as neu- Arg2, iNOS, and eNOS (green) and nuclei (blue) and imaged at 3600 trophils (Supplemental Fig. 2). original magnification to represent the unique staining patterns associated with each enzyme. Each panel presents an independent set of epithelioid Myeloid cell populations in macaque and human granulomas macrophages in the macrophage region. Scale bar, 20 mm. (B) Coex- pression of Arg1 (red) and iNOS (green) in CD163+ macrophages (blue, In this study, we imaged granulomas representing the types com- top panel) and in cells with segmented neutrophil-like nuclei (arrowheads, monly seen in cynomolgus macaques (5) with active TB to deter- bottom panel). Image acquired at 3600 original magnification. mine whether different, antigenically defined populations of The Journal of Immunology 777 Downloaded from

FIGURE 2. Granulomas contain higher levels of arginase and NOS activity and gene expression than uninvolved lung tissue. Arginase and NOS enzyme activity is from independent experiments using tissues from different animals. (A) Relative transcript quantification in which granuloma-containing tissues (n = 8) are compared against relative transcript abundance in uninvolved tissues (n = 4) from four animals. Data represent the mean 6 SEM. (B) Arginase activity was measured from four uninvolved tissues and six granulomas from five monkeys. Statistical comparison by the Mann–Whitney U test. (C) NOS http://www.jimmunol.org/ activity was measured from 7 uninvolved tissues (2) and 35 granulomas (+) from 6 animals (A, m23210; B, m21410; C, m22210; D, m5210; E, m22510; F, m22910) showing a trend in which involved tissues have more functional enzyme than uninvolved tissue. (D) Aggregated data from (C) indicating granulomas have significantly higher mean NOS activity than uninvolved tissues. Statistical comparison by the Mann–Whitney U test. (E) Immunohis- tochemical staining of a necrotic granuloma showing strong nitrotyrosine staining at the epithelioid macrophage–caseum interface (indicated by a dashed line) with significant numbers of macrophages and some cells having segmented neutrophil-like nuclei (arrows) that stain positively for nitrotyrosine (red). Scale bar, 10 mm. macrophages exist in different microenvironments and how the levels of organization (Fig. 3B). The position of iNOS+ cells was position of these cells correlate with bacterial localization. In ad- similar to that observed for HAM56 expression; less organized, dition to markers of different macrophage populations, we exam- nonnecrotic granulomas had randomly distributed iNOS expres- by guest on September 26, 2021 ined the NOS and arginase expression by region to determine how sion (Fig. 3C), whereas increasingly organized granulomas had microenvironments correlate with macrophage function. Macro- foci of iNOS expression that was consistent with the position of phage activation states in primates are complex and poorly under- epithelioid macrophages (data not shown). Similarly, eNOS ex- stood, so although these markers may reflect a functional capacity, pression most strongly correlated with the presence and location we realize they cannot fully describe the full spectrum of macro- of epithelioid macrophages (Fig. 3D). Arg1-expressing cells were phage polarization. We also imaged fibrocalcific granulomas as- identified but did not localize to any particular region in these sociated with clinically latent TB to compare the organization and granulomas. Relatively few Arg2-expressing cells were identified, population structure of macrophage subsets in healed lesions with and most of these appeared to be in macrophages at the granu- active lesions from animals that poorly controlled TB. To compare loma’s outer edge and in neutrophils dispersed throughout the the nonhuman primate data with human data, we obtained deiden- granuloma (Fig. 3E). tified samples from patients undergoing lung resection for recal- citrant TB; these patients are likely to have had long courses of Caseous necrotic and suppurative granulomas infection and may have undergone several rounds of treatment. Necrotic and suppurative granulomas (Fig. 4) have similar features Under these circumstances, the pathology can be more complex (e.g., well-defined lymphocyte cuffs and epithelioid macrophage- than in macaques and can include extensive fibrosis. In fact, lung rich regions), but caseous granulomas have noncellular necrotic tissue samples obtained contained multiple granulomas that were centers and suppurative granulomas have centers nearly com- often highly fibrotic; we selected nonnecrotic and necrotic granu- pletely infiltrated by neutrophils. Although we consider necrotic lomas with the lowest amount of fibrosis for imaging and were and suppurative granulomas to be defined morphotypes, they are mindful that these lesions represent those from chronic, poorly grouped together in this study because of their similarities. controlled disease hosts. CD163+ macrophages were most abundant in peripheral regions of granulomas adjacent to, or inside, the lymphocyte cuff (Figs. 4B, Nonnecrotic granulomas 4G, 5A, 5D). Clusters of strongly CD11c+CD68+CD163+ alveolar Nonnecrotic granulomas were characterized by dense macrophage macrophage-like cells were often immediately adjacent to the populations without necrotic areas (Fig. 3A). CD68+ and CD163+ lymphocyte cuff (Fig. 5A, arrowheads). Epithelioid macrophages macrophages were most abundant at the periphery, although were CD1632 or CD163dim but often strongly expressed CD68 macrophages with lower expression of these markers could be (Fig. 4D, 4I, 5A, 5C) and CD11c (Fig. 5B). HAM56+ foamy mac- found throughout these granulomas (Fig. 3B, 3D). Neutrophils rophages were most abundant on the rim of the caseous or neu- were randomly distributed throughout nonnecrotic granulomas trophilic center (Fig. 4B, 4G) with more elongate cellular mor- (Fig. 3B). HAM56+ foamy macrophages were present but were phology extending perpendicular to the center of the granuloma. especially abundant in central regions of granulomas with higher Calprotectin+ neutrophils were present in the lymphocyte cuff and 778 MYELOID CELL NOS AND ARGINASE ISOFORMS IN PRIMATE GRANULOMAS Downloaded from

FIGURE 3. Macrophage phenotypes and distribution in nonnecrotic granulomas from macaques with active TB. Individual panels show serial 5-mm– http://www.jimmunol.org/ thick sections of a representative nonnecrotic granuloma. (A) H&E staining (top panel) with a pseudocolored representation (bottom panel) indicating lymphocyte-rich (cyan) and epithelioid macrophage-rich (purple) regions. Black box indicates the region depicted at higher magnification in (C)–(E). (B) Macrophage-specific stains including HAM56 (red), CD163 (green), and calprotectin-stained neutrophils (blue). White box indicates the region depicted at higher magnification in (C)–(E). (C) iNOS (green) and Arg1 (red) expression with nuclei (blue). (D) CD68 (red) and eNOS (green) expression with nuclei (blue). (E) Arg2 (green) expression with nuclei (blue). Scale bars, 100 mm. macrophage-rich regions but were most abundant in areas near the These granulomas commonly contain mineralized material sur- HAM56+ foamy macrophages adjacent to the caseum (Fig. 4B) and rounded by fibrotic tissue and limited numbers of lymphocytes and centers of suppurative granulomas (Fig. 4G). Arg1 expression was macrophages (Fig. 6A). Many of the cells in the fibrotic region by guest on September 26, 2021 observed throughout the cellular regions of granulomas (Fig. 4C, surrounding mineralized centers expressed CD163 (Fig. 6B). These 4H), but was most strongly expressed by cells in the lymphocyte cuff granulomas contained very few neutrophils (Fig. 6B), HAM56+ (Fig. 5F). Epithelioid macrophages and lymphocyte cuff macro- (Fig. 6B) and CD68+ (Fig. 6D) macrophages, or eNOS-expressing phages also expressed iNOS (Figs. 4C, 4H, 5E), with more iNOS macrophages (Fig. 6D). iNOS and Arg1 were expressed in the cells signal observed in lymphocyte cuff region. The lymphocyte cuff closest to the mineralized material (Fig. 6C). Arg2 expression was region is significantly more cellular than the epithelioid macrophage minimal, but strong signal was noted at the fibrosis–mineral in- region (mean density, 6373 nuclei/mm2 versus 4459 nuclei/mm2, terface (Fig. 6E). respectively; p = 0.0008, Mann–Whitney U test; n = 9 granulomas) Bacteria are present in multiple areas in the granuloma and contains a residual population of epithelial cells that are iNOS rich; consequently, this figure may overrepresent macrophage iNOS The localization of bacterial populations is poorly understood for expression in the lymphocyte cuff region. When the ratio of iNOS/ primate granulomas. We stained granulomas for the presence of Arg1 signal intensity from epithelioid macrophages and lymphocyte mycobacterial cell wall components with auramine-rhodamine cuff regions was calculated, epithelioid macrophages expressed with overlaid H&E-stained features to correlate the positions of significantly more iNOS relative to Arg1 than cells in the lymphocyte bacteria and different macrophage populations. Ongoing studies cuff region (Fig. 5G), suggesting that Arg1 expression differentiates in our laboratory where homogenized granulomas are plated for NO-generating capacity of macrophages in the epithelioid macro- bacterial culture indicate that numbers of bacteria per granuloma phage and lymphocyte cuff regions. are variable and generally low, ranging from sterile granulomas to 6 Strong eNOS expression was also noted in epithelioid macro- 10 bacteria/granuloma (J.L.F. and P.L.L., manuscript in prepa- phages, with the majority of eNOS associated with the plasma ration). M. tuberculosis were often not abundant in granulomas, membrane (Fig. 4D, 4I). We also observed smaller cells in the with most granulomas having either no or very small numbers of lymphocyte cuff that were strongly positive for cytoplasmic eNOS visible bacteria per 5-mm tissue section. In granulomas in which but did not stain for either macrophage or endothelial markers bacteria were visible, bacilli were present in a variety of locations, (CD31, von Willebrand factor; data not shown). Arg2 expression including in epithelioid macrophages (Fig. 7A), intermixed with was largely associated with neutrophils at the border of the caseum neutrophils at the caseum–macrophage interface (Fig. 7A), and in and in the center of suppurative granulomas (Fig. 4E, 4J). giant cells at the granuloma’s periphery (Fig. 7B). Bacteria were most commonly present as small groups of individual bacilli as- Fibrocalcific granulomas sociated with necrotic appearing cells at the caseum–macrophage Small numbers of fibrocalcific granulomas are commonly found in interface. Small numbers of individual bacilli were also occa- latently infected hosts, but also in active TB, and are likely to rep- sionally visible deep in the necrotic regions of caseous lesions resent the successful outcome of an effective immune response. (Fig. 7B). Auramine-rhodamine–stained objects that did not ap- The Journal of Immunology 779 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Macrophage phenotypes and distribution in necrotic and suppurative granulomas from macaques with active TB. Individual panels show serial 5-mm–thick sections of representative necrotic and suppurative granulomas. H&E staining (top panel) of necrotic (A) and suppurative (F) granulomas with pseudocolored representation (bottom panel), indicating lymphocyte-rich (cyan), epithelioid macrophage-rich (purple), and necrotic [yellow, (A)] or [suppurative yellow, (F)] regions. Black boxes indicate the regions depicted at higher magnification in (C)–(E) and (G)–(J). (B and G) Macrophage-specific stains including HAM56 (red), CD163 (green), and calprotectin-stained neutrophils (blue). White box indicates the region depicted at higher magnification in (C)–(E). (C and H) iNOS (green) and Arg1 (red) expression with nuclei (blue). (D and I) CD68 (red) and eNOS (green) expression with nuclei (blue). (E and J) Arg2 (green) expression with nuclei (blue). Scale bars, 100 mm. pear to be intact bacteria were also occasionally visible in epi- Nonnecrotic and necrotic granulomas from humans are similar thelioid macrophages adjacent to foamy macrophages with elon- to cynomolgus macaques with respect to macrophage subset, gated morphologies consistent with foamy HAM56+ cells (Fig. 7C). NOS, and arginase localization Interestingly, fibrotic granulomas from latent disease contained a The organization of macrophage subsets in nonnecrotic and ne- ring of auramine-rhodamine–stained mycolic acid at the fibroblast- crotic human granulomas was grossly similar to macaque gran- collagen–transformed caseum interface (Fig. 7D), suggesting per- ulomas. In nonnecrotic human granulomas (Fig. 8A), CD163+ sistence and diffusion of bacterial Ag after most of the bacteria macrophages were present throughout the granuloma, with cells in appeared to have been cleared. the outer regions commonly coexpressing HAM56 (Fig. 7B). 780 MYELOID CELL NOS AND ARGINASE ISOFORMS IN PRIMATE GRANULOMAS

FIGURE 5. Region-specific expression of macrophage markers iNOS and Arg1 in necrotic granulomas. (A) A necrotic granuloma stained for CD11c (red), CD68 (green), and CD163 (blue) showing distinct stratification of cell populations. Arrowheads indicate clusters of CD11c+CD68+CD163+ alveolar Downloaded from macrophage-like cells. Original magnification 3100. Scale bar, 100 mm. (B–G) Image analysis of necrotic granulomas comparing fluorescence signal intensity of epithelioid macrophage and lymphocyte cuff regions in necrotic granulomas from four monkeys [(B)–(D), n = 37 fields from 18 granulomas; (E)–(G), n = 30 fields from 18 granulomas]. The fields imaged for (B)–(D) are different from panels (E)–(G) but come from granulomas in the same tissue section. (B) CD11c. (C) CD68. (D) CD163. (E) iNOS. (F) Arg1. (G) Ratio of iNOS/Arg1 signal intensity showing decreased Arg1 expression in epithelioid macrophage region relative to the lymphocyte cuff. Pairwise comparisons by the Wilcoxon matched-pairs signed-rank test. *p = 0.062, **p , 0.0001.

Necrotic granulomas (Fig. 8F) had large numbers of CD163+ granulomas strongly expressed CD68 (Fig. 8D, 8I). Clusters of http://www.jimmunol.org/ HAM56+ macrophages in the peripheral tissue, whereas cells in macrophages in the peripheral tissue surrounding these granulo- the lymphocyte cuff were more likely to be CD163+HAM562 mas were HAM56+CD68+CD163+ (arrows, Fig. 8G, 8I), whereas (Fig. 8G). Although HAM56 staining adjacent to the caseum was these clusters in macaques were HAM562CD68+CD163+ (data observed, it was not as intense or as distinct as it was in macaque not shown). Calprotectin-expressing neutrophils were present, and granulomas. Epithelioid macrophages in nonnecrotic and necrotic their distribution in nonnecrotic granulomas was similar to that by guest on September 26, 2021

FIGURE 6. Macrophage phenotypes and distribution in fibrocalcific granulomas from macaques with latent TB. Individual panels show serial 5-mm– thick sections of a representative necrotic granuloma. (A) H&E (top panel) with pseudocolored representation (bottom panel) indicating the outer fibrotic region (cyan), fibrocalcific interface (purple), and central region containing mineralized material (yellow) that shattered during cutting. Dashed line indicates portion of the fibrocalcific interface that is separated from the rest of the granuloma and is present as an artifact in (A). Black box indicates the region depicted at higher magnification in (C)–(E). (B) Macrophage-specific stains including HAM56 (red), CD163 (green), and calprotectin-stained neutrophils (blue). Gray line outlines the tissue edge denoted by the dashed line in (A) where the fibrocalcific interface has separated from the surrounding tissue and reflected over the other side. White box indicates the region depicted at higher magnification in (C)–(E). (C) iNOS (green) and Arg1 (red) expression with nuclei (blue). (D) CD68 (red) and eNOS (green) expression with nuclei (blue). (E) Arg2 (green) expression with nuclei (blue). Scale bars, 100 mm. The Journal of Immunology 781 Downloaded from http://www.jimmunol.org/

FIGURE 7. Bacteria and bacterial Ags can be detected in granulomas from active and latent disease. Auramine-rhodamine images (red images overlaid on grayscale H&E backgrounds) indicating the presence of mycobacteria in granulomas come from regions indicated by white boxes in the colored H&E- stained granuloma. Auramine-rhodamine stains come from the same tissue section as the H&E-stained image. (A) A necrotic granuloma with large numbers of neutrophils infiltrating into the caseum showing bacteria in epithelioid macrophages (top panel) and admixed with macrophage and neutrophils (bottom panel). (B) A necrotic granuloma with bacilli in giant cells in the granuloma periphery (top panel) and in the caseum (bottom panel). (C) A necrotic by guest on September 26, 2021 granuloma showing mycobacterial Ags and bacilli in epithelioid macrophages (top panel) and adjacent to foamy macrophages and mycobacterial Ags in the caseum (bottom panel). (D) A fibrotic granuloma from an animal with latent TB showing residual mycobacterial Ags at the interface of highly fibrotic tissue (top and bottom panels). Scale bars, 200 mm. observed in macaque granulomas (Fig. 8B). Although we ob- surrounding the spatial organization of macrophage subsets in served neutrophils in necrotic granulomas, they were not as nu- granulomas and whether macrophages have microenvironment- merous as they were in macaque granulomas and did not appear to specific homeostatic or bactericidal functions. Much of what we accumulate at the macrophage–caseum interface (Fig. 8G). know about granuloma macrophages comes from animal models iNOS was expressed at low levels by macrophages distributed that may not represent the spectrum of pathology seen in humans or throughout human nonnecrotic granulomas or adjacent to caseum in has been derived from cells removed from the context of the necrotic granulomas (Fig. 8C, 8H). Unidentified cells expressing granuloma. To address these questions, we used immunohisto- high levels of iNOS were present in regions of nonnecrotic granu- chemistry to clarify the interplay of microenvironment and mac- lomas and in the lymphocyte cuff of necrotic granulomas. Similarly rophage biology by identifying macrophage subsets and arginase stained cells were observed in some macaque granulomas. CD68+ and NOS expression in granulomas from cynomolgus macaques, macrophages in human granulomas frequently expressed eNOS a nonhuman primate that recapitulates human TB (4). We found (Fig. 8D, 8I), and clusters of HAM56+CD68+CD163+ macrophages that granulomas have macrophage subsets that are stratified into in the tissue adjacent to granulomas stained particularly strong for pro- and anti-inflammatory regions with the implication that this eNOS (arrows). Arg1 expression was similar that seen in macaque organization may limit immunopathogenic antimicrobial activity granulomas: Arg1 staining was visible throughout cellular regions to bacteria-rich microenvironments by surrounding them with a of granulomas with little evidence for region-specific expression layer of cells with anti-inflammatory phenotypes. and Arg1 expression frequently occurred in cells that also expressed The importance of NOS in human TB is controversial (36–38). iNOS (Fig. 8C, 8H). As has been previously reported (25), there was In mice, elimination of NO leads to higher bacterial numbers and little Arg2 expressed in human granulomas. Human granulomas decreased survival time (9, 38). M. tuberculosis has limited sen- contained scattered Arg2-expressing cells, but Arg2 staining did not sitivity to NO-mediated killing (39–41), and mice with functional appear to be as tightly correlated with neutrophils as it was in ma- iNOS still die of TB, demonstrating that nitrogen radical pro- caque granulomas (Fig. 8E, 8J). duction alone does not correlate with protection. Humans with TB also express iNOS (12–14), but even though they do not generate Discussion NO as vigorously as mice, the human immune system is better at Large populations of macrophages are a prominent feature of containing M. tuberculosis, and most infections do not progress to tuberculous granulomas, yet there are many unanswered questions active TB. Our data demonstrate that macaque macrophages can 782 MYELOID CELL NOS AND ARGINASE ISOFORMS IN PRIMATE GRANULOMAS Downloaded from

FIGURE 8. Macrophage phenotypes and distribution in nonnecrotic and necrotic human granulomas. (A–E) indicate images of a nonnecrotic granuloma. (F–J) indicate images of a necrotic granuloma. Black boxes (H&E images) and white boxes (HAM56, CD163, calprotectin images) denote areas shown in the higher magnification Arg1/iNOS, CD68/eNOS, and Arg2 panels. (A and F) H&E staining. (B and G) Macrophage-specific stains including HAM56 (red), CD163 (green), and calprotectin-stained neutrophils (blue). (C and H) iNOS (green) and Arg1 (red) expression with nuclei (blue). (D and I) CD68 (red) and eNOS (green) expression with nuclei (blue). Arrows indicate clusters of CD68+eNOS+ cells adjacent to the granuloma. (E and J) Arg2 (green) expression with nuclei (blue). Scale bars, 50 mm. http://www.jimmunol.org/ express NOS, and granulomas have higher eNOS and iNOS ex- healing macrophages (52). It has been demonstrated that conver- pression than uninfected lung tissue. Epithelioid macrophages can sion of arginine to proline in anti-inflammatory macrophages is be associated with M. tuberculosis bacilli in granulomas and iNOS dependent on Arg1 (53), consistent with the notion that arginase- and eNOS expression, with low levels of Arg1 expression, sug- derived ornithine may promote synthesis of collagen (leading to gests that NO production by these cells is an active component of fibrosis) in tuberculous granulomas. However, ablation of mac- the anti–M. tuberculosis response in macaques. Fibrocalcific rophage Arg1 expression resulted in increased fibrosis in mice granulomas associated with latent M. tuberculosis infection also infected with Schistosoma mansoni (20), indicating that fibrosis by guest on September 26, 2021 had iNOS-positive cells, implying that even successful immune may not be enhanced by Arg1 in all circumstances. Arginase responses during latency retain low levels of iNOS expression. expression is correlated with decreased protection against acute This population of iNOS-expressing cells remains in place, pre- M. tuberculosis infection in mice (23, 24), likely reflecting de- sumably either because stimulus is provided by residual myco- pletion of L-arginine substrate for NO synthesis. Protective im- bacterial Ags or the low numbers of viable bacilli that may be mune responses against M. tuberculosis may require both present in these lesions. The surprising amount of eNOS in proinflammatory macrophages with bactericidal activity and pro- granulomas in active disease brings up the question of whether it healing anti-inflammatory macrophages to limit immunopathol- participates in protection against M. tuberculosis. Its localization ogy. Moreover, primate granulomas are highly organized, and to plasma membranes instead of phagosome-like structures sug- protection would be contingent upon appropriate spatial expres- gests it may not be appropriately positioned to target intracellular sion of NOS and arginase expression. Because of this, we hy- bacteria. Alternatively, eNOS produces superoxide when uncou- pothesized Arg12NOS+ macrophages would be present in bacilli- + 2 pled if tetrahydrobiopterin or L-arginine are limiting (42, 43) or rich microenvironments and Arg1 NOS macrophages along the through protein kinase Cz–mediated processes following exposure outer margins. Instead, we found substantial coexpression of NOS to hypochlorous acid (44). Superoxide in the presence of NO and arginase throughout necrotic granulomas and elevated Arg1 generates peroxynitrite, which is lethal to M. tuberculosis (45). expression in the lymphocyte cuff region, indicating the ratio of There remains much to be learned about epithelioid macrophage NOS/arginase expression is most likely to be a factor determining biology in situ, and eNOS expression may serve alternative functional macrophage polarity in primates. The paucity of Arg1 functions that are not directly bactericidal but still contribute to expression in epithelioid macrophages also suggests that compe- protection. However, NO may also downregulate immune responses tition for L-arginine by Arg1 may be a critical posttranscriptional in the granuloma; for example, by inhibiting T cell functions as has determinant of macrophage polarity and NOS activity in primate been demonstrated in other experimental systems (20, 46–48) tuberculous granulomas. The abundance of L-arginine–using or by limiting inflammasome activation and subsequent IL-1b enzymes in granulomas may also lead to L-arginine depletion. secretion (49). Consequences of this depletion on T cell function may include Arginases can compete with NOS for L-arginine, thereby downregulation of T cell TCR-z expression (46, 47), activation (47, downregulating NO production and generating L-ornithine, an 48), proliferation (47, 48) and cytokine secretion (20, 47). Thus, amino acid that can be used for proline synthesis (18), which is arginase and NOS expression may decrease proinflammatory T cell used for synthesis of proline-rich proteins such as collagen in responses and modulate macrophage function. granulomas, wound healing, and fibrotic tissues or for polyamine Macrophage diversity and spatial organization of cells within synthesis (18). In mice, Arg1 expression occurs in M2-polarized granulomas are significant yet underappreciated aspects of the (anti-inflammatory) macrophages (16, 50, 51) and in wound biology of TB. Alveolar macrophages in cynomolgus macaques are The Journal of Immunology 783 predominately CD11c+CD68+CD163+, but macrophage phenotypes type in a particular microenvironment and techniques to promote in the granuloma are considerably more complex. CD163 expres- specific macrophage phenotypes may provide important avenues for sion delineated two macrophage subsets: CD11c+CD68+CD163+/ immunotherapeutic treatment of TB. CD682CD163+ cells and CD68+CD163dim/CD68+CD1632 cells. CD163 expression has been identified as a marker of alternative Acknowledgments activation (M2) (51, 54), and the abundance of these cells in outer We thank Jennifer Linderman and Nicholas Clifone for critical reading of regions of granulomas are phospho-STAT3 positive (data not the manuscript and helpful discussions. We also thank the patients and staff shown), an indicator of IL-10 signaling in alternatively activated of National Masan Tuberculosis Hospital for time and participation in our macrophages (55, 56), and have a lower ratio of iNOS/Arg1 ex- studies and the Flynn laboratory for helpful discussions and excellent veter- pression relative to epithelioid macrophages suggests these cells inary staff. may not be actively bactericidal. In contrast, CD11c+CD68+CD1632 epithelioid macrophages are present in regions with the largest Disclosures number of bacteria or bacterial Ags and had classically activated The authors have no financial conflicts of interest. NOS-expressing phenotypes. HAM56, a reported marker for foamy macrophages (57–59), was also associated with cells at the References epithelioid macrophage–caseum interface, a position similar to the 1. Lawn, S. D., and A. I. Zumla. 2011. Tuberculosis. Lancet 378: 57–72. location of Oil Red O–stained macrophages in human granulomas 2. 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Similarly, granulo- Boca Raton, FL, p. 53–81. mas from cynomolgus macaques with poorly controlled TB can 7. Marino, S., J. J. Linderman, and D. E. Kirschner. 2011. A multifaceted approach to modeling the immune response in tuberculosis. Wiley Interdiscip Rev Syst Biol contain large numbers of neutrophils (5), oftentimes in close prox- Med 3: 479–489. imity to bacilli. M. tuberculosis is resistant to neutrophil-mediated 8. Ray, J. C., J. L. Flynn, and D. E. Kirschner. 2009. Synergy between indi- killing (61, 64), and neutrophils in the airways of TB patients vidual TNF-dependent functions determines granuloma performance for controlling Mycobacterium tuberculosis infection. J. Immunol. 182: 3706– by guest on September 26, 2021 commonly contain replicating M. tuberculosis (65). Moreover, 3717. neutrophil cytosol is rich in calprotectin, an antimicrobial peptide 9. Adams, L. B., M. C. Dinauer, D. E. Morgenstern, and J. L. 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