Mycobacterial Trehalose 6,6′-Dimycolate–Induced M1-Type Inflammation
2019; Elsevier BV; Volume: 190; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2019.10.006
ISSN1525-2191
AutoresT Nguyen, John d’Aigle, Luis Chinea, Zainab Niaz, Robert L. Hunter, Shen‐An Hwang, Jeffrey K. Actor,
Tópico(s)Adolescent and Pediatric Healthcare
ResumoMurine models of Mycobacterium tuberculosis (Mtb) infection demonstrate progression of M1-like (proinflammatory) and M2-like (anti-inflammatory) macrophage morphology following primary granuloma formation. The Mtb cell wall cording factor, trehalose 6,6′-dimycolate (TDM), is a physiologically relevant and useful molecule for modeling early macrophage-mediated events during establishment of the tuberculosis-induced granuloma pathogenesis. Here, it is shown that TDM is a major driver of the early M1-like macrophage response as seen during initiation of the granulomas of primary pathology. Proinflammatory cytokines tumor necrosis factor-α, IL-1β, IL-6, and IL-12p40 are produced in lung tissue after administration of TDM to mice. Furthermore, CD11b+CD45+ macrophages with a high surface expression of the M1-like markers CD38 and CD86 were found present in regions of pathology in lungs of mice at 7 days post-TDM introduction. Conversely, only low phenotypic marker expression of M2-like markers CD206 and EGR-2 were present on macrophages. These findings suggest that TDM plays a role in establishment of the M1-like shift in the microenvironment during primary tuberculosis. Murine models of Mycobacterium tuberculosis (Mtb) infection demonstrate progression of M1-like (proinflammatory) and M2-like (anti-inflammatory) macrophage morphology following primary granuloma formation. The Mtb cell wall cording factor, trehalose 6,6′-dimycolate (TDM), is a physiologically relevant and useful molecule for modeling early macrophage-mediated events during establishment of the tuberculosis-induced granuloma pathogenesis. Here, it is shown that TDM is a major driver of the early M1-like macrophage response as seen during initiation of the granulomas of primary pathology. Proinflammatory cytokines tumor necrosis factor-α, IL-1β, IL-6, and IL-12p40 are produced in lung tissue after administration of TDM to mice. Furthermore, CD11b+CD45+ macrophages with a high surface expression of the M1-like markers CD38 and CD86 were found present in regions of pathology in lungs of mice at 7 days post-TDM introduction. Conversely, only low phenotypic marker expression of M2-like markers CD206 and EGR-2 were present on macrophages. These findings suggest that TDM plays a role in establishment of the M1-like shift in the microenvironment during primary tuberculosis. Dogma in tuberculosis pathology is the notion that induction of a strong T helper 1 lymphocyte (Th1) phenotype is critical in maintaining protective health during primary mycobacterial infection.1Orme I.M. Robinson R.T. Cooper A.M. The balance between protective and pathogenic immune responses in the TB-infected lung.Nat Immunol. 2015; 16: 57-63Crossref PubMed Scopus (183) Google Scholar,2Jasenosky L.D. Scriba T.J. Hanekom W.A. Goldfeld A.E. 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Macrophage heterogeneity and plasticity in tuberculosis.J Leukoc Biol. 2019; 106: 275-282Crossref PubMed Scopus (50) Google Scholar The primary stages of mycobacterial infection manifest with a robust proinflammatory response to effectively mitigate organism dissemination. Indeed, these host responses induce hemorrhagic inflammation and vascular occlusion,12Turken O. Kunter E. Sezer M. Solmazgul E. Cerrahoglu K. Bozkanat E. Ozturk A. Ilvan A. Hemostatic changes in active pulmonary tuberculosis.Int J Tuberc Lung Dis. 2002; 6: 927-932PubMed Google Scholar,13Sezer M. Ozturk A. Ilvan A. Ozkan M. Uskent N. The hemostatic changes in active pulmonary tuberculosis.Turk J Haematol. 2001; 18: 95-100PubMed Google Scholar that coincide with changes to fibrinolytic activity and thrombosis,14Kager L.M. Blok D.C. Lede I.O. Rahman W. Afroz R. Bresser P. van der Zee J.S. Ghose A. Visser C.E. de Jong M.D. Tanck M.W. Zahed A.S. Alam K.M. Hassan M. Hossain A. Lutter R. Veer C.V. Dondorp A.M. 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Multiple models of the TDM-induced pulmonary granulomatous response have been studied in mice, all of which have common features to those identified in early acute primary immunopathology of the human host. Initially, Bloch and Noll17Bloch H. Noll H. Studies on the virulence of Tubercle bacilli; the effect of cord factor on murine tuberculosis.Br J Exp Pathol. 1955; 36: 8-17PubMed Google Scholar (1955) utilized cord factor to mimic pathologies seen in early primary human disease, including changes to fibrinolytic activity12Turken O. Kunter E. Sezer M. Solmazgul E. Cerrahoglu K. Bozkanat E. Ozturk A. Ilvan A. Hemostatic changes in active pulmonary tuberculosis.Int J Tuberc Lung Dis. 2002; 6: 927-932PubMed Google Scholar,13Sezer M. Ozturk A. Ilvan A. Ozkan M. Uskent N. The hemostatic changes in active pulmonary tuberculosis.Turk J Haematol. 2001; 18: 95-100PubMed Google Scholar and thrombosis.14Kager L.M. Blok D.C. Lede I.O. Rahman W. Afroz R. Bresser P. van der Zee J.S. Ghose A. Visser C.E. de Jong M.D. Tanck M.W. Zahed A.S. Alam K.M. Hassan M. Hossain A. Lutter R. Veer C.V. Dondorp A.M. Meijers J.C. van der Poll T. Pulmonary tuberculosis induces a systemic hypercoagulable state.J Infect. 2015; 70: 324-334Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Perez et al18Perez R.L. Roman J. Staton Jr., G.W. Hunter R.L. Extravascular coagulation and fibrinolysis in murine lung inflammation induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.Am J Respir Crit Care Med. 1994; 149: 510-518Crossref PubMed Scopus (48) Google Scholar successfully repeated these experiments using purified TDM; Donnachie et al19Donnachie E. Fedotova E.P. Hwang S.A. Trehalose 6,6-dimycolate from mycobacterium tuberculosis induces hypercoagulation.Am J Pathol. 2016; 186: 1221-1233Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar and Hwang et al20Hwang S.A. Byerly C.D. Actor J.K. Mycobacterial trehalose 6,6'-dimycolate induced vascular occlusion is accompanied by subendothelial inflammation.Tuberculosis (Edinb). 2019; 116S: S118-S122Crossref PubMed Scopus (5) Google Scholar built on this using molecular tools to further examine early events in extravascular coagulation. Alternative models of the primary TB response were developed using administered TDM to initiate a synchronized transient granulomatous response21Geisel R.E. Sakamoto K. Russell D.G. Rhoades E.R. In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Guerin is due principally to trehalose mycolates.J Immunol. 2005; 174: 5007-5015Crossref PubMed Scopus (158) Google Scholar, 22Perez R.L. Roman J. Roser S. Little C. Olsen M. Indrigo J. Hunter R.L. Actor J.K. Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.J Interferon Cytokine Res. 2000; 20: 795-804Crossref PubMed Scopus (74) Google Scholar, 23Welsh K.J. Abbott A.N. Hwang S.A. Indrigo J. Armitige L.Y. Blackburn M.R. Hunter Jr., R.L. Actor J.K. A role for tumour necrosis factor-alpha, complement C5 and interleukin-6 in the initiation and development of the mycobacterial cord factor trehalose 6,6'-dimycolate induced granulomatous response.Microbiology. 2008; 154: 1813-1824Crossref PubMed Scopus (56) Google Scholar; these models are extremely effective to assess monocyte-macrophage factors involved in primary granuloma pathogenesis. Indeed, modifications to this model have allowed further insights into how macrophages utilize required receptors to initiate the cytokine cascades,24Bowdish D.M. Sakamoto K. Kim M.J. Kroos M. Mukhopadhyay S. Leifer C.A. Tryggvason K. Gordon S. Russell D.G. MARCO, TLR2, and CD14 are required for macrophage cytokine responses to mycobacterial trehalose dimycolate and Mycobacterium tuberculosis.PLoS Pathog. 2009; 5: e1000474Crossref PubMed Scopus (235) Google Scholar and how these events can push the macrophages to elicit hypersensitive responses.25Guidry T.V. Hunter Jr., R.L. Actor J.K. Mycobacterial glycolipid trehalose 6,6'-dimycolate-induced hypersensitive granulomas: contribution of CD4+ lymphocytes.Microbiology. 2007; 153: 3360-3369Crossref PubMed Scopus (38) Google Scholar,26Yamagami H. Matsumoto T. Fujiwara N. Arakawa T. Kaneda K. Yano I. Kobayashi K. Trehalose 6,6'-dimycolate (cord factor) of Mycobacterium tuberculosis induces foreign-body- and hypersensitivity-type granulomas in mice.Infect Immun. 2001; 69: 810-815Crossref PubMed Scopus (89) Google Scholar The common link between the different models is the relatively rapid initiated proinflammatory macrophage responses elicited in vivo by TDM. Here, the authors desired to further examine the nature of the macrophage phenotype response to TDM, and determine the histopathological evidence to support a hypothesis linking TDM to the presence of recruited macrophage polarization to an M1-like phenotype. Evidence presented in this report uses histologic markers and flow cytometry, immunostaining, and enzyme-linked immunoassay methods to link TDM as a driver for the M1-like macrophage phenotype during recruited induction of the primary granulomatous pathology. Female C57BL/6 mice (Envigo, Houston, TX) were 5 to 6 weeks of age, and weighed approximately 20 g, at study initiation. Animal work was performed at the University of Texas Health Science Center and was approved by the animal welfare committee, according to protocols detailed in approved documents HSC-AWC-16-0140 and HSC-AWC-17-0089. Mycobacterial-derived TDM (cord factor) (Enzo Life Sciences, Farmingdale, NY) was solubilized in hexane/ethanol at a ratio of 9:1. Material was evaporated by a stream of air. The TDM oil/water emulsion was prepared as previously described.23Welsh K.J. Abbott A.N. Hwang S.A. Indrigo J. Armitige L.Y. Blackburn M.R. Hunter Jr., R.L. Actor J.K. A role for tumour necrosis factor-alpha, complement C5 and interleukin-6 in the initiation and development of the mycobacterial cord factor trehalose 6,6'-dimycolate induced granulomatous response.Microbiology. 2008; 154: 1813-1824Crossref PubMed Scopus (56) Google Scholar Briefly, evaporated TDM (25 μg/mouse) was homogenized in Drakeol (2 μL/mouse) (Penreco, Indianapolis, IN). Then 48 μL/mouse of Dulbecco's phosphate buffered solution 1× (cellgro; Corning, Corning, NY) with 0.2% Tween-80 (Mallinckrodt, Hazelwood, MO) was added; the mixture was homogenized in a glass tube with a Teflon pestle for 1 minute to produce an oil/water emulsion. The TDM was intravenously given at a volume of 100 μL per animal. Control mice received material formulated with no addition of TDM in oil. Emulsion-only controls did not exhibit inflammation or exhibit cytokines production at the times described here, as was previously reported and detailed.22Perez R.L. Roman J. Roser S. Little C. Olsen M. Indrigo J. Hunter R.L. Actor J.K. Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.J Interferon Cytokine Res. 2000; 20: 795-804Crossref PubMed Scopus (74) Google Scholar,27Guidry T.V. Olsen M. Kil K.S. Hunter Jr., R.L. Geng Y.J. Actor J.K. Failure of CD1D-/- mice to elicit hypersensitive granulomas to mycobacterial cord factor trehalose 6,6'-dimycolate.J Interferon Cytokine Res. 2004; 24: 362-371Crossref PubMed Scopus (35) Google Scholar All mice were sacrificed at times indicated (7 days after injection of formulated material). Mice were sacrificed, and lungs were immediately perfused with a solution of 1 mmol/L EDTA in Dulbecco's phosphate buffered solution. Lungs were weighed, sectioned, and evaluated for pathology and histologic results. Tissues were fixed in 10% buffered formalin for histology (Fisher Scientific, Pittsburg, PA). Specimens were processed by the Histology Laboratory at the University of Texas Health Science Center McGovern Medical School (Houston, TX); tissues were embedded in paraffin blocks and then 5-μm-thick sections were subsequently stained with hematoxylin (Surgipath, Richmond, IL) and eosin (Richard-Allen Scientific, Kalamazoo, MI). The authors' laboratory utilizes an accepted lung weight index (LWI) that calculates as an approximation of lung inflammation intensity. The following equation was used for calculation of the gross tissue inflammation due to TDM-induced pathology, as used in prior studies22Perez R.L. Roman J. Roser S. Little C. Olsen M. Indrigo J. Hunter R.L. Actor J.K. Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.J Interferon Cytokine Res. 2000; 20: 795-804Crossref PubMed Scopus (74) Google Scholar,28Pelletier M. Forget A. Bourassa D. Gros P. Skamene E. Immunopathology of BCG infection in genetically resistant and susceptible mouse strains.J Immunol. 1982; 129: 2179-2185PubMed Google Scholar,29Collins F.M. Congdon C.C. Morrison N.E. Growth of mycobacterium bovis (BCG) in T lymphocyte-depleted mice.Infect Immun. 1975; 11: 57-64Crossref PubMed Google Scholar:LWI=Lungweight(g)×1000Mouseweight (g)/1010(1) High-resolution scanned images of hematoxylin and eosin–stained slides were scanned for computerized analysis of lung inflammation using Motic Digital Side Assistant software version 1.0.7.44 (Kowloon Bay, Kowloon, Hong Kong). Quantitation of inflammation was performed in two steps using ImageJ software version 1.52o (NIH, Bethesda, MD; https://imagej.nih.gov/ij). Lung area was initially quantified by separation of the image's scale from background. Minimum and maximum values for hue, saturation, and brightness of the image were set as follows: 120, 255; 0, 255; and 0, 255, respectively. Total cell area measurement was calculated using a modified version of the procedure detailed in the online ImageJ stained-sections example directory (NIH, https://imagej.nih.gov/ij/docs/examples/stained-sections/index.html, last accessed September 1, 2019), where the peak threshold was set at 164 for all digitized slides analyzed. Methods were similar to previously published materials.30Schneider C.A. Rasband W.S. Eliceiri K.W. NIH Image to ImageJ: 25 years of image analysis.Nat Methods. 2012; 9: 671-675Crossref PubMed Scopus (34949) Google Scholar Lung inflammation was calculated as a percentage of total area occupied by cell area; values were averaged within treatment groups and normalized to that of group nontreated controls. A weighed section of lung was excised, then homogenized, then incubated at 37°C and 5% CO2 for 4 hours in Dulbecco's Modified Eagle's medium containing 50 μg/mL l-Arginine, 50 μg/mL HEPES, 100 μg/mL penicillin, and 50 μg/mL gentamycin, and 10% fetal bovine serum. Collected supernatants were spun to remove debris, then assessed by enzyme-linked immunoassay. Production of tumor necrosis factor-α (TNF-α), IL-1β, IL-12p40, IL-6, TGF-β, and IL-10 was determined by the manufacturer's instructions (DuoSet kits; R&D Systems, Minneapolis, MN). Supernatants to detect TGF-β were pretreated with a 1:5 supernatant:1N HCl ratio, then neutralized with the same volume of 1.2N NaOH/0.5 mol/L HEPES. The average of duplicate wells was determined using a standard curve produced by reactivity to the manufacturer's supplied recombinant molecules. Detection sensitivity limit was at least 32 pg/mL, according to the manufacturer's product details. The large right lobe of each lung was collected and fixed in 10% buffered formalin. The fixed lung tissue was stained with hematoxylin and eosin using standard procedures. Assessment was performed using immunohistochemistry for integrin family member CD11b (Ab01114-23.0; Absolute Antibody, Wilton, UK),31Yue Y. Huang W. Liang J. Guo J. Ji J. Yao Y. Zheng M. Cai Z. Lu L. Wang J. IL4I1 is a novel regulator of M2 macrophage polarization that can inhibit T cell activation via L-tryptophan and arginine depletion and IL-10 production.PLoS One. 2015; 10: e0142979Crossref PubMed Scopus (57) Google Scholar diluted at 1:2000, was performed according to modification of the manufacturer's instructions (20 minutes at low pH), and subsequently visualized using standard horseradish peroxidase techniques and 3,3′-diaminobenzidine chromogen using Dako reagents (Dako, Agilent, Santa Clara, CA). In a similar manner, M1-like marker CD38 (14-0381-02; Invitrogen, Thermo Fisher Scientific, Waltham, MA), diluted at 1:1000, was used for visualization on serial slide sections. Hematoxylin-counterstained slides were viewed by a trained pathologist, with descriptive results obtained in an experimentally blinded manner (R.L.H.). Lungs were extracted from wild-type and TDM treated mice, homogenized by hand, and underwent a 30-minute incubation with collagenase/hyaluronidase (StemCell Technologies, Vancouver, BC, Canada) and DNAse I (Sigma-Aldrich, St. Louis, MO) in sterile filtered RPMI 1640 medium with 1% 100× pen/strep and 2.5% HEPES (Thermo Fisher Scientific), and 5% heat-inactivated fetal bovine serum (Corning). Enzymatic digestion was quenched with addition of the RPMI mixture before lung tissue was plunged through a 70-μm cell strainer. Cells were pelleted at 4°C for 5 minutes at 450 × g. Cell pellets were resuspended in a 70% Percoll (17-0891-01; GE Healthcare, Chicago, IL) gradient and underlain by 40% Percoll followed by centrifugation at 500 × g for 20 minutes at 22°C with no break and slow acceleration. Cells were collected at the 70–40 Percoll interface before being resuspended in a sterile filtered PBS mixture containing 2% heat-inactivated fetal bovine serum and 1 mL of 0.5 mol/L EDTA (Thermo Fisher Scientific). Surface markers chosen to delineate murine M1-like and M2-like macrophages were based on published data from Jablonski et al,32Jablonski K.A. Amici S.A. Webb L.M. Ruiz-Rosado Jde D. Popovich P.G. Partida-Sanchez S. Guerau-de-Arellano M. Novel markers to delineate murine M1 and M2 macrophages.PLoS One. 2015; 10: e0145342Crossref PubMed Scopus (545) Google Scholar later detailed by Orecchioni et al.33Orecchioni M. Ghosheh Y. Pramod A.B. Ley K. Macrophage polarization: different gene signatures in M1(LPS+) vs. classically and M2(LPS-) vs. alternatively activated macrophages.Front Immunol. 2019; 10: 1084Crossref PubMed Scopus (661) Google Scholar Similar findings were identified in human monocyte-derived macrophages.34Amici S.A. Young N.A. Narvaez-Miranda J. Jablonski K.A. Arcos J. Rosas L. Papenfuss T.L. Torrelles J.B. Jarjour W.N. Guerau-de-Arellano M. CD38 is robustly induced in human macrophages and monocytes in inflammatory conditions.Front Immunol. 2018; 9: 1593Crossref PubMed Scopus (108) Google Scholar Cells were stained in accordance with the manufacturer's recommendation, using Live/Dead Aqua stain (L34966; eBioscience, San Diego, CA) for 15 minutes, then FC (antibody receptor) blocked with Anti-Mo CD16/CD32 (14-0161-86; eBioscience) for 15 minutes. Staining used the following antibodies against specific receptors: CD38 (102728; BioLegend, San Diego, CA) APC/Cy7, CD86 (105014; BioLegend) phycoerythrin (PE)/Cy7, CD206 (141704; BioLegend) fluorescein isothiocyanate, EGR2 (17-6691-82; eBioscience) APC, CD11b (101208; BioLegend,) PE, and CD45 (48-0451-82; eBioscience) eFluor 450 for 20 minutes. Cells were fixed with 2% paraformaldehyde and then evaluated on a Beckman Coulter Cytoflex S flow cytometer (B75442; Beckman Coulter, Indianapolis, IN). Data were then analyzed using FlowJo software version 10 (Becton Dickinson, Franklin Lakes, NJ). Collected data were compared across groups, and against naive mice or mice challenged with vehicle formulated without TDM. Analysis used an unpaired t-test, or used one-way analysis of variance. The differences between means were considered significant at a level of P ≤ 0.05. Generated data points were compiled using GraphPad Prism software version 5.03 (San Diego, CA) and are presented as a representative value obtained from multiple experimental repeats (sets of 2 or 3). Experiments had an N of 4 to 6 mice. C57BL/6 mice injected with Mtb-derived TDM (25 μg/mouse) in oil/water given intravenously developed inflammation in lung tissue (Figure 1). Broad lung inflammation was assessed as LWI at 7 days after i.v. TDM challenge; this time point reflects peak granulomatous response induced by TDM.22Perez R.L. Roman J. Roser S. Little C. Olsen M. Indrigo J. Hunter R.L. Actor J.K. Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.J Interferon Cytokine Res. 2000; 20: 795-804Crossref PubMed Scopus (74) Google Scholar Acute treatment increased LWI significantly. TDM-treated mice had an average LWI of 1.60 ± 0.49 units, compared with naive controls (0.97 ± 0.03 units; P ≤ 0.05). The LWI was similar to computerized assessments of lung inflammation. Analysis of digitized lung histograms post-TDM administration confirmed significant parenchymal inflammation and presence of granulomas throughout pulmonary tissue. Mice administered i.v. TDM showed a focal accumulation of macrophages at day 7 post-injection (Figure 1). The pathologic reactivity demonstrated widespread inflammation and severely reduced open alveolar space. Small focal hemorrhagic petechiae were present as part of the inflammatory response. Lymphocytic infiltration to lung tissue occurred around regions where granulomas coincided with vasculature. Slight hemorrhage was present throughout the tissue. There was visual evidence of occlusion of intermediate or small blood vessels. Activated macrophages with intracellular vesicles were predominant in regions of reactivity; limited-to-negligible accumulation of lymphocytes occurred within the focal response. General edema was not a major component of the response. Naive (no TDM) mice did not demonstrate changes to lung architecture. Lungs from control mice exhibited normal pulmonary parenchyma; there were no noticeable cellular infiltrates, and limited presence of monocytic or leukocytic foci. Administration of TDM results in a strong proinflammatory response.23Welsh K.J. Abbott A.N. Hwang S.A. Indrigo J. Armitige L.Y. Blackburn M.R. Hunter Jr., R.L. Actor J.K. A role for tumour necrosis factor-alpha, complement C5 and interleukin-6 in the initiation and development of the mycobacterial cord factor trehalose 6,6'-dimycolate induced granulomatous response.Microbiology. 2008; 154: 1813-1824Crossref PubMed Scopus (56) Google Scholar Cytokine assessment by enzyme-linked immunoassay confirmed these findings. Lungs were examined at 7 days post-administration of TDM. Significant production of proinflammatory mediators TNF-α, IL-1β, and IL-12p40 was observed relative to control mice; IL-6 was also elevated in the TDM-treated group (Figure 2). Anti-inflammatory mediating cytokines were also evaluated; whereas there was a minor increase in production of TGF-β, it was not a significant change. There was no change, relative to controls, in IL-10 production (Figure 2). The extent of macrophage phenotypic polarization was investigated using flow cytometry. Lungs treated with TDM were dissociated, and individual cells were further examined for the presence of M1-like and M2-like surface markers. Populations were initially gated on infiltrating monocytic macrophages (CD11bhiCD45hi).35Koeniger T. Kuerten S. Splitting the "unsplittable": dissecting resident and infiltrating macrophages in experimental autoimmune encephalomyelitis.Int J Mol Sci. 2017; 18: E2072Crossref PubMed Scopus (15) Google Scholar,36Sheng J. Ruedl C. Karjalainen K. Most tissue-resident macrophages except microglia are derived from fetal hematopoietic stem cells.Immunity. 2015; 43: 382-393Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar Figure 3 depicts data collected for the M1-like markers CD38 and CD86, along with assessment for expression of the M2-like marker CD206 and the early growth response gene-2 (EGR-2). Treatment with TDM resulted in an overall accumulation of CD11bhiCD45hi cells that expressed higher M1-like markers, but not the M2-like surface proteins. Specifically, CD38 was present on 39.50 ± 3.33% on infiltrating macrophages, which was significantly elevated when compared with the nontreated wild-type group controls (7.76 ± 2.24%; P ≤ 0.001). Additionally, CD86 was present on 19.34 ± 1.48% macrophages, compared with nontreated controls (5.31 ± 0.61%; P ≤ 0.001). Figure 3 also lists the values for the markers examined. CD14 was also highly elevated in this population (57.74 ± 4.06%; data not shown). Immunohistochemical staining allowed localization of the CD11b+ macrophage population to regions of granulomatous response (Figure 4A), and specifically to areas of inflammation. Furthermore, staining for the CD38 surface glycoprotein marker on serial sections demonstrated the presence of the M1-like population in a pattern overlapping that for CD11b, coinciding with regions of high macrophage activation within the granulomas architecture (Figure 4B). The CD11b marker is present throughout the inflammatory foci, with diffuse staining in most of the monocytic cells. There is also a number of CD11b+ cells that are heavily stained; the presence of the CD38 marker appears to coincide with cells also expressing the higher levels of CD11b. Of note, not all macrophages within the granuloma exhibit CD38. Cells were also stained with anti-CD206 and anti–EGR-2; however, the presence of these M2-markers was not detected (data not shown). Taken together with the flow analysis, this suggests that the macrophages recruited to the focal inflammation during the pulmonary granulomatous response induced by the TDM are of the M1-like phenotype. The establishment of the Mtb-induced granuloma is a complex interaction of multiple cell phenotypes responding to a myriad of antigenic stimuli.37Dorhoi A. Kaufmann S.H. Versatile myeloid cell subsets contribute to tuberculosis-associated inflammation.Eur J Immunol. 2015; 45: 2191-2202Crossref PubMed Scopus (51) Google Scholar A multitude of studies have focused on mechanisms underlying development of the induced inflammatory response leading to development of this pathology. Clearly, the influence of any one specific molecule may not form an essential pathological bias in vivo during infection. However, a deficiency in production of purified cord factor, trehalose 6,6′-dimycolate,38Copenhaver R.H. Sepulveda E. Armitige L.Y. Actor J.K. Wanger A. Norris S.J. Hunter R.L. Jagannath C. A mutant of Mycobacterium tuberculosis H37Rv that lacks expression of antigen 85A is attenuated in mice but retains vaccinogenic potential.Infect Immun. 2004; 72: 7084-7095Crossref PubMed Scopus (44) Google Scholar or manipulation of its structure,3
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