Trehalose 6,6-Dimycolate from Mycobacterium tuberculosis Induces Hypercoagulation
2016; Elsevier BV; Volume: 186; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2015.12.019
ISSN1525-2191
AutoresElizabeth Donnachie, Elena P. Fedotova, Shen-An Hwang,
Tópico(s)Syphilis Diagnosis and Treatment
ResumoTuberculosis (TB) remains a global health concern. Trehalose 6′6-dimycolate (TDM) activates innate inflammation and likely also stimulates chronic inflammation observed during disease progression. Noninfectious models using purified TDM oil/water emulsions elicit pathologic findings observed in patients with TB. We introduce a new TDM model that promotes inflammatory lung pathologic findings and vascular occlusion and hemorrhage. C57BL/6 and BALB/c mice were injected with 10 μg of i.p. TDM in light mineral oil (TDM-IP). At day 7, another injection of 10 μg of i.v. TDM in oil/water emulsion was given (TDM-IV). The i.p./i.v. TDM (TDM-IVIP) group was compared with mice injected once with i.v. or i.p. TDM. The responses to TDM-IP, TDM-IV, or TDM-IPIV were consistent between mouse strains. Mice that received TDM-IV and TDM-IPIV had inflammatory pathologic findings with increases in inflammatory and T-cell cytokines, and the TDM-IPIV group had further enhancement of IL-10 and granulocyte-macrophage colony-stimulating factor. The TDM-IPIV group had increased CD4+ T cells in lung tissue, significantly increased coagulation, decreased clot formation time, and increased maximum clot firmness. Masson's trichrome staining revealed increased deposition of collagen in the occluded vasculature. TDM-IPIV promotes a hypercoagulopathy state, independent of inflammation. This new model argues that TDM is sufficient to generate the hypercoagulopathy observed in patients with TB. Tuberculosis (TB) remains a global health concern. Trehalose 6′6-dimycolate (TDM) activates innate inflammation and likely also stimulates chronic inflammation observed during disease progression. Noninfectious models using purified TDM oil/water emulsions elicit pathologic findings observed in patients with TB. We introduce a new TDM model that promotes inflammatory lung pathologic findings and vascular occlusion and hemorrhage. C57BL/6 and BALB/c mice were injected with 10 μg of i.p. TDM in light mineral oil (TDM-IP). At day 7, another injection of 10 μg of i.v. TDM in oil/water emulsion was given (TDM-IV). The i.p./i.v. TDM (TDM-IVIP) group was compared with mice injected once with i.v. or i.p. TDM. The responses to TDM-IP, TDM-IV, or TDM-IPIV were consistent between mouse strains. Mice that received TDM-IV and TDM-IPIV had inflammatory pathologic findings with increases in inflammatory and T-cell cytokines, and the TDM-IPIV group had further enhancement of IL-10 and granulocyte-macrophage colony-stimulating factor. The TDM-IPIV group had increased CD4+ T cells in lung tissue, significantly increased coagulation, decreased clot formation time, and increased maximum clot firmness. Masson's trichrome staining revealed increased deposition of collagen in the occluded vasculature. TDM-IPIV promotes a hypercoagulopathy state, independent of inflammation. This new model argues that TDM is sufficient to generate the hypercoagulopathy observed in patients with TB. Tuberculosis (TB) remains a serious health burden to the world's population. The World Health Organization estimates that approximately one-third of the world's populations are infected with Mycobacterium tuberculosis, the causative pathogen of TB disease. In 2013, there were 9 million cases of TB disease and 1.5 million deaths (Centers for Disease Control and Prevention, http://www.cdc.gov/tb/statistics/default.htm, last accessed September 24, 2015). The pathology of TB disease is a complex interaction between pathogen and host immune responses. Understanding these specific mechanisms will lead to improving current therapeutics and vaccines. M. tuberculosis possesses several pathogen components that can stimulate the innate and adaptive immune response. Trehalose 6′6-dimycolate (TDM), the major glycolipid on the cell wall of M. tuberculosis, is a strong inducer of host immunity.1Hunter R.L. Armitige L. Jagannath C. Actor J.K. TB research at UT-Houston–a review of cord factor: new approaches to drugs, vaccines and the pathogenesis of tuberculosis.Tuberculosis (Edinb). 2009; 89: S18-S25Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Geisel 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 The acute immune response to TDM peaks at day 7 with innate lymphocytes and macrophage activation.3Welsh K.J. Hunter R.L. Actor J.K. Trehalose 6,6'-dimycolate–a coat to regulate tuberculosis immunopathogenesis.Tuberculosis (Edinb). 2013; 93: S3-S9Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar During the acute TDM response, several inflammatory cytokines are produced, including tumor necrosis factor (TNF)-α, IL-6, and IL-1β.4Perez 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, 5Welsh 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 Innate cytokines, such as IL-12 and IL-10, produced by activated macrophages are also increased in response to TDM.6Indrigo J. Hunter Jr., R.L. Actor J.K. Influence of trehalose 6,6'-dimycolate (TDM) during mycobacterial infection of bone marrow macrophages.Microbiology. 2002; 148: 1991-1998Crossref PubMed Scopus (91) Google Scholar, 7Indrigo J. Hunter Jr., R.L. Actor J.K. Cord factor trehalose 6,6'-dimycolate (TDM) mediates trafficking events during mycobacterial infection of murine macrophages.Microbiology. 2003; 149: 2049-2059Crossref PubMed Scopus (177) Google Scholar Recently, IL-17 has also been observed to be increased in lung tissue in TDM-injected mice.8Saitoh T. Yano I. Kumazawa Y. Takimoto H. Pulmonary TCR gammadelta T cells induce the early inflammation of granuloma formation by a glycolipid trehalose 6,6'-dimycolate (TDM) isolated from Mycobacterium tuberculosis.Immunopharmacol Immunotoxicol. 2012; 34: 815-823Crossref PubMed Scopus (6) Google Scholar The acute response to TDM resembles the acute phase of M. tuberculosis infection, eliciting macrophage/dendritic cell innate responses to promote development of the adaptive T-cell response. The adaptive response to TDM evokes strong lymphocyte inflammation, promoting large clusters of leukocyte infiltration into lung tissue and high levels of interferon (IFN)-γ. This adaptive response can be transferred to nonsensitized hosts by CD4+ T cells,9Guidry T.V. Hunter Jr., R.L. Actor J.K. CD3+ cells transfer the hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate in mice.Microbiology. 2006; 152: 3765-3775Crossref PubMed Scopus (21) Google Scholar, 10Guidry 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 and it requires the presence of CD1.10Guidry 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, 11Guidry 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 The adaptive response is dominated with high levels of IFN-γ, IL-2, and IL-12, eliciting a strong T-cell helper type 1 response.12McMullen A.M. Hwang S.A. O'Shea K. Aliru M.L. Actor J.K. Evidence for a unique species-specific hypersensitive epitope in Mycobacterium tuberculosis derived cord factor.Tuberculosis (Edinb). 2013; 93: S88-S93Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Thus, TDM is capable of generating an adaptive T-cell helper type 1 immunity and the antigen-specific memory that is observed in M. tuberculosis infection. Macrophages, professional antigen-presenting cells, carry surface recognition molecules for M. tuberculosis components and TDM that allow phagocytosis of M. tuberculosis and TDM particles and generate an innate immune response. The immune mechanism of TDM is mapped to macrophage inducible C-type lectin, dectin-3, and additional C-type lectin receptors to activate NF-κB.13Miyake Y. Toyonaga K. Mori D. Kakuta S. Hoshino Y. Oyamada A. Yamada H. Ono K. Suyama M. Iwakura Y. Yoshikai Y. Yamasaki S. C-type lectin MCL is an FcRgamma-coupled receptor that mediates the adjuvanticity of mycobacterial cord factor.Immunity. 2013; 38: 1050-1062Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 14Schoenen H. Huber A. Sonda N. Zimmermann S. Jantsch J. Lepenies B. Bronte V. Lang R. Differential control of Mincle-dependent cord factor recognition and macrophage responses by the transcription factors C/EBPbeta and HIF1alpha.J Immunol. 2014; 193: 3664-3675Crossref PubMed Scopus (46) Google Scholar, 15Zhao X.Q. Zhu L.L. Chang Q. Jiang C. You Y. Luo T. Jia X.M. Lin X. C-type lectin receptor dectin-3 mediates trehalose 6,6'-dimycolate (TDM)-induced Mincle expression through CARD9/Bcl10/MALT1-dependent nuclear factor (NF)-kappaB activation.J Biol Chem. 2014; 289: 30052-30062Crossref PubMed Scopus (84) Google Scholar, 16Bowdish 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 Macrophage activation by TDM induces an inflammatory response, leading to production of TNF-α.6Indrigo J. Hunter Jr., R.L. Actor J.K. Influence of trehalose 6,6'-dimycolate (TDM) during mycobacterial infection of bone marrow macrophages.Microbiology. 2002; 148: 1991-1998Crossref PubMed Scopus (91) Google Scholar In addition, TDM protects M. tuberculosis from macrophage intracellular killing mechanisms,7Indrigo J. Hunter Jr., R.L. Actor J.K. Cord factor trehalose 6,6'-dimycolate (TDM) mediates trafficking events during mycobacterial infection of murine macrophages.Microbiology. 2003; 149: 2049-2059Crossref PubMed Scopus (177) Google Scholar creating a protective niche for organism growth and antigen production. These unique attributes of TDM, stimulating host inflammation and protecting M. tuberculosis against host cell killing, are thought to be major contributors to the presence of foamy macrophages. These highly activated macrophages in TB disease are hypothesized to be a result of continuous stimulation by M. tuberculosis antigens, specifically TDM. The clinical lung pathologic findings of TB disease remain largely unknown. Although no animal model is capable of reproducing all the clinical components, each animal model is capable of reproducing at least one aspect of the disease observed in human patients. In the 1950s, a mouse model using repeated i.p. injections of TDM in oil17Bloch 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 along with subsequent reports published in 1970s and 1980s revealed that TDM can induce pneumonitis, accompanied with pronounced hemorrhage in the lung tissue.18Goren M.B. Cord factor revisited: a tribute to the late Dr. Hubert Bloch.Tubercle. 1975; 56: 65-71Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 19Sakamoto Y. Goren M.B. Kirkpatrick C.H. Phenotypes of infiltrating cells in trehalose dimycolate-induced interstitial pneumonitis.Infect Immun. 1989; 57: 2098-2106PubMed Google Scholar, 20Seggev J. Goren M.B. Carr R.I. Kirkpatrick C.H. Interstitial and hemorrhagic pneumonitis induced by mycobacterial trehalose dimycolate.Am J Pathol. 1982; 106: 348-355PubMed Google Scholar This TDM-induced pneumonitis has been observed in lung tissues of patients who died of active TB disease. This finding suggests that lipid pneumonia is an important part of TB disease.21Hunter R.L. Actor J.K. Hwang S.A. Karev V. Jagannath C. Pathogenesis of post primary tuberculosis: immunity and hypersensitivity in the development of cavities.Ann Clin Lab Sci. 2014; 44: 365-387PubMed Google Scholar To incorporate these 2 TDM pathologic findings, immune responses and hemorrhagic pneumonia, into a single model, a new model was developed that incorporated the TDM in oil i.p. with the TDM in oil/water emulsion i.v. This new TDM protocol stimulates immune responses that result in development of both inflammation and pneumonitis in lung tissue, simulating the pathologic findings observed during prolong M. tuberculosis infection. This new model can be used as a noninfectious model to investigate the mechanisms of several pathologic findings observed during M. tuberculosis infection. Female C57BL/6 mice and female BALB/c mice (both 6 weeks; both from Harlan Laboratories, Indianapolis, IN) weighing approximately 20 g initially were used. All in vivo experiments were conducted under approved guidelines of the animal ethics committee at the University of Texas, Health Science Center at Houston (HSC-AWC-13-123). M. tuberculosis–derived TDM (cord factor) was purchased from Santa Cruz Biotechnology (Dallas, TX). Drakeol 6VR, light mineral oil, was purchased from Penreco (Dickinson, TX). TDM (10 μg per mouse) was solubilized in hexane:ethanol (ratio of 9:1), the appropriate amount was aliquoted, and hexane:ethanol evaporated under stream of air. TDM (10 μg per mouse) in oil was prepared by homogenizing in Drakeol (100 μL per mouse) with a glass tube and Teflon pestle for 1 minute. The TDM 10 μg per mouse dose was chosen because it was the dose that did not induce severe physical distress in the C57BL/6 and BALB/c mice from preliminary studies (animal husbandry observations). The TDM in oil formulation was injected i.p. The TDM oil/water emulsion was prepared as previously described.5Welsh 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, 22Abbott A.N. Guidry T.V. Welsh K.J. Thomas A.M. Kling M.A. Hunter R.L. Actor J.K. 11beta-hydroxysteroid dehydrogenases are regulated during the pulmonary granulomatous response to the mycobacterial glycolipid trehalose-6,6'-dimycolate.Neuroimmunomodulation. 2009; 16: 147-154Crossref PubMed Scopus (17) Google Scholar The evaporated TDM (10 μg per mouse) was homogenized in Drakeol (2 μL per mouse) for 1 minute. To the TDM-Drakeol mixture, 48 μL per mouse of 1× Dulbecco's phosphate-buffered solution (DPBS; Cellgro; Mediatech Inc, Manassas, VA) with 0.2% Tween 80 (Mallinckrodt, Hazelwood, MO) was added and homogenized for another 2 minutes. The TDM oil/water emulsions were injected i.v. Controls with no TDM in oil and oil/water formulations were also prepared. C57BL/6 and BALB/c mice were treated as follows: i) control: naive, no injections; ii) oil i.p.: no TDM, oil only, injected i.p.; iii) oil/water i.v.: no TDM, oil/water emulsion, injected i.v.; iv) TDM i.p. (TDM-IP): 10 μg per mouse of TDM in Drakeol (100 μL per mouse) injected i.p.; v) TDM i.v. (TDM-IV): 10 μg per mouse of TDM in oil/water emulsion (100 μL per mouse) injected i.v.; and vi) TDM i.p./i.v. (TDM-IVIP: 10 μg per mouse of TDM in Drakeol (100 μL per mouse) injected i.p. and, after 7 days, 10 μg per mouse TDM in Drakeol (100 μL per mouse) were injected i.v. The second TDM injection was given on day 7, at the height of the primary TDM immune response. All mice were sacrificed at day 7 after the last TDM or control formulation injections. On sacrifice, lungs were perfused with 1 mmol/L EDTA in DPBS, weighed, and sectioned to evaluate pathologic findings, cytokine production, and immune cell typing by flow cytometry. Lung weight index (LWI)5Welsh 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, 9Guidry T.V. Hunter Jr., R.L. Actor J.K. CD3+ cells transfer the hypersensitive granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate in mice.Microbiology. 2006; 152: 3765-3775Crossref PubMed Scopus (21) Google Scholar, 10Guidry 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, 11Guidry 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, 22Abbott A.N. Guidry T.V. Welsh K.J. Thomas A.M. Kling M.A. Hunter R.L. Actor J.K. 11beta-hydroxysteroid dehydrogenases are regulated during the pulmonary granulomatous response to the mycobacterial glycolipid trehalose-6,6'-dimycolate.Neuroimmunomodulation. 2009; 16: 147-154Crossref PubMed Scopus (17) Google Scholar, 23Actor J.K. Indrigo J. Beachdel C.M. Olsen M. Wells A. Hunter Jr., R.L. Dasgupta A. Mycobacterial glycolipid cord factor trehalose 6,6'-dimycolate causes a decrease in serum cortisol during the granulomatous response.Neuroimmunomodulation. 2002; 10: 270-282Crossref PubMed Scopus (13) Google Scholar was calculated as a rough measure of lung inflammation intensity using the following equation:LWI=Lungweight(g)×1000Mouseweight(g)/1010(1) A section of lung tissue was homogenized and placed into 2 mL of Dulbecco's modified Eagle's medium containing 50 mg/L of l-arginine, 50 mg/L of HEPES, 10% fetal bovine serum, 100 μg/mL of penicillin, and 50 μg/mL of gentamicin (Sigma-Aldrich, St. Louis, MO). Samples were incubated for 4 hours at 37°C with 5% CO2. The resulting supernatants were analyzed by enzyme-linked immunoassay (ELISA). Levels of cytokines in cell supernatants were measured by a sandwich ELISA according to the manufacturer's instructions (R&D Systems, Minneapolis, MN). Absorbance was read at 570 nm with background subtracted at 450 nm on a plate reader (Molecular Devices, Menlo Park, CA). The lower range limit for detection sensitivity was 16 to 32 pg/mL. Section of lung tissue is processed into a single-cell suspension. Lung section was cut into tiny pieces (<1 mm) and digested in a solution of 1 mg/mL of collagenase type II and 30 μg/mL DNase (Sigma-Aldrich) in 1× DPBS at 37°C, in a shaking incubator, for 1 hour. After digestion, glass slides were used to breakup tissue clumps. Once the bigger clumps settle to the bottom of the tube, the remaining cell suspension was pelleted at 350 × g for 5 minutes. The cell pellet was resuspended in flow staining buffer (1% bovine serum albumin in 1× DPBS). Identification of surface molecule expression by flow cytometric analysis (mean of 20,000 events per sample) was conducted as previously described.24Hwang S.A. Actor J.K. Lactoferrin modulation of BCG-infected dendritic cell functions.Int Immunol. 2009; 21: 1185-1197Crossref PubMed Scopus (39) Google Scholar, 25Hwang S.A. Kruzel M.L. Actor J.K. Influence of bovine lactoferrin on expression of presentation molecules on BCG-infected bone marrow derived macrophages.Biochimie. 2009; 91: 76-85Crossref PubMed Scopus (33) Google Scholar Briefly, samples were blocked by incubating purified anti-CD16/32 (Fc block; BD Bioscience, San Diego, CA) on ice for 15 minutes. Then, samples were stained with CD4 fluorescein isothiocyanate, CD8 phycoerythrin, CD3 allophycocyanin, CD11b–peridinin chlorophyll–Cy5.5, Gr1-Pacific Blue, and NK1.1-Cy7 (eBioscience, San Diego, CA) on ice for approximately 1 hour. Stained samples were fixed with 4% paraformaldehyde on ice for 15 minutes. Stained and fixed samples were stored in 500 μL of staining buffer. Samples were read on a Gallios Flow Cytometer (Beckman-Coulter, Atlanta, GA). Analysis was conducted with Cyflogic version 1.2.1. The large right lobe of the mouse lung was collected and fixed in 10% buffered formalin. For histologic analysis, the lung was sectioned (5 μm thick) and stained with hematoxylin and eosin (H&E) or Masson's trichrome as per standard procedures. The number of plaques per lung section was counted under the microscope. Formalin-fixed, paraffin-embedded human lung samples from our collection were sectioned (5 μm thick) and stained with H&E and acid fast. Images were taken under light microscopy and color balanced with Adobe Photoshop. Whole blood was taken from TDM-IPIV–injected C57BL/6 and BALB/c mice into a tube with 3.2% citrate to achieve a final dilution of nine parts blood with one part anticoagulant. Thromboelastometry was performed using ROTEM software version 1.6.0 (Pentapharm, GmbH, Munich, Germany) δ analyzers. The instrument monitors changes in the viscoelastic properties of whole blood as the clot is formed by measuring the impedance of a pin attached to a rotating shaft (±4.75°) connected to a spring. Clotting in whole blood was initiated via the intrinsic pathway by the addition of reagents supplied by the manufacturer (in-TEM, diluted 1/10 with saline, and 0.2 M CaCl2; TEM Systems Inc, Durham, NC). Data obtained were compared across groups and against naive mice or mice challenged with vehicle formulation without TDM, then analyzed by an unpaired t-test or one-way analysis of variance with the Tukey post-hoc test; differences in the means were significant at a level of P ≤ 0.05. Data are presented as a compilation of all experimental repeats (two or three). Each experiment had three to six mice. To examine TDM-induced inflammation in lung tissue, C57BL/6 and BALB/c mice were injected with M. tuberculosis–derived TDM, comparing among several different methods: i) TDM-IP, ii) TDM-IV, and iii) TDM-IPIV. Controls included naive mice, light mineral oil (no TDM) only i.p., and oil/water emulsion (no TDM) only i.v. Naive and vehicle (no TDM)–injected mice had no noticeable lung pathologic findings, with normal parenchyma and little to no noticeable cellular infiltrates (Supplemental Figure S1). C57BL/6 mice injected with TDM-IP had minimal lung pathologic findings with very small clusters of leukocytes and thickening of alveoli walls. BALB/c mice injected with TDM-IP also had few to no lung pathologic findings that appeared to be mostly normal with no obvious thickening of alveoli walls. Mice injected with TDM-IV had inflamed lung pathologic findings, similar to published data, and small granuloma clusters mainly composed of myeloid cells and thickened alveoli walls. A more enhanced inflammatory response was observed in C57BL/6 mice compared with BALB/c mice. The difference between the two mouse strains became more prominent with TDM-IPIV. Although BALB/c mice lungs appeared similar in pathologic findings to the TDM-IV–injected mice, C57BL/6 mice had an increase in inflammation, larger leukocyte clusters with widespread alveoli thickening, and accompanying hemorrhage. The severity of lung hemorrhage in C57BL/6 mice was not always consistent across all individuals. However, every C57BL/6 mouse given TDM-IPIV had small hemorrhagic regions, and at least one mouse from each experimental repeat manifested widespread hemorrhage across the entire lung section. The other noticeable finding was that the mice given TDM-IV and TDM-IPIV (C57BL/6 and BALB/c) had signs of vascular occlusion (Figure 1). Gross lung observations clearly revealed the extensive hemorrhage in C57BL/6 TDM-IPIV group compared with the C57BL/6 TDM-IV, BALB/c TDM-IV, and BALB/c TDM-IPIV groups (Figure 2).Figure 2Gross lung pathologic findings of mice injected with trehalose 6′6-dimycolate (TDM). The left lobe of lung from C57BL/6 and BALB/c mice was collected from mice injected with i.v. TDM (TDM-IV) and i.p./i.v. TDM (TDM-IPIV) at day 7. The lungs were fixed in 10% formalin. Photograph was taken under normal lighting conditions with a white background. One representative lung is shown per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Overall lung inflammation was measured as LWI. For both C57BL/6 and BALB/c mice, TDM-IV treatment increased LWI significantly against the TDM-IP and non-TDM groups (Figure 3). Interestingly, both clonal strains that received TDM-IPIV had a wider range of LWI that was significantly decreased compared with TDM-IV in C57BL/6 and BALB/c mice. Whole-lung cytokine environments were examined for inflammation (TNF-α, IL-6, IL-1β), T-cell activity (IFN-γ and IL-17), and T-cell helper type 1 differentiation (IL-12p40 and IL-10). In mice that did not receive TDM, no cytokine production of all analytes was found. Only mice given the oil/water vehicle produced any measurable cytokine levels but were significantly lower than the TDM-injected groups (Supplemental Table S1). In general, C57BL/6 mice produced higher levels (approximately 10×) of cytokines compared with BALB/c mice in response to TDM. In all cytokines measured, the mice injected with TDM-IV and TDM-IPIV induced significantly higher levels of cytokines compared with the TDM-IP group. Only IL-6 revealed no difference among the TDM-IP, TDM-IV, and TDM-IPIV groups. The C57BL/6 mice had an increase in IL-1β in the TDM-IV and TDM-IPIV groups compared with the TDM-IP group (Figure 4A). In the BALB/c mice, the IL-1β level in the TDM-IPIV group was decreased compared with the TDM-IV group (Figure 4B), with both groups having increased levels compared with TDM-IP. C57BL/6 and BALB/c mice given TDM-IPIV and TDM-IV induced significantly higher levels of IFN-γ in the lung tissue. Although both C57BL/6 and BALB/c mice given TDM-IV had an increase in lung IL-17 levels compared with TDM-IP, only C57BL/6 mice given TDM-IPIV produced significantly higher IL-17 levels compared with TDM-IV (Figure 4). Production of IL-12p40 and IL-10 in C57BL/6 mice was increased as follows: TDM-IP < TDM-IV < TDM-IPIV (Figure 4A). In BALB/c mice, IL-l2p40 production increased in both the TDM-IV and TDM-IPIV groups compared with the TDM-IP group (Figure 4B). IL-10 production in BALB/c mice was significantly increased in the same trend as in the C57BL/6 mice: TDM-IP < TDM-IV < TDM-IPIV (Figure 4). Granulocyte-macrophage colony-stimulating factor production in C57BL/6 mice was higher overall compared with BALB/c mice (Figure 4A), but only the BALB/c mice had an increase in GM-CSF from TDM-IPIV mice compared with the TDM-IP and TDM-IV mice (Figure 4B). The cytokine levels from C57BL/6 and BALB/c mice indicated that the different TDM injections induced a varied inflamed lung environment. Infiltrating leukocytes drawn by TDM and/or the inflamed environment can contribute to progression and modulation of the immune response. To investigate whether the cytokine environment correlated to the leukocyte population, multicolor flow analysis of immune cell markers was examined. Naive, oil vehicle i.p., and oil/water emulsion i.v. controls all had relatively low percentages of T cells, macrophage, and neutrophil populations in the lung. Only the oil/water vehicle i.v. induced an increase in macrophage and neutrophils (Supplemental Table S2). With the use of forward and side scatterplot, lymphocytes and myeloid cells were differentiated (confirmed by lack of CD3+ in the myeloid population). Both C57BL/6 and BALB/c mice had the same trend in lymphocyte and myeloid percent makeup. Both mouse strains had increase lymphocyte populations: TDM-IV > TDM-IPIV > TDM-IP. Consequently, the increase in lymphocyte population correlated with a decrease in myeloid cell population, as reflected by the changes in the myeloid population: TDM-IP > TDM-IPIV > TDM-IV (Figure 5A). The lymphocyte population was delineated into CD3+ and CD3− populations. The TDM-IP mice (C57BL/6 and BALB/c) had a balanced population of CD3+ and CD3− groups. The TDM-IV and TDM-IPIV mice had significantly increased CD3− population and decreased CD3+ population (Figure 5B). The CD3+ population was then split into CD4+ and CD8+ populations. In both strains of mice, the TDM-IV and TDM-IPIV mice had decreased CD3+CD4+ cells compared with the TDM-IP group (Figure 5C). The myeloid population can be divided into macrophages (CD11b+GR1−) and neutrophils (CD11b+GR1+). No differences in neutrophil numbers were observed in all TDM C57BL/6 mice; however, the TDM-IPIV group had an increase in macrophages compared with the TDM-IV and TDM-IP groups. In BALB/c mice, both the TDM-IV and TDM-IPIV groups had increased macrophages in the lungs compared with TDM-IP. In addition, BALB/c mice given TDM-IV and TDM-IPIV had decreased neutrophil numbers compared with the TDM-IP group (Figure 5D). Little to no cells that expressed NK1.1 were CD3+,
Referência(s)