Active transforming growth factor-β is associated with phenotypic changes in granulomas after drug treatment in pulmonary tuberculosis
© DiFazio et al. 2016
Received: 13 January 2016
Accepted: 25 April 2016
Published: 27 April 2016
Tuberculosis (TB) chemotherapy clears bacterial burden in the lungs of patients and allows the tuberculous lesions to heal through a fibrotic process. The healing process leaves pulmonary scar tissue that can impair lung function. The goal of this study was to identify fibrotic mediators as a stepping-stone to begin exploring mechanisms of tissue repair in TB.
Hematoxylin and eosin staining and Masson’s trichrome stain were utilized to determine levels of collagenization in tuberculous granulomas from non-human primates. Immunohistochemistry was then employed to further interrogate these granulomas for markers associated with fibrogenesis, including transforming growth factor-β (TGFβ), α-smooth muscle actin (αSMA), phosphorylated SMAD-2/3, and CD163. These markers were compared across states of drug treatment using one-way ANOVA, and Pearson’s test was used to determine the association of these markers with one another.
TGFβ and αSMA were present in granulomas from primates with active TB disease. These molecules were reduced in abundance after TB chemotherapy. Phosphorylated SMAD-2/3, a signaling intermediate of TGFβ, was observed in greater amounts after 1 month of drug treatment than in active disease, suggesting that this particular pathway is blocked in active disease. Collagen production during tissue repair is strongly associated with TGFβ in this model, but not with CD163+ macrophages.
Tissue repair and fibrosis in TB that occurs during drug treatment is associated with active TGFβ that is produced during active disease. Further work will identify mechanisms of fibrosis and work towards mitigating lung impairment with treatments that target those mechanisms.
KeywordsTuberculosis Drug treatment Transforming growth factor-β Collagen I
With eight million new cases and 1.5 million deaths annually worldwide, tuberculosis (TB) is one of the humanity’s greatest health threats . Granulomas, the pathologic hallmark of TB, are well-circumscribed organized collections of host immune cells that form in response to the inhalation of aerosols containing Mycobacterium tuberculosis (Mtb)—the causative agent of TB. Although granulomas can function to kill or contain Mtb, they can also serve as a niche for growth and persistence of the organism [2–4]. Granulomas often feature a necrotic center and are thus dubbed as necrotizing or caseous, while granulomas lacking this necrosis are said to be non-necrotizing. Granulomas feature epithelioid macrophages, elongated cells with larger nuclei, surrounded by other macrophages and lymphocytes . Bacteria can be found in epithelioid macrophages and in caseum . Uncontrolled replication leads to dissemination of the bacteria and formation of new granulomas. However, some granulomas can restrain bacterial dissemination or even develop locally sterilizing immunity. As a result, these granulomas are often fibrotic and can contain a calcified core (referred to as fibrocalcific lesions) . A mixture of necrotizing and collagenous lesions is typical of the secondary pulmonary tuberculosis and is referred to as fibrocaseous disease [6, 7]. Although the host factors that lead to control or dissemination of a single granuloma are unclear, we have demonstrated that various types of granulomas and outcomes exist within a single non-human primate, similar to humans [6, 8].
Tissue fibrosis can result from a wound healing response that includes fibroblast activation and recruitment, production of extracellular matrix materials, and distortion of the normal tissue architecture. The most common extracellular matrix component is collagen I, which is the most fibrous form of collagen and represents about 84 % of the collagen produced by fibroblasts . Fibrosis can be caused by a local inflammatory response, and fibrosis-related pathogenesis is associated with dysfunction of many organs, including lungs, liver, and kidneys [10–12]. Transforming growth factor-β (TGFβ) is the main cytokine implicated in fibrogenesis, although other cytokines are implicated, including TNF, IL-6, IL-10, IL-13, and IL-17 [13–18]. TGFβ is produced in a latent form (L-TGFβ) and can be activated through the plasmin protease pathway, CD36 and thrombospondin (TSP), reactive oxygen and nitrogen species, hypoxia, low pH, and matrix metalloproteases . Active TGFβ utilizes type 1 and 2 TGFβ receptors, signaling through a variety of intermediaries, including phosphorylated SMAD-2/3 . Through these intermediaries, TGFβ stimulates differentiation of fibroblasts into myofibroblasts that then produce alpha-smooth muscle actin (αSMA), a key indicator of and contributor to fibrotic pathogenesis . TGFβ has been observed and measured in pulmonary fibrosis and, in the lung, is produced by alveolar macrophages, fibrocytes, and lung epithelial cells [22–25]. Alveolar macrophages from humans with pulmonary fibrosis display an alternatively activated (M2) phenotype, and induction and maintenance of M2 macrophages is critical to pathology in pulmonary fibrosis . M2 macrophages are also major producers and activators of TGFβ .
Although several types of pulmonary fibrosis have been characterized and studied, fibrosis in tuberculosis is not well-understood. Significant pulmonary impairment was observed in 59 % of patients with TB disease , half of whom had less than 50 % of their original forced vital capacity . This loss of pulmonary function resulted in 177 subjects losing 1189 disability adjusted-life years . Lung function does not improve over the course of chemotherapy , and this chronic impairment increases incrementally with the number of TB episodes experienced in a progressive manner . The main course of treatment for post-tuberculosis lung damage is pulmonary rehabilitation, which has mixed results , highlighting the need for more targeted therapies to resolve TB-induced fibrosis and scarring. Since macrophages produce TGFβ in pulmonary fibrosis, and are a major cellular component of granulomas , macrophages may be important contributors to fibrosis in TB lesions . The environment of the granuloma may contain almost all of the conditions that activate TGFβ, including hypoxia , nitrogen radicals , and metalloproteases , so it is likely that the disease process activates TGFβ locally at the site of infection. Cutaneous TB lesions in humans have been noted as centers of fibrosis, with lesions containing active TGFβ . Patients with TB have peripheral blood monocytes and alveolar macrophages that produce and active more TGFβ than cells from healthy controls [35, 36]. TGFβ has also been observed directly in granulomas from human TB patients by immunohistochemistry .
Drug treatment for M. tuberculosis infection [38, 39] is a lengthy process that slowly clears bacterial burden in the lung and induces tissue repair in TB-affected lung. The factors that promote fibrotic resolution of tuberculous granulomas are poorly understood. This has been a challenging topic to address because of difficulties associated with studying human TB and a lack of appropriate mouse models demonstrating the granuloma structures seen in humans. Our laboratory previously published that drug-treated macaques with TB had fibrotic granulomas, and the fibrotic granulomas were most often sterile , representing a successful outcome of drug treatment. Since macaques recapitulate the spectrum of granuloma types and infection outcomes seen in humans, they represent a useful system for studying the process of drug-associated fibrosis. Understanding the fibrotic processes that occur in TB may provide insights into treatments to safely resolve residual lung fibrosis during or after drug therapy. The objective of this study was to determine how the cell types and molecules associated with pulmonary fibrosis differ between granulomas associated with active TB and fibrotic changes after chemotherapy. This study will open up further exploration of the fibrogenic mechanisms, with the aim of developing treatments to minimalize or reverse scarring after drug treatment.
Macrophages experience spindloid transformation in tuberculous granulomas
Granulomas exhibit a range of collagen deposition before and after drug treatment
Tuberculous granulomas bear signs of TGFβ-driven fibrosis
Active Mtb infection and disease activates TGFβ and suppresses SMAD-2/3 signaling
TGFβ is strongly associated with collagen I expression
The goal of this study was to identify molecular and cellular markers associated with fibrotic resolution in experimental tuberculosis infection. Tuberculosis produces a wide range of pulmonary pathologies through infection and disease, which typically result in chronic fibrocaseous disease [6, 7]. Drug treatment reduces bacterial burden and allows for tissue repair, although this does not quickly alter the pathology remaining in the lung as demonstrated by computed tomography in both human and non-human primates . Chemotherapy additionally leaves individuals vulnerable to relapse; more than half of smear positive TB cases have been previously treated for TB . Relapse was originally thought to be cause by endogenous sources of Mtb remaining after drug treatment, but 75 % of relapse cases in a high TB burden cohort were exogenously reinfected . Why is this population prone to reinfection after successful chemotherapy? The risk of developing active TB is about 4.5 times higher in patients with chronic interstitial lung disease and idiopathic pulmonary fibrosis [47, 48]. This could be due to the specific pathology of fibrotic lung tissue as it may promote colonization and establishment of infection . The residual scar tissue left after drug therapy and loss of normal lung architecture could play roles in increasing the risk of relapse. Therefore, understanding the mechanism of fibrogenesis in TB and ameliorating tissue damage after drug treatment could reduce the risk of reinfection and help shrink the pool of infected individuals.
Several cell types are likely to play significant roles in driving fibrotic processes in granulomas, but it has been difficult to identify the specific cells that are the most important drivers of fibrosis. Histologic assessment can be used to evaluate the cellular responses to infection from a morphological perspective and can provide insights into lesion development and resolution. Identifying the cellular origin of some spindle-type cells in granulomas is not always possible, but histologic examination can provide important inferences as to whether these cells arose from epithelioid macrophages or collagen-producing fibroblasts. Based on our histologic observations and immunohistochemical analyses, we hypothesize that collagen-producing fibroblast-like cells may originate from several sources, and in some cases not from fibroblasts recruited from nearby tissues, but from non-fibroblast-like cells that have undergone a process similar to endothelial–mesenchymal progression. Additionally, circulating monocyte-derived cells (fibrocytes) can have phenotypes reminiscent of the phenotypes we have identified [50, 51]. Given the ability of some circulating monocytes to differentiate into fibroblast-like cells instead of macrophages, the question of whether subsequent differentiation between macrophages and fibroblasts can occur should be considered. The wide range in collagen presence and the appearance of various collagenous phenotypes suggest that different mechanisms for fibrotic resolution occur in tuberculous granulomas. Some granulomas manifest both peripheral and central fibrous organization, while others demonstrate only central collagenization, or none at all. Future studies should seek to correlate these divergent repair mechanisms to lesion-specific Mtb burden to determine efficacy of clearance and elucidate the immunologic processes of each.
Fibrosis occurs in TB lesions during and after drug treatment. The goal of this project was to determine the cell types and molecules associated with fibrosis in non-human primates with TB. We provide additional evidence of activated TGFβ being present in lesions from M. tuberculosis-infected lung tissue. Collagen production in active disease and TB chemotherapy is strongly associated with TGFβ, suggesting its role as the chief cytokine driving tissue repair. Future studies will seek to further explore these results mechanistically with hopes of developing adjunct treatment to minimize, or possibly reverse, scar formation after TB chemotherapy.
The Institutional Animal Care and Use Committee of University of Pittsburgh approved all experiments. The animals were housed and maintained in accordance with standards established in the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals.
Cynomolgus macaques (4–9 years of age) imported from China (Valley Biosystems, Sacramento, CA) were used for these studies (n = 21). Monkeys were infected via bronchoscope with 25 CFU of M. tuberculosis Erdman strain. Using published criteria, monkeys were determined to have active or latent TB by 6–8 months post-infection and were randomized to treatment or non-treatment groups; treatment was initiated when monkeys developed active TB, as determined by clinical and microbiologic signs [43, 55].
The samples described in the current study were obtained from macaques in a previously published study from our laboratory [40, 42]. Monkeys with active disease were as follows: untreated (n = 9, 110 granulomas), treated with isoniazid (INH) and rifampin (RIF) for 1 month (n = 2, 22 granulomas) or 2 months (n = 5, 91 granulomas), or treated with INH, RIF, and metronidazole (MTZ) for 1 month (n = 3, 154 granulomas) or 2 months (n = 5, 93 granulomas).
Necropsies were performed as previously described [35, 51]. Individual granuloma and lung samples were taken from each monkey from sites of infection and surrounding tissue. Portions of these samples were homogenized into single cell suspension before storing at −80 °C for ELISA and hydroxyproline assays, while another portion was formalin-fixed paraffin-embedded for histology.
Formalin-fixed paraffin-embedded tissue sections were cut and stained with Harris hematoxylin modified (Sigma-Aldrich, St. Louis, MO) and eosin Y solution (Sigma-Aldrich, St. Louis, MO). Slides were deparaffinized in deionized water. Slides were then stained with hematoxylin for 3 min. Slides were rinsed under running tap water, rinsed with 70 % ethanol, and then stained with eosin for 3 min. Slides were rinsed and dehydrated in ethanol, cleared in xylene, and then mounted. Criteria for characterizing granulomas were based on size and shape, type of granuloma, and cellular composition. A veterinary pathologist who is an expert in macaque tuberculosis (ECK) performed all histologic analyses.
Formalin-fixed paraffin-embedded tissue sections were stained with Masson’s trichrome to identify connective tissue, muscle, and collagen fibers (Sigma-Aldrich, St. Louis, MO). Slides were deparaffinized to deionized water. Slides were then immersed in Bouin’s solution overnight at room temperature to intensify the subsequent staining. Slides were washed with tap water then stained with Harris hematoxylin solution (Sigma-Aldrich, St. Louis, MO) for 5 min. Slides were washed again in running tap water for 5 min, rinsed in deionized water, and stained in Biebrich scarlet-acid fuchsin for 5 min. Slides were rinsed in deionized water and placed in phosphotungstic and phosphomolybdic acid solution for 5 min. Slides were moved to Aniline Blue solution for 5 min and then placed in 1 % acetic acid solution for 2 min. Slides were rinsed in deionized water, dehydrated through alcohol, cleared in xylene, and then mounted.
Tissue sections were stained for collagen I (rabbit polyclonal, Abcam, Cambridge, MA, 1:50 dilution), pSMAD-2/3 (goat polyclonal, Santa Cruz Biotechnology, Dallas, TX, 1:10 dilution), CD163 (mouse clone 10D6, Neomarkers, Fremont, CA, 1:30 dilution), L-TGFβ (goat polyclonal, R&D Systems, Minneapolis, MN, 1:10 dilution), TGFβ (chicken polyclonal, R&D Systems, Minneapolis, MN, 1:10 dilution), and αSMA (mouse clone 1A4, Thermo Fisher, Pittsburgh, PA, 1:100 dilution). Antigen retrieval and staining were done as previously described . Briefly, formalin-fixed paraffin-embedded tissues samples were deparaffinized in xylene and rehydrated in ethanol. Samples were then placed into a pressure cooker with boiling antigen retrieval buffer (Tris–HCl, EDTA, Tween-20) for 7 min. After allowing for the slides to cool, the sections were blocked with 2 % fetal bovine serum in phosphate-buffered saline for 30 min. Antibodies and fluorescent tags were incubated on each sample for 1 h with washes in between with phosphate-buffered saline with 0.2 % Tween-20. Prolong Gold Mounting Medium with DAPI (Invitrogen) was then applied to the slides, which were then cured in the dark overnight before being imaged.
Quantification of histology
Trichrome- and H&E-stained sections were imaged using Provis fluorescent microscope (Olympus America, Center Valley, PA) and fluorescently stained slides visualized with a FluoView 1000 confocal microscopes (Olympus). For images used for quantitative imaging, care was taken to keep the camera settings constant between granulomas and animals. These images were then saved as 24-bit TIFF files and read into the language R via the package “EBImage” from Bioconductor (http://www.bioconductor.org/packages/release/bioc/html/EBImage.html). For the trichrome slides, the blue channel only was isolated. Red, green, and blue channels were pulled from the immunohistochemistry (IHC) slides. The median pixel intensity for each channel was then saved and exported to Microsoft Excel (Microsoft, Redmond, WA).
Active TGFβ-1 in granuloma homogenates was measured by using a commercial ELISA (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Briefly, a high-affinity protein-binding plate was coated with a capture antibody overnight and blocked with assay diluent for an hour before adding standards and undiluted granuloma homogenates for overnight incubation. TGFβ was detected the next day using a biotinylated detection antibody and streptavidin-HRP, and the absorbance was immediately measured at 450 nm. Total protein of the same samples was quantified by Pierce BCA Protein Assay (Thermo Scientific, Pittsburgh, PA), where granuloma homogenates were added to BCA Working Reagent, and the absorbance measured at 562 nm after 30 min at 37 °C. Levels of TGFβ were normalized to total protein in granuloma homogenates.
Collagen was detected in homogenized granulomas by using a commercial hydroxyproline kit (Sigma-Aldrich, St. Louis, MO). Briefly, samples were mixed with hydrochloric acid and hydrolyzed at 120 °C for 3 h. These samples and standards were then transferred to a 96-well plate and dried. Chloramine T/oxidation buffer was added to wells and incubated at room temperature for 5 min. To this, diluted DMAB reagent was added and incubated at 60 °C for 90 min. Absorbance at 560 nm was then measured. Levels of collagen were normalized to total protein in granuloma homogenates.
Quantitative data from Masson’s trichrome staining and immunohistochemical staining were visualized using Prism (Graphpad, La Jolla, CA). Analysis of these data was done using the tests indicated, typically a one-way ANOVA and a multiple comparison test for either parametric or non-parametric data. p values were significant if less than 0.05. Further analysis of the immunohistochemical staining was performed with JMP (SAS, Cary, NC). Correlations between markers in histological stains were performed using the multivariate function, and scatter plots were generated. Significance was determined using pairwise correlations with the strength of the relationship given as Pearson’s r.
hematoxylin and eosin
transforming growth factor-β
α-smooth muscle actin
We would like to thank the members of the Flynn lab for their support and constructive criticisms, especially Pauline Maiello for the statistical queries and Chelsea Chedrick for the figure preparation. We would also like to thank the Center for Biologic Imaging and the Department of Microbiology and Molecular Genetics at the University of Pittsburgh School of Medicine for the use of their Provis light microscope and Fluoview 1000 confocal microscope, respectively. This study was funded by NIH HL110811.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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