article: Bone marrow-derived mesenchymal stem cells (BMMSCs) have been consider as a promising therapy in fibrotic diseases. Experimental models suggest that BMMSCs may be used as an alternative therapy to treat chemical- or physical-induced pulmonary fibrosis. We investigated the anti-fibrotic potential of BMMSCs in an experimental model of lung fibrosis by infection with Paracoccidioides brasiliensis. BMMSCs were isolated and purified from BALB/c mice using standardized methods. BALB/c male mice were inoculated by intranasal infection of 1. 5x106 P. brasiliensis yeasts. Then, 1x106 BMMSCs were administered intra venous at 8th week post-infection (p. i.). An additional group of mice was treated with itraconazole (ITC) two weeks before BMMSCs administration. Animals were sacrificed at 12th week p. i. Histopathological examination, fibrocytes counts, soluble collagen and fibrosis-related genes expression in lungs were evaluated. Additionally, human fibroblasts were treated with homogenized lung supernatants (HLS) to determine induction of collagen expression. Histological analysis showed an increase of granulomatous inflammatory areas in BMMSCs-treated mice. A significant increase of fibrocytes count, soluble collagen and collagen-3α1, TGF-β3, MMP-8 and MMP-15 genes expression were also observed in those mice. Interestingly, when combined therapy BMMSCs/ITC was used there is a decrease of TIMP-1 and MMP-13 gene expression in infected mice. Finally, human fibroblasts stimulated with HLS from infected and BMMSCs-transplanted mice showed a higher expression of collagen I. In conclusion, our findings indicate that late infusion of BMMSCs into mice infected with P. brasiliensis does not have any anti-fibrotic effect; possibly because their interaction with the fungus promotes collagen expression and tissue remodeling.
  Bone marrow-derived mesenchymal stem cells (BMMSCs) are adult stem cells capable of both renew themselves and differentiate in vitro into multiple cell lineages [1]. These cells can also modulate the inflammatory response and induce tissue regeneration through release of cytokines, chemokines, growth factors and genetic material (e. g. miRNAs) [2–6]. Likewise, their microbicidal properties have been already described [7–10]. Cell-based therapies in regenerative medicine using syngeneic or autologous BMMSCs are considered a promising approach, because they do not induce tissue rejection and exert a localized effect than the systemic classical pharmacological strategies [11]. BMMSCs have also been evaluated in animal models of acute lung injury induced by chemicals, such as bleomycin [12,13] and hydrochloric acid (HCl) [14]. These studies have shown that BMMSCs secrete cytokines, chemokines, growth factors and extracellular matrix proteins, and that can influence the magnitude and quality of the immune response (e. g. modulating the inflammatory response), and promote tissue repair. Likewise, BMMSCs can differentiate into pulmonary stromal cells (e. g. lung fibroblasts and myofibroblasts) [12,14,15]. Pulmonary fibrosis (PF) is a process characterized by excessive deposition of collagen and extracellular matrix components that results in a pathological remodeling of the pulmonary architecture. Thus, patients with PF exhibit radiographic, but also functional and clinical alterations in the lung [16]. From the pathological perspective, PF is a dynamic process involving immune system cells and soluble factors including leukotrienes, cytokines (IFNγ, TNFα, IL1, IL4, IL6, IL17), chemokines (CCL2, CCL3, CXCL12), reactive oxygen species (ROS), growth factors [platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF) ], and membrane-bounded and soluble molecules such as prostaglandins, metalloproteinases (and their tissue inhibitors), among others [17]. An imbalance between pro-fibrotic responses and anti-inflammatory and pro-tissue repair agents, results in the differentiation and activation of myofibroblasts which once activated produce abundant amounts of collagen, thus inducing fibrosis of the pulmonary parenchyma [18]. The participation of all the previous components in the PF has been extensively studied in animal models [16,18,19]. Pulmonary fibrosis can be induced by microbial agents including the dimorphic fungal pathogen Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), disease that is considered one of the most important endemic systemic mycosis in South America and Central America [20–23]. Brazil, Colombia and Venezuela are the countries with the highest number of cases reported so far, with an estimated of 10 million people infected [20,21]. The chronic form of PCM is the most frequent clinical presentation (90% of the cases), and it is characterized by a granulomatous inflammatory response with fibrosis development and loss of respiratory function, which is observed in 60% of the patients [22]. Itraconazole (ITC) is the treatment of choice in PCM [23]. Nonetheless, it exerts a fungistatic effect against P. brasiliensis in vivo, and it does not attenuate the pulmonary alterations induced by the fungal infection, including fibrosis [24,25]. Animal models of PCM have allowed characterization of the mechanisms involved in the development of pulmonary fibrosis, and evaluate diverse strategies to treat it. Thus, combined therapies including pentoxifylline plus ITC [26], or an anti- neutrophil monoclonal antibody alone or in combination with ITC [27,28], have showed that such treatment strategies reduced substantially the PF. However, the potential risk for using immunosuppressive drugs or biological agents, mainly in host with other unknown latent infections, should be considered [29]. The objective of this study was to investigate the regenerative effect of BMMSC on PF induced by the fungal pathogen P. brasiliensis in an in vivo experimental model of PCM; nonetheless, our findings indicated that these cells impaired the anti-fibrotic effect and by the contrary, an exacerbated fibrotic process was observed.
  BMMSCs were obtained from four weeks-old BALB/c mice from the breeding colony maintained at the Corporación para Investigaciones Biológicas (CIB, Medellín-Colombia). The BMMSCs isolation and purification protocols were adapted from a protocol previously described by Rojas et al [12]. Briefly, mice were anesthetized with a solution of ketamine (80 mg/kg) and Xylazine (8 mg/kg) via intramuscular. Femurs and tibias were removed and bone marrow cells were isolated by flushing with Dulbecco' s Modified Eagle Medium (DMEM) -low glucose (GIBCO, Invitrogen Corporation, Carlsbad, CA, USA) containing penicillin/streptomycin1% (vol/vol) (GIBCO). Cells were transferred to cell culture flasks (Eppendorf, Hamburg, Germany) with DMEM-low glucose supplemented with 10% (vol/vol) fetal bovine serum (FBS) (GIBCO) and nonessential amino acids 1% (vol/vol) (GIBCO), followed by incubation at 37°C in 5% CO2. Non-adherent cells were removed after 48 hours, and maintained in standard culture media for 7 days. In order to exclude hematopoietic stem cells and leucocytes, a magnetic bead-based mouse cell depletion kit (Miltenyi Biotec, Bergisch Gladbach, Germany) containing anti-CD45, anti-CD11b, anti-CD5, anti-Gr1 (Ly-6/C), and anti-Ter 119 monoclonal antibodies was used. The BMMSC surface markers expression profile was determined by flow cytometry. The following antibodies were used: isothiocyanate (FITC) anti-CD45 (BD Pharmingen, San Diego, CA, USA), phycoerythrin (PE) -Cy5-anti-CD44, allophycocyanin (APC) anti-CD105, PE-Cy7-anti-CD106, APC-anti-TER-119, Pacific blue-anti-SCA-1, and PE-anti-CD73 (Biolegend, San Diego, CA, USA). Cells were analyzed using a FACS Canto II system (BD Biosciences, San Jose, CA, USA) and FlowJo V10 software (FlowJo, LLC, Data Analysis software, Ashland, OR, USA). In addition, a differentiation assay to demonstrated the BMMSCs plasticity (differentiation to chondrogenic, adipogenic and osteogenic lineages) was performed using a differentiation commercial kit, and following the manufacturer’s instructions [StemPro (Waltham, MA, USA) ]. Finally, the purified cells were kept in standard culture media until the day of transplant. This study was carried out following the Colombian (Law 84/1989, Resolution No. 8430/1993), European Union, and Canadian Council on Animal Care regulations. The protocol was approved by the Institutional Ethics Committee of the CIB (Acta No. 95). A highly virulent strain of P. brasiliensis (Pb18) was used in order to develop the experimental pulmonary fibrosis model as described previously [28]. Briefly, BALB/c male mice (8 weeks old) were intranasally infected with 1. 5 x 106 P. brasiliensis yeast cells contained in 60 μl of phosphate-buffered saline (PBS). The total inoculum was split into two equal doses, which were instilled within a 5–10 minutes (min) period. Non-infected (control) mice were inoculated with 60 μl of PBS. Infected and non-infected mice were intravenously injected with 1x106 BMMSCs at 8th week post-challenged given in a single dose. Six week post-inoculation, an additional group of infected animals was treated with 100 μl of Itraconazole (ITC) oral solution (Sporanox, Janssen-Cilag S. A. , Mexico) administered at a dose of 1mg/day in order to achieve serum levels equal to 1 μg/mL. The above treatment was administrated daily and uninterruptedly for 6 weeks by gavage. All animals included in the various experimental groups were sacrificed at week 12th p. i. and their lungs harvest for further studies. Lungs of mice were removed, homogenized and sequentially filtered through 70 and 40μm sterile cell strainers (Thermo Fisher Scientific Inc, Waltham, MA, USA) in RPMI cell culture medium plus 1% (vol/vol) FBS (Sigma-Aldrich, Saint Louis, MO, USA). Cells suspension were centrifuged at 500 G, 10°C for 10 min, and red blood cells were lysed using the ACK Lysing Buffer (GIBCO). Viability of the cells was determined by trypan blue exclusion test with samples being used if they were 95% of viable. Cells were resuspended in RPMI plus 10% FBS and counted using a hemocytometer. Fc receptors were blocked using a purified rat anti-mouse CD16/CD32 (BD Pharmigen, San Diego, CA, USA). Then, cells were treated with Cytofix/Cytoperm and Perm/Wash solution (BD Pharmigen, San Diego, CA, USA) [28]. Fibrocytes were determined using FITC anti-collagen I (Rocklad inc Limerick USA), PE anti-CD45 (Biolegend San Diego USA), and APC anti-CD34 (BD Pharmingen, San Diego USA). Anti-mouse IgG-FITC (Rocklad), anti-mouse IgG2aκ-PE (Biolegend) and anti-mouse IgG1κ-APC (BD) were used as isotype controls. The stained cell suspensions were fixed with FACS buffer/1% (vol/vol) PFA (Carlo Erba, Barcelona, Spain). Assays were performed using a FACS Canto II system (BD Biosciences, San Jose, CA, USA), while information analysis were done using FlowJo V10 (FlowJo, LLC, Data Analysis software, Ashland, OR, USA). Fibrocyte population was analyzed as follows: (a) cell events in region 1 (R1) were gated by forward scatter versus side scatter areas; (b) CD45+ events were gated from R1 by side scatter area versus CD45 staining to establish the R2 region, from which (c) cell events were gated to determine fibrocytes by collagen 1+ (intracellular) and CD34+ (surface). The number of fibrocytes was determined by multiplying the percentage of the gated population by the total number of leukocytes (CD45+ population). Lungs were processed and analyzed as described by Puerta-Arias et al [28]. Briefly, lungs were perfused with 1X PBS to wash out red blood cells. Tissue fixation was completed in a 4% buffered formalin solution. Then, fixed tissues were embedded in paraffin and sections stained with Masson trichrome, and examined using a Nikon Eclipse Ci-L microscope—Nikon DS-Fi2 digital camera. A morphometric analysis was performed using NIS Elements 4. 30. 02 Laboratory Image Software (Nikon Instruments Inc. , Melville, USA). The percentage of occupied area by the inflammatory response was calculated by dividing the total inflamed area, which includes cellular infiltrates and granulomatous lesions by the total area of the lung. Homogenized lung suspensions were treated with acid neutralizing reagent (0. 5M acetic acid, 0. 1 mg/ml pepsin) (Sigma-Aldrich, Saint Louis, MO, USA). Then, colorimetric detection of soluble collagen content was performed according to the manufacturer' s protocol of a sircol collagen assay kit (Biocolor, Northern Ireland, U. K.). A calibration curve was constructed using bovine collagen-I in the range of 1–10 μg. Human lung fibroblasts were obtained from Rojas’ Lab repository, collected under an established protocol from the University of Pittsburgh Center for Organ Research Involving Decedents (CORID). Cultures of human fibroblasts (2x104 cells/200uL, pass 4) were treated with soluble lungs supernatants (protein concentration 10ug/mL) from all experimental groups, for 24h at 37°C. Then, fibroblast activity was determined by measuring the expression of collagen type-I gen using reverse transcriptase real-time-PCR (RT-qPCR) assays, as previously described [29]. As controls, we used PBS and TGF-β [ (5ng/ml final concentration) Peprotech Rocky Hill, United States]. All real time PCR assays were performed as previously described [28]. Briefly, RNA was obtained from lungs of mice using Trizol (Invitrogen, Carlsbad, CA, USA). Samples were treated with DNase I (Thermo Fisher Scientific Inc, Waltham, MA, USA), and cDNA was synthesized using 500ng of total RNA using cDNA synthesis kit for RT-qPCR according to the manufacturer’s instructions (Thermo Fisher Scientific Inc, Waltham, MA, USA). Real-time PCR was done using Maxima EVAGreen/Fluorescein qPCR Master Mix according to the manufacturer’s instructions (Applied Biological Materials ABM Inc, Richmond, Canada). The CFX96 Real-Time PCR Detection System (Bio-Rad, Headquarters Hercules, California, USA) was employed to measure gene expression levels. Melting curve analysis was performed after the amplification phase of real time PCR assays to eliminate the possibility of non-specific amplification or primer-dimer formation. Validation of housekeeping genes for normalization mRNA expression was performed before gene expression analysis. Expression of fibrosis-related genes encoding for collagen, transforming growth factor beta (TGF-β), matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP) were evaluated. Fold changes in the target gene mRNA expression were quantified relative to glycer-aldehyde-3-phosphate dehydrogenase (GAPDH the housekeeping gene previously defined) [28]. Each experiment was repeated twice using 5 mice per each one of the groups with gene expression analysis being conducted by triplicate. Data analysis was performed using Graph Pad Prism software version 7 (GraphPad Software, Inc. , La Jolla, CA, USA). Normality for all values was calculated by the Shapiro-Wilk test and when comparisons between three or more groups were required, the ANOVA test was employed. On the other hand, comparisons between two specific groups were determined by Student-t test. Mean and standard error of the mean (SEM) were calculated for all analyses. We considerate P<0. 05 values as significant.
  We determined the granulomatous inflammatory areas through histopathological analysis in lung of experimental mice. We observed that lungs of P. brasiliensis infected-mice developed a granulomatous inflammatory response with collagen fibers surrounding granulomas (Fig 1A). Interestingly, administration of BMMSCs in infected mice showed an exacerbation of the inflammatory process, with a higher granulomatous inflammation and fibrosis areas with loss of parenchyma (Fig 1B). In contrast, the infected animals treated with ITC alone showed inflammatory and fibrotic responses similar to those infected and non-transplanted mice (Fig 1C). Moreover, the combined administration of BMMSCs/ITC in P. brasiliensis-infected mice considerably decreased the inflammatory response and fibrosis (Fig 1D), in comparison with those infected animals that only received cell-based therapy. A morphometric analysis revealed that occupied area by granulomatous inflammation in infected and transplanted mice was twice higher when compared with infected non-treated mice (p<0. 001), or three time that infected and BMMSCs/ITC-treated animals (p<0. 001) (Fig 1E). There was statistically significant difference in the average of occupied area by granulomas between infected and ITC-treated mice and those that received combined therapy (p<0. 005). Fibrocytes are bone marrow-derived fibroblast progenitor cells that have been implicated in tissue remodeling or repairing process, including the development of fibrosis. Following flow cytometry analysis we found a significantly increased number of fibrocytes (CD45+/CD34+/Collagen1+) in lungs from infected mice (p<0. 005) (Fig 2) relative to PBS instilled controls. Moreover, infected and BMMSCs-treated animals showed almost twice the number of fibrocytes when compared with infected non-treated mice (p<0. 005) (Fig 2). Interestingly, ITC treatment, in combination with BMMSCs, reduced the fibrocytes counts, versus P. brasiliensis infected mice (p<0. 001) or infected BMMSCs-treated animals (p<0. 001) (Fig 2). Collagen is considered the most important extracellular matrix protein involved in fibrosis. Accordingly, we determined the effect of BMMSCs therapy on lung soluble collagen content. Significantly increased levels of collagen in lungs from mice infected with P. brasiliensis were observed when compared with PBS instilled animals (p<0. 005) (Fig 3). Infected and BMMSCs-treated mice exhibited an increased significantly in collagen content relative to infected non-treated animals (p<0. 001) (Fig 3). Remarkably, ITC treatment reduced the amount of soluble collagen in the lungs from both, P. brasiliensis infected-mice (p<0. 005), or those with BMMSCs transplantation (p<0. 005) (Fig 3). To assessment the capability of lung homogenized to activate human lung fibroblasts, we performed in vitro assays stimulating fibroblasts with lung supernatants from experimental animals. We observed that lung supernatants from infected and BMMSCs-treated mice induced a higher expression of the gene encoding for collagen type-I in human fibroblast, in comparison with the respective homogenized lung supernatants from the infected non-treated mice (p<0. 005) (Fig 4). The homogenized lung supernatants from mice infected with P. brasiliensis and treated with ITC, alone or in combination with BMMSCs transplantation, in human fibroblast also induced a collagen gene expression similar to that found in human fibroblast stimulated with lung homogenates from infected non-treated-mice (Fig 4). Our next step was to determine if the BMMSCs administration affects the related-fibrotic response genes expression in lungs from mice infected with P. brasiliensis. We observed that P. brasiliensis infected-mice showed a higher expression of almost all genes evaluated (Col1α3, Col3α1, TGF-β1, TGF-β3, MMP-8, MMP-12, MMP-13, MMP14, TIMP-1 and TIMP-2) when compared with uninfected mice. Moreover, after BMMSCs treatment, a significantly higher expression of Col3α1, TGF-β3, MMP-8, and MMP-15 was observed in comparison with those P. brasiliensis infected-mice, while a reduction on MMP-13 gene expression was also observed (Fig 5). Infected mice that received the ITC treatment showed a slight but higher expression of the MMP-15 and TIMP-2 genes relative to infected non-treated animals. Remarkably, the combined therapy BMMSCs/ITC induced a synergistic reduction of Col3α1, TGF-β-3, MMP-8, MMP-12, and TIMP-1, as well as an increase of TIMP-2 gene expression, when compared to infected mice that received cell transplantation (Fig 5).
  Overall, our results demonstrated an exacerbating effect of the BMMSCs therapy on pulmonary fibrosis induced by P. brasiliensis infection. We hypothesized that this outcome could be triggered by either the interaction with P. brasiliensis compounds or by the inflammatory microenvironment induced by this process. Nonetheless, the combined therapy ITC/BMMSCs showed promising results since synergistically it reduced TIMP-1 and MMP-13. Thus, the use of BMMSC under different conditions or combined with other treatments (e. g. ITC) opens the possibility to new therapeutic approaches for this type of fibrosis resulting from an infectious disease. PCM is considered a neglected tropical disease mostly affecting low income individuals who live in underdeveloped Latin American rural regions where the technology and the resources needed to administer the immunotherapeutic measures here suggested would probably not be available. Nonetheless, the implementation of cellular therapies is progressing and the prospects are to arrive in a few years to the administration of autologous bone marrow or stem cells obtained from adipose tissues even in these regions.
summary: This is the first study that evaluates the effect of BMMSCs therapy for lung fibrosis induced by the fungal pathogen Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis, one of the most important systemic endemic mycosis diagnosed in South America and Central America. Our findings showed an impaired anti-fibrotic effect of BMMSCs transplantation. This effect could be triggered by either the chronic inflammatory microenvironment induced by P. brasiliensis or by a direct interaction between BMMSCs and the fungus, resulting in an exacerbation of the pulmonary fibrosis. In fact, the pro-fibrotic effect exerted by BMMSCs was toned-down by the usage of the antifungal ITC.
keywords: medicine and health sciences
  pathology and laboratory medicine
  pathogens
  immunology
  fibrosis
  microbiology
  fibroblasts
  collagens
  animal models
  developmental biology
  model organisms
  signs and symptoms
  stem cells
  connective tissue cells
  experimental organism systems
  paracoccidioides brasiliensis
  mesenchymal stem cells
  fungal pathogens
  research and analysis methods
  mycology
  inflammation
  animal cells
  proteins
  medical microbiology
  gene expression
  microbial pathogens
  connective tissue
  biological tissue
  mouse models
  immune response
  biochemistry
  diagnostic medicine
  cell biology
  anatomy
  genetics
  biology and life sciences
  cellular types
title: Impaired anti-fibrotic effect of bone marrow-derived mesenchymal stem cell in a mouse model of pulmonary paracoccidioidomycosis