Mesenchymal stromal cell exosomes prevent

Exosome isolation, purification, and characterization.
(A) Concentrated ...

Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF).

 

Exosome isolation, purification, and characterization.
(A) Concentrated ...

 

Mesenchymal stromal cell exosomes prevent and revert experimental pulmonary fibrosis through modulation of monocyte phenotypes

Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF). The aim of this study was to test the therapeutic effects of extracellular vesicles produced by human BM MSCs (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate mechanisms of action. Adult C57BL/6 mice were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently, or for reversal models, at day 7 or 21. Experimental groups were assessed at day 7, 14, or 28. Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented or reversed by MEx treatment. MEx modulated lung macrophage phenotypes, shifting the proportions of lung proinflammatory/classical and nonclassical monocytes and alveolar macrophages toward the monocyte/macrophage profiles of control mice. A parallel immunomodulatory effect was demonstrated in the BM. Notably, transplantation of MEx-preconditioned BM-derived monocytes alleviated core features of pulmonary fibrosis and lung inflammation. Proteomic analysis revealed that MEx therapy promotes an immunoregulatory, antiinflammatory monocyte phenotype. We conclude that MEx prevent and revert core features of bleomycin-induced pulmonary fibrosis and that the beneficial actions of MEx may be mediated via systemic modulation of monocyte phenotypes.
 
Introduction

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive respiratory disease that is characterized by clinical features such as shortness of breath, hypoxemia, radiographically evident pulmonary infiltrates, and continuing accumulation of fixed fibrosis (13). Arguably, the complex interplay between immune cell subsets, coupled with an incomplete understanding in disease pathophysiology, have contributed to the paucity of successful therapies (46). In turn, IPF remains a fatal disease with, at present, a 5-year survival rate of less than 10% from the time of diagnosis (79). Thus, with no effective therapy for either the prevention or treatment of IPF, the need for new therapies is paramount.

To this end, novel approaches are required to address this multifactorial progressive disease. Interestingly, in experimental models of pulmonary fibrosis, mesenchymal stromal/stem cell (MSC) therapy has shown promise, reducing lung collagen deposition, improving Ashcroft score, and decreasing inflammatory markers in bronchoalveolar lavage (1014). Despite such physiological improvements in the recipient lung following MSC transplantation, there is a burgeoning awareness that the mechanism of therapeutic action is predominantly paracrine. Indeed, one of the major therapeutic modalities identified in the MSC secretome are extracellular vesicles (EVs), including exosomes, the EV subset that is generated through the endocytic/endosomal pathway (1518). Recently, we and others have shown that i.v. delivery of purified human MSC-derived exosomes (MEx) has provided substantial functional and immunomodulatory benefits in several experimental models of lung disease (151920) characterized by low levels of fibrosis. In this study, we utilize the murine bleomycin–induced lung injury model to investigate the therapeutic and immunomodulatory capacity of MEx on IPF pathology, a disease with prominent features of fibrosis.

Results

Purification, isolation, and characterization of exosomes. Exosomes were isolated from fraction 9 (F9) of concentrated cell culture supernatants after flotation on an iodixanol cushion (Figure 1A). Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that both human BM MSCs and human dermal fibroblasts (HDFs) gave rise to a heterogeneous exosome population that occupied a diameter of ~35–150 nm and exhibited the typical morphological features of exosomes (Figure 1, B and C). Purified MEx and fibroblast-derived exosome (FEx) fraction (F9) had comparable particle counts (8.6 ± 1 × 10 10 and 9.5 ± 1 × 10 10, respectively) (Figure 1A, data for FEx not shown). Immunoblots demonstrated that all exosome preparations were positive for established exosome markers (CD63, ALIX, Flotillin-1 [FLOT1], and CD9) and lacked the cellular marker GM130 (Figure 1D and Supplemental Figure 3; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.128060DS1).

Exosome isolation, purification, and characterization.Figure 1

Exosome isolation, purification, and characterization. (A) Concentrated conditioned media (CM) was floated on an iodixanol (IDX) cushion gradient, and the purify exosome fraction was isolated from fraction 9 (F9; mesenchymal stromal/stem cell–extracellular vesicles/exosomes [MEx] density ~ 1.16–1.18 g/mL). Nanoparticle tracking analysis (NTA) and protein concentration was used to assess exosome concentration and particle/protein ratio in the IDX cushion (12 × 1 mL fractions), respectively. (B) Transmission electron microscopy images demonstrating heterogeneous vesicle morphology (scale bar: 500 nm) . (C) Size distribution and particle concentration was measured by NTA. (D) The IDX cushion gradient fractions were analyzed by Western blot (fractions 1–6 and 7–12, side by side), using antibodies to proteins representing exosome markers. Equivalent volume of each fraction was loaded per lane. Representative images are shown. Flotillin 1 (FLOT1), ALIX, and tetraspanins (CD63, CD9) were enriched in F9. GM130 (cytoplasmic marker) was absent in F9.

A bolus dose of MEx prevents and reverts bleomycin-induced pulmonary fibrosis. We used the murine bleomycin–induced experimental model of pulmonary fibrosis to assess the therapeutic capacity of MEx (21). First, to evaluate the preventive effect of MEx, 14-week-old mice received a single dose (endotracheal administration) of bleomycin (3 U/kg) or saline (vehicle control) on day 0. Mice that received bleomycin were compared with an untreated control group. Concurrent with bleomycin administration (day 0), treatment groups received a single i.v. dose of MEx, FEx, or exosome-free iodixanol vehicle. Mice were sacrificed at day 14, and lung sections were assessed for quantification of fibrosis (Masson’s trichrome) and collagen content (schematic shown in Figure 2A). Bleomycin-exposed mice demonstrated a histological pattern akin to human IPF, characterized by severe septal thickening and prominent fibrosis (Figure 2B). Accordingly, compared with the control group, animals that received bleomycin presented with a greater Ashcroft score (1.76 ± 0.6 vs. 7.2 ± 0.3, P < 0.001, Figure 2C), coupled with an elevated degree of collagen deposition (2.18 ± 0.09 vs. 3.02 ± 0.23 mg/mL, P < 0.05, Figure 2D). A single i.v. dose of MEx dramatically blunted the bleomycin-induced pulmonary fibrosis, improved the Ashcroft score (2.7 ± 0.6, P < 0.0001), and restored collagen content to levels similar to their untreated-counterparts (2.18 ± 0.15 mg/mL, P < 0.05). FEx and exosome-free iodixanol served as biologic and treated-vehicle controls, respectively, and had no effect on pulmonary fibrosis, Ashcroft score, or collagen deposition (P > 0.05, Figure 2, B–D).

A bolus dose of MEx prevents bleomycin-induced pulmonary fibrosis.Figure 2

A bolus dose of MEx prevents bleomycin-induced pulmonary fibrosis. (A) Fourteen-week-old mice (C57BL/6 strain) received endotracheal bleomycin (3 U/kg) or 0.9% normal saline on day 0 (control). Concurrently, treated groups received a bolus i.v. dose of MEx (Bleo+MEx), FEx (Bleo+FEx), or iodixanol (Bleo+IDX). Mice were sacrificed on day 14. The cross symbol represents animal harvest. (B) Lung sections were stained with Masson’s trichrome. Images were taken at ×100 magnification. Bleomycin, Bleo+FEx, and Bleo+IDX showed architectural destruction, alveolar septal thickening, and fibrotic changes. Administration of MEx to bleomycin-challenged mice substantially reduced fibrosis and alveolar distortion. Scale bar: 100 μm. (C) Lung fibrosis was measured at day 14 by Ashcroft score. (D) Collagen deposition was assessed by Sircol assay and represented as mg/mL of left lung homogenate. (E) Data are representative of 3 independent experiments, mean ± SD. n = 3–4 per experimental group; each symbol represents 1 mouse. *P < 0.05; ****P < 0.0001, 1-way ANOVA followed by Fisher’s LSD post hoc analysis. (F and G) MEx therapy decreases apoptosis. Annexin V/PI staining in whole lungs shows an increase in apoptosis (annexin V+PI) in bleomycin-exposed mice compared with control and bleomycin+MEx mice. TUNEL staining in whole lung sections shows increase in apoptosis (green) in the bleomycin-exposed group of mice compared with control and bleomycin+MEx. Nuclei were stained with DAPI. Images obtained at ×20 magnification. MFI quantified using ImageJ software and normalized for DAPI. Data are representative of 2 independent experiments, mean ± SD. n = 6–8 per group; each symbol represents 1 mouse. *P < 0.05; **P < 0.01 vs. bleomycin-exposed mice. One-way ANOVA followed by Fisher’s LSD post hoc analysis. MEx, mesenchymal stromal/stem cell–extracellular vesicles/exosomes; FEx, Human dermal fibroblast exosomes; IDX, iodixanol; Bleo, bleomycin; PI, propidium iodide.

Given the critical role of apoptosis in the pathogenesis of fibrotic lung diseases (2223), we also investigated the antiapoptotic effect of MEx in the lung parenchyma. Here, both flow cytometric analysis of annexin V and TUNEL staining in whole lung sections revealed that bleomycin control mice presented with elevated levels of whole lung apoptosis compared with untreated control animals and that MEx treatment efficiently reduced the degree of apoptosis (Figure 2, E–G).

In addition to the preventive capacity of MEx, we also assessed the ability of MEx to revert bleomycin-induced pulmonary fibrosis after the injury occurred. Here, following endotracheal bleomycin instillation at day 0, we administered a bolus dose of MEx at day 7 and assessed experimental groups at day 14 (schematic shown in Figure 3A). Notably, a single MEx dose substantially reverted bleomycin-induced pulmonary fibrosis, improved the Ashcroft score (P < 0.0001), and restored collagen content to levels akin to their untreated counterparts (P < 0.05, Figure 3, B–D).

MEx therapy reverts bleomycin-induced pulmonary fibrosis.Figure 3

MEx therapy reverts bleomycin-induced pulmonary fibrosis. (A and E) MEx were administered 7 days or 21 days after the administration of bleomycin, and mice were sacrificed on day 14 or 28. Cross symbol represents animal harvest. (B and F) Lung sections were stained with Masson’s trichrome. Images were taken at ×100 magnification. Scale bar: 100 μm. Representative lung sections from control, Bleomycin, and Bleo+MEx mice. (CDG, and H) Lung sections were assessed for collagen deposition (C and G) and histology (D and H). Data represent mean ± SD. Data in AD are representative of 3 independent experiments, n = 3–4 per experimental group. Data in EG are representative of 2 independent experiments, n = 5–6 per experimental group; each symbol represents 1 mouse. *P < 0.05; **P < 0.01; ****P < 0.0001. One-way ANOVA followed by Fisher’s LSD post hoc analysis.

We also sought to test the capacity of MEx to revert core features of bleomycin-induced pulmonary fibrosis at a late rescue time point. Here, mice that received bleomycin (day 0) were given a bolus MEx dose at day 21 and assessed at day 28 (schematic shown in Figure 3E). Compared with bleomycin control animals, late-rescue MEx treatment significantly reduced the bleomycin-induced elevation in collagen content (P < 0.0001, Figure 3, F and G), although no difference was noted in the Ashcroft score (P > 0.05, Figure 3, F and H).

MEx treatment modulates alveolar macrophage and monocyte populations in the lung. Alveolar macrophages (alveolar MΦ) and infiltrating monocytes play a pivotal role in pulmonary inflammation and in the development and progression of fibrosis (2426). To investigate changes in immune cell populations following bleomycin-induced lung injury, we performed whole lung cytometric analysis at days 7 and 14 in animals that received MEx at day 0. On day 7, compared with control mice, we noted a decrease in the proportion of alveolar MΦ and nonclassical monocytes (defined as CD45+CD11bCD11c+CD64+ cells and CD45+ CD11b+MHCIICD64lo/intCCR-2Ly6clo cells, respectively), coupled with a concomitant increase in the number of proinflammatory classical monocytes (defined as CD45+CD11b+MHCIICD64lo/intCCR-2+Ly6chi cells) in bleomycin-exposed animals. MEx therapy effectively rescued all cell populations, increasing the levels of alveolar MΦ (P < 0.01) and nonclassical monocytes (P < 0.05), while decreasing the number of classical monocytes comparable with controls (P < 0.001, Figure 4A).

MEx therapy modulates alveolar macrophage and monocyte populations in the lFigure 4

MEx therapy modulates alveolar macrophage and monocyte populations in the lung. (A and B) Flow cytometry was used to assess whole lung monocyte and alveolar macrophage (alveolar MΦ) at day 7 (A) and day 14 (B). (C) Classical monocytes (Mo) were defined as CD45+CD11b+MHCIICD64CCR-2+Ly6Chi. Nonclassical monocytes were defined as CD45+CD11b+MHCIICD64CCR-2Ly6Clo. Representative gating strategy of alveolar MΦ (CD45+ CD11b CD11c+ CD64+ cells), classical Mo, and nonclassical Mo. The gating strategy was performed according to fluorescence minus one controls (Supplemental Figure 1). Data are representative of 3 independent experiments, mean ± SD, n = 4–5 per experimental group; each symbol represents 1 mouse. *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA followed by Fisher’s LSD post hoc analysis.

Paradoxically, at day 14 (Figure 4B), we found that the proportion of alveolar MΦ was increased (P < 0.05), while the number of classical monocytes were reduced in bleomycin-treated animals, compared with controls. Again, MEx therapy shifted the alveolar MΦ and monocyte profiles toward that of their untreated counterparts. Importantly, cytometric analysis showed that the overall percentage of CD45+ monocytes did not change across the 3 experimental groups at either time point (day 7 and day 14). Representative gating strategy is shown in Figure 4C.

Administration of MEx modulates whole lung inflammation. To investigate the effect of MEx on pulmonary inflammation, whole lung mRNA levels were assessed at days 7 and 14. We noted that gene expression levels of proinflammatory cytokines that were typically associated with activation of MΦ, such as Ccl2 and arginase 1 (Arg1), were dramatically elevated in mice that received bleomycin compared with control animals at both 7 and 14 days), and that this bleomycin-induced elevation was reduced by MEx administration (P < 0.05, Figure 5, A and B). Il6 showed a similar trend, but the difference did not reach statistical significance between groups (P > 0.05). Tgfb expression was similar at day 7 and day 14 between all experimental groups (P > 0.05; Figure 5, A and B). In accordance, immunofluorescence analysis of lung tissue sections showed a marked increase in the expression of CD206 and ARG1 proteins in bleomycin-exposed mice, and it was effectively reduced by MEx therapy to levels akin to the control group (P < 0.01; Figure 5, C and D). As expected, bronchoalveolar lavage (BAL) protein content was increased in bleomycin-exposed mice as opposed to control. MEx therapy decreased the BAL total protein content (P < 0.05, Figure 5E).

Administration of MEx modulates whole lung inflammation.Figure 5

Administration of MEx modulates whole lung inflammation. (A and B) Whole lung qPCR demonstrated an increase in the mRNA expression levels of Ccl2 and Arginase1 (Arg1) at days 7 and 14 in bleomycin-challenged animals compared with untreated control mice. This was ameliorated by MEx treatment. (C and DIl6 expression showed a similar trend, but statistical significance was not achieved. Levels of Tgfb remained unchanged between the groups. Results are expressed relative to control expression (fold change). Immunofluorescence analysis of lung sections using antibodies against ARG1 (green) and CD206 (red) . Nuclei staining performed with DAPI (blue). Images obtained at ×10 magnification. Mean fluorescence intensity (MFI) normalized for cell number (DAPI stain). Analysis performed by ImageJ software. Mean ± SD, n = 5–8 per group; each symbol represents 1 mouse. *P < 0.05; ** P < 0.01. One-way ANOVA followed by Fisher’s LSD post hoc analysis. (E) BAL protein content was decreased after MEx treatment. Mean ± SD, n = 3–4 per group; each symbol represents 1 mouse. *P < 0.05; **P < 0.01. One-way ANOVA followed by Fisher’s LSD post hoc analysis. BAL, bronchoalveolar lavage.

MEx therapy modulates monocyte population in the BM. Recruited monocytes from the BM to the lung have been associated with the pathophysiology of pulmonary fibrosis (2427) and considering our finding that MEx therapy was associated with modulation of monocyte populations in the lung, we next investigated if i.v.-delivered MEx exert immunomodulatory actions directly on BM progenitors. Here, mice were sacrificed at day 7 and myeloid cells in the BM were assessed by flow cytometry.

Interestingly, bleomycin-exposed mice presented with dramatically reduced levels of nonclassical monocytes present in the BM compared with control animals (14.18 % ± 2.7 vs 32.3 % ± 5.5, P < 0.05, respectively). A single dose of MEx therapy administered concomitantly with bleomycin, shifted the BM nonclassical monocyte profile (27.57 % ± 5.7, P < 0.05) toward that of their untreated counterparts. Moreover, the classical monocyte population in the bleomycin-exposed group was greater (67.8 % ± 1.7) than the control group (50.1 % ± 3.2, P < 0.001), and effectively suppressed by MEx therapy (57.5 % ± 3.9, P < 0.05, Figure 6A). Notably, cytometric analysis showed that the proportion of total monocytes did not change across the 3 experimental groups (Figure 6).

MEx modulate monocyte phenotype in the BM.Figure 6

MEx modulate monocyte phenotype in the BM. (A) To investigate the systemic effects of MEx, we analyzed the myeloid cell profile of the BM at day 7 by flow cytometry. Classical monocytes (Classical Mo) were defined as CD45+CD11b+MHCIICD64CCR-2+Ly6Chi. Nonclassical monocytes (Nonclassical Mo) were defined as CD45+CD11b+MHCIICD64CCR-2Ly6Clo. (B) Representative gating strategy. Data represents mean ± SD, n = 5–8 per group; each symbol represents 1 mouse *P < 0.05; *** P < 0.001. One-way ANOVA followed by Fisher’s LSD post hoc analysis.

MEx therapy reprograms monocytes to a nonclassical phenotype. Since MEx therapy was associated with a “prohomeostatic” shift in lung MΦ/monocyte phenotype and a concomitant rescued BM nonclassical monocyte population, we sought to determine whether MEx therapy affords such robust physiological changes in the lung by directly modulating the BM-myeloid/monocyte (My/Mo) cell lineage phenotype. To investigate the modulatory effect of MEx on My/Mo populations, we preconditioned these cells with MEx ex vivo. Here, primary myeloid cells were isolated from the BM of WT (healthy) FVB mice aged 6–8 weeks. At day 0 of in vitro culture, the population of myeloid cells was heterogeneous with monocyte lineage accounting for approximately 24% of the cells in culture (BM-derived myeloid cells [BMDMy], data not shown). Cells were subsequently cultured for 3 days in the presence of MΦ colony stimulating factor (M-CSF) to drive the monocyte/MΦ lineage (28). Myeloid cells were treated with MEx or cell culture (MEx-free) medium alone (schematic shown in Figure 7A) and confirmed to be CD45+ and CD11b+ by flow cytometry (>90%, Figure 7B). There was no difference in the number of viable cells and the degree of apoptosis between MEx treated or media (control) treated myeloid cells (data not shown). To understand the impact of MEx on My/Mo cells, we next profiled this cell lineage by performing proteome analysis after MEx treatment using Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). To this end, we treated the My/Mo cells on day 1 and 2 with MEx (Figure 7A) or FEx (control) and harvested cells at day 3 for protein extraction. Analysis of the proteomic data revealed a signature composed of 84 features whose abundance varied significantly between MEx-treated My/Mo cells and FEx-treated controls (FDR < 0.25, Figure 7C). Interestingly, MEx treatment was associated with lower abundance of multiple proinflammatory proteins such as those belonging to the MAPK and the serpin family. On the other hand, MEx increased the abundance of proregulatory proteins such as Mrc1 and CerS2. We next set out to test if this proteomic signature was indicative of either a classical or a nonclassical phenotype in MEx-treated monocytes using publicly available transcriptome data of Ly6cpos and Ly6cneg BM-derived monocytes (BMDMo) (29). Indeed, gene set testing revealed that genes encoding for the proteins overrepresented in the MEx-treated group were overexpressed in nonclassical Ly6cneg monocytes (P < 0.001), whereas genes encoding for the proteins under-represented in MEx-treated My/Mo cells were overexpressed in classical Ly6cpos monocytes (P < 0.001) (Figure 7D and Supplemental Figure 2). These results provide compelling evidence that the protective effects induced by MEx treatment are, at least in part, mediated by reprogramming of BMDMo to a nonclassical phenotype.

MEx therapy reprograms monocytes to a nonclassical (Ly6Cneg) phenotype.Figure 7

MEx therapy reprograms monocytes to a nonclassical (Ly6Cneg) phenotype. (A) BM-derived myeloid cells (BMDMy) were isolated from 6- to 8-week-old FVB mice, cultured ex vivo for 3 days to drive the monocytic lineage, and treated with MEx (MEx dose: 1 × 106 MSC equivalents) or media alone on day 1 (D1) and day 2 (D2). (B) Flow cytometric analysis of BM-derived monocytes (BMDMo) after 3 days of culture showed that > 90% were CD45+CD11b+ cells. Student’s t test (2 tailed) . To assess the direct effect of MEx on monocyte phenotype, BMDMo were treated with 2 doses of MEx or FEx on days 1 and 2, and cells were processed for liquid chromatography–tandem mass spectrometry (LC-MS/MS) on day 3, as shown in A. (C) Eighty-four peptides where deemed differentially abundant in MEx-treated BMDMo vs. FEx-treated controls at FDR < 0.25. (D) Gene set analysis revealed that genes encoding for peptides abundant in MEx-treated BMDMo were, on average, overexpressed in the Ly6Cneg monocytes (P < 0.001), whereas genes encoding for peptides abundant in the FEx-treated BMDMo were, on average, overexpressed in the Ly6Cpos monocytes (P < 0.001) . Transcriptome data were obtained from GSE95411.

Transplantation of BMDMo preconditioned with MEx prevents bleomycin-induced pulmonary fibrosis. Since our proteomic data indicated a primary effect of MEx on monocyte phenotypes within the BMDMy populations, we sought to address whether the promodulatory effect of MEx on BMDMo is responsible for the prevention of fibrosis. We performed adoptive transfer experiments of BMDMo that were preconditioned with MEx (as shown in the schematic of Figure 8A) ex vivo and delivered into the bleomycin-induced pulmonary fibrosis model. Mice that received 2 doses of BMDMo on days 0 and 3 after bleomycin injection were sacrificed at day 14 and lungs were assessed for histology and collagen content (Figure 8). Compared with bleomycin control mice, the group that received BMDMo preconditioned with MEx (BMDMo+MEx) presented with a drastically improved Ashcroft score (7.2 ± 0.3 vs. 3.5 ± 1, P < 0.0001, respectively) coupled with lower total collagen levels (3.02 ± 0.23 vs. 0.68 ± 0.41 mg/mL, P < 0.001, respectively). Interestingly, mice that received BMDMo control (not preconditioned with MEx, [BMDMo+Media]), presented with evidence of partially reduced fibrosis, as assessed by the Ashcroft score (P < 0.05), however collagen deposition was similar between the 2 experimental groups (Figure 8, D and E).

Transplantation of BM-derived monocytes preconditioned with MEX prevents blFigure 8

Transplantation of BM-derived monocytes preconditioned with MEX prevents bleomycin-induced pulmonary fibrosis. (A) We explored the effects of ex vivo treated BM-derived monocytes and alveolar macrophages (alveolar MΦ) in the prevention of fibrosis. BMDMo were stained with lipophilic dye (DiI) on day 3 (D3) and i.v. administered at a 1:1 ratio on days 0 and 3 to C57BL/6 mice following endotracheal instillation of bleomycin. Mice were sacrificed at day 14. Cross symbol represents animal assessment. (B) DiI-labeled BMDMo were detected in the lung 14 days after injection, while no signal was seen in the lung of mice that received cell-free dye (vehicle). Images obtained at ×20 magnification. Arrow marks the DiI-labeled monocytes. (CE) Pulmonary fibrosis was ameliorated in mice that received monocytes that were preconditioned with MEx, while alveolar MΦ had little effect. Inserts were taken at ×100 magnification. Data are representative of at least 2 independent experiments. Mean ± SD, n = 4–5 per experimental group; each symbol represents 1 mouse. Between-group comparison: *P < 0.05; ** P < 0.01; ***P < 0.001; ****P < 0.0001. One-way ANOVA followed by Fisher’s LSD post hoc analysis. Scale bar: 100 μm. DiI, 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindocarbocyanine Perchlorate (“DiI”; DiIC18(3)).

To explore if the antifibrotic effect of MEx is due to resident alveolar MΦ, we also administered alveolar MΦ preconditioned with MEx (alveolar MΦ+MEx) endotracheally following bleomycin instillation. Again, alveolar MΦ were sourced from bronchoalveolar lavage fluid (BALF) obtained from WT (healthy) FVB mice aged 6–8 weeks. Importantly, we did not detect any amelioration of fibrosis in mice who received preconditioned alveolar MΦ compared with the bleomycin group (Figure 8, C, D, and E). To further assess the inflammatory changes in the lung after the administration of MEx-preconditioned BMDMo, we quantified the Cd68- and Retnla-expressing MΦ in the pulmonary parenchyma using immunofluorescent staining. Mice that received monocytes and were preconditioned with MEx showed reduced pulmonary MΦ numbers compared with BMDMo plus Media–treated littermates (P < 0.05, Figure 9).

Transplantation of BM-derived monocytes preconditioned with MEx prevents blFigure 9

Transplantation of BM-derived monocytes preconditioned with MEx prevents bleomycin-induced pulmonary inflammation. (A and B) Pulmonary inflammation was analyzed at day 14 after the injection of MEx-preconditioned BMDMo (see details in Figure 8) by counting Cd68- and Retnla-expressing macrophages in the pulmonary parenchyma. Mice that received monocytes that were preconditioned with MEx showed reduced macrophage numbers compared with BMDMo+media–treated littermates. Images were taken at ×200 magnification. Arrows mark inflammatory macrophages in alveolar spaces. Data are representative of at least 2 independent experiments, n = 3–5 per experimental group. Mean ± SD; each symbol represents 1 mouse. Between-group comparison: *P < 0.05, Student’s t test (2-tailed). Scale bar: 100 μm.

 

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