R1R2 peptide ameliorates pulmonary fibrosis in mice through fibrocyte migration and differentiation

<div><p>Circulating fibrocytes play a key role in the pathogenesis of pulmonary fibrosis. Fibrocytes are bone marrow-derived leukocytes, which enter the lungs in response to their chemoattractant CXCL12 and differentiate into fibroblasts or myofibroblasts, leading to excess deposition of the collagen-rich extracellular matrix. Matrix metalloproteinase (MMP)-9 and MMP-2, secreted by fibrocytes, degrade the subendothelial basement membrane and promote fibrocyte influx into the lungs. Here, we demonstrate that R1R2, a novel peptide derived from the bacterial adhesin SFS, attenuates pulmonary fibrosis by preventing the differentiation of fibrocytes into myofibroblasts and by reducing the invasion of fibrocytes through basement membrane-like proteins. Moreover, our findings reveal dual regulation of R1R2 on MMP-9 through reduced enzymatic activity on gelatin and increased cleavage of CXCL12. These data suggest that R1R2 has potent anti-fibrotic effects against pulmonary fibrosis.</p></div

R1R2 reduces collagen type I content in the fibrotic lung tissues.

<p>(A, B) Confocal images of immunofluorescence staining for type I collagen 14 days after bleomycin administration, accompanied by R1R2 and the scrambled peptide (20 μM). Bar, 50 μm. (C, D) Representative immunoperoxidase images showing in a bleomycin-treated lung 14 days after bleomycin instillation, accompanied by R1R2 and the scrambled peptide administration (20 μM). (E) Quantification of the percentage of type I collagen⁺ area in a fibrotic lung 14 days after bleomycin instillation (Saline: n = 7, Bleo+Scrambled: n = 7, Bleo+R1R2: n = 5). Data are expressed as the mean ± SEM. *** <i>P</i> < 0.001, one-way analysis of variance followed by a Bonferroni <i>t</i> test.</p

R1R2 disables the proteolytic activity of MMP-9.

<p>(A) Equal amounts of R1R2, the scrambled peptide, or bovine serum albumin (BSA; 20 μg/mL) were coated onto 96-well plates; fibronectin (FN), collagen (Col), or phosphate-buffered saline (PBS) were added into the wells, and the interaction between the peptides was evaluated by measuring the absorbance at 405 nm. **** <i>P</i> < 0.0001, one-way analysis of variance (ANOVA) followed by a Bonferroni <i>t</i> test. (B) Biotinylated R1R2 or scrambled peptide (20 μg/mL) were added into 96-well plates coated with catalytic domain or full-length proteins (FLPs) of MMP-9 and MMP-2. Bound R1R2 and the scrambled peptide were quantified at 405 nm by using a solid-phase binding assay following the incubation of anti-biotin antibodies. **** <i>P</i> < 0.0001 by t-test. (C, D) Enzymatic activity levels of catalytic domain (C) or FLPs (D) of MMP-9 were determined using a fluorescence gelatinolytic assay in the presence of 10 times the molar ratio of R1R2 or the scrambled peptide. (E, F) Enzymatic activity of MMP-9 was evaluated using a fluorescence gelatinolytic assay in the presence of 300 μg/mL (0.9 μM) FN and 20 μM R1R2 or the scrambled peptide. (G, H) Enzymatic activity of catalytic domain (G) or FLPs (H) of MMP-2 was determined using a fluorescence gelatinolytic assay in the presence of 10 times the molar ratio of R1R2 or scrambled peptide. Data are expressed as the mean ± SD. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, and *** <i>P</i> < 0.001 by <i>t</i> test.</p

R1R2 decreases CXCR4⁺ cell accumulation in the bleomycin-induced fibrotic lung tissues.

<p>(A)-(C) Immunohistochemistry (IHC) for CXCR4 in a normal or bleomycin-treated lung 14 days after bleomycin instillation, accompanied by R1R2 and the scrambled peptide administration (20 μM). (D) Quantitative analysis of immunostaining intensities of CXCR4 in a fibrotic lung 14 days after bleomycin instillation (n = 7). Data are expressed as the mean ± SEM. *<i>P</i> < 0.05 and **<i>P</i> < 0.01, one-way analysis of variance followed by a Bonferroni <i>t</i> test. (E)-(H) Confocal images of immunofluorescence staining for CXCR4 (red) and CD31 (green) in a fibrotic lung 14 days after bleomycin instillation, accompanied by R1R2 and the scrambled peptide (20 μM). Sections were counterstained with DAPI. (I)-(N) Confocal images of immunofluorescence staining for CXCR4 (red) and SMAA (green) in a bleomycin-treated lung 14 days after bleomycin treatment. The boxed portion of the low magnification images (I)-(K) is presented at higher magnification (L)-(N) below. A capillary is delineated by dashed lines. Sections were counterstained with DAPI.</p

R1R2 reduces collagen content in bleomycin-induced fibrotic lung tissues.

<p>(A) Collagen content was biochemically measured using a hydroxyproline assay in the lung 14 days after bleomycin instillation, accompanied by 20 μM R1R2 and the scrambled peptide (Saline: n = 6, Bleo+Scrambled: n = 8, Bleo+R1R2: n = 6). Data are expressed as the mean ± standard error of the mean (SEM). ** <i>P</i> < 0.01, one-way analysis of variance (ANOVA) followed by a Bonferroni <i>t</i> test for subsequent pairwise comparison. (B–D) Picrosirius red staining of lung sections from the scrambled peptide or R1R2. Quantification of the percentage of the area (B), which is yellow/orange/green (Saline: n = 7, Bleo+Scrambled: n = 7, Bleo+R1R2: n = 7). Data are expressed as the mean ± standard error of the mean (SEM). *** <i>P</i> < 0.001, one-way ANOVA followed by a Bonferroni <i>t</i> test. Representative images of picrosirius red staining in the fibrotic lungs of the scrambled group (C) and R1R2 group (D). Bar, 250 μm.</p

R1R2 attenuates the presence of myofibroblasts.

<p>(A)-(C) Representative immunostaining images showing smooth muscle α-actin positive (SMAA⁺) myofibroblasts in a normal or bleomycin-treated lung 14 days after bleomycin instillation, accompanied by R1R2 and the scrambled peptide administration (20 μM). Bar, 30 μm. Arrows and double arrows indicate SMAA⁺ myofibroblasts. (D) Quantitative analysis of immunostaining intensities of SMAA in a fibrotic lung 14 days after bleomycin instillation (n = 7). Data are expressed as the mean ± SEM. * <i>P</i> < 0.05 and ****<i>P</i> < 0.0001, one-way analysis of variance followed by a Bonferroni <i>t</i> test.</p

R1R2 increases CXCL12 degradation by MMP-9.

<p>CXCL12 degradation was determined by SDS-PAGE, followed by Coomassie blue staining. Lane 1: marker. Lane 2: MMP-9 (0.1 μg) was incubated with CXCL12 (2 μg) at room temperature for 3h. Lanes 3 and 4: MMP-9 was incubated with CXCL12 at room temperature for 3h with the scrambled peptide (100 μg) and R1R2 (100 μg), respectively. Lane 5 and 6: scrambled peptide and R1R2 maintained at room temperature for 3h. Lanes 7: MMP-9 was incubated with R1R2 at room temperature for 3h. The smear at the bottom of the gel indicated the slightly degraded products of proteins.</p

R1R2 regulates fibrocyte differentiation into myofibrblasts.

<p>(A, B) Western blot analyses of CXCR4 and SMAA expression in the isolated fibrocytes treated with R1R2 or the scrambled peptide for 24 hours (h). Data are expressed as the mean ± standard deviation (SD). *<i>P</i> < 0.05 by <i>t</i> test.</p

R1R2 impedes fibrocyte infiltration into the bleomycin-induced fibrotic lung tissues.

<p>(A) Fibrocytes were isolated and sorted by flow cytometry after staining for CD45, type I collagen (collagen I), and CXCR4. Fibrocytes were identified as CD45⁺, type I collagen⁺, and CXCR4⁺. (B) Cell numbers of CD45⁺/collagen I⁺/CXCR4⁺ fibrocytes are presented through flow cytometry analysis after R1R2 and scrambled peptide treatment in the bleomycin-induced fibrotic lung tissues (n = 6). Data are expressed as the mean ± SEM. **<i>P</i> < 0.01 by <i>t</i> test.</p

R1R2 impairs the invading capacity of fibrocytes through the BM by reducing the proteolytic activity of MMP-9.

<p>(A, B) CD45⁺, type I collagen⁺, and CXCR4⁺ fibrocytes isolated from bleomycin-treated lungs (14 days) were seeded in the upper chamber and treated with R1R2 or the scrambled peptide (1000 nM) for 24 h. CXCL12 (20 ng/mL) or 10% fetal bovine serum (FBS) were added in the bottom wells. CXCL12-induced fibrocyte invasion through Matrigel (A). Data are expressed as relative fluorescence units (RFU). The invasive capacities through Matrigel-coated inserts between R1R2- and scrambled peptide-treated fibrocytes are shown. Results were normalized for FBS-induced fibrocyte invasion. ** <i>P</i> < 0.01 by <i>t</i> test. Quantitative CXCL12-induced fibrocyte transwell migration (B). * <i>P</i> < 0.05 by <i>t</i> test. (C) Isolated fibrocytes were treated with R1R2 or scrambled peptide for 24 h. Gelatin zymography of the conditioned medium is shown. (D) MMP-9 gene expression by fibrocytes treated with R1R2 and the scrambled peptide. Gene expression was evaluated by quantitative real-time polymerase chain reaction. Data are expressed as the mean ± SD.</p