Fig 1.
Genetic deletion of S1pr2 prevents bleomycin–induced lung fibrosis.
The lungs (A-E) and BALF (F-I) were collected from mice on day 33 after starting saline or bleomycin administration and analyzed. (A) Representative images at low magnification (x40) of Sirius Red–stained whole lung sections from saline–or bleomycin (Bleo)–administered wild-type (WT) and S1pr2-/- mice. Scale bar: 600 μm. (B) Quantification of fibrotic areas in whole lung sections from WT and S1pr2-/- mice. (C) Representative images at high magnification (x400) of immunostaining using anti-PDGFRα antibody and Sirius Red–stained lung sections from WT and S1pr2-/- mice. Scale bar:100 μm. (D) Representative fluorescent images of EGFP fluorescence (green) and nuclear staining (DAPI, blue) in the lung from Col1α2−EGFP and Col1α2−EGFP:S1pr2-/- mice. Magnification: x400. Scale bar: 50 μm. (E) mRNA expression levels of Fibronectin and Collagen1a1 in the lung from WT and S1pr2-/- mice. (F-I) Total protein concentrations (F), total cell numbers (G), Macrophage numbers (H) and soluble collagen levels (I) in BALF from WT and S1pr2-/- mice. The individual values and the mean ± SEM are shown. N = 5−16 from three to seven independent experiments. *P <0.05, **P <0.01, ***P<0.001, ****P<0.0001 and n.s. no significance (two-way ANOVA followed by the Bonferroni post-test).
Fig 2.
Expression of S1PR2 in the lung of S1pr2LacZ/+ mice.
(A) X-gal stained brain, heart, lung, liver, spleen and kidney from 8-week old WT and S1pr2LacZ/+ mice. (B) X-gal stained lung sections of S1pr2LacZ/+ mice on day 33 after starting administration of saline or bleomycin (Bleo). Magnification: x400. Scale bar: 100 μm. (C) X-gal stained and anti–Mac3 immunostained lung sections of S1pr2LacZ/+ mice on day 33. Magnification: x1000. Scale bar: 100 μm. (D) X-gal stained alveolar macrophages in the BALF smear from bleomycin–administered S1pr2LacZ/+ mice. Magnification, x1000. Scale bar, 100 μm. In (B) and (D), the tissue sections were counterstained by nuclear fast red. In (C) and (D), the arrowheads denote X-gal-stained macrophages. The presented data represent three independent experiments.
Fig 3.
S1pr2 deletion in bone marrow–derived cells suppresses bleomycin–induced lung fibrosis in mice.
Wild-type (WT) and S1pr2-/- mice had been transplanted with BM from either WT or S1pr2-/- mouse donors. The lungs (A, B) and BALF (C-E) were collected from BM–chimeric wild-type or S1pr2-/- mice. on day 33 after starting administration of saline or bleomycin (Bleo) and analyzed. (A) Representative images of Sirius Red–stained whole lung sections Magnification: x40. Scale bar: 600 μm. (B) Quantification of lung fibrotic lesions using image analysis of Sirius Red–stained lung sections. (C-E) Soluble collagen levels (C), total cell numbers (D), and macrophage numbers (E) in BALF. The details of BM transplantation are indicated on each image in (A) and at the bottoms of (B) to (E). The individual values and the mean ± SEM are shown. N = 5−13 from five independent experiments. *P <0.05, **P <0.01, ***P<0.001 and n.s. no significance (two-way ANOVA followed by the Bonferroni post-test).
Fig 4.
A CSF-1 receptor inhibitor inhibits bleomycin–induced lung fibrosis in mice.
The CSF-1 receptor inhibitor BLZ945 (200 mg/kg, open circles), vehicle (20% Captisol) (closed circles) or saline (None group, closed triangles) were administered into wild-type mice given bleomycin once daily by oral gavage. Control wild-type mice (closed square) received i.p. injection of saline. The lungs (A) and BALF (B-E) were collected on day 25 after starting bleomycin administration and analyzed. (A) Representative tissue images of Sirius Red–stained lung sections (Left). Magnification, x400. Scale bar, 100 μm. Quantification of fibrotic areas in Sirius Red–stained lung sections (Right). N, none; V, vehicle; B, BLZ945. (B-E) Macrophage numbers (B), soluble collagen levels (C), total protein concentrations (D), and total cell numbers (E) in BALF. The individual values and the mean ± SEM are shown. N = 4−7 from the single experiments, except the saline-administered wild-type control mice in which N was 20 from multiple experiments in (A) and 2 in (B-E). *P <0.05 (one-way ANOVA followed by the Bonferroni post-test).
Fig 5.
S1pr2 deletion suppresses the expression of profibrotic genes in BALF cells.
The mRNA expression levels of M1 and M2 markers and related genes (A-N) in BALF cells from wild-type (WT) and S1pr2-/- mice on days 7, 22 and 33 after starting administration of bleomycin or saline were determined by qPCR. The open and solid bars indicate the values of saline–and bleomycin (Bleo)–administered mice, respectively. The individual values and the mean ± SEM are shown. Each sample was prepared from the pooled cells obtained from 3–4 mice. N = 3−6 from three independent experiments. *P <0.05, **P <0.01 and ***P<0.001 (two-way ANOVA followed by the Bonferroni post-test).
Table 1.
List of chemokines, cytokines, macrophage-related and extracellular matrix-related genes upregulated by more than 2.0-fold and downregulated by less than 0.5-fold in S1pr2-/- BALF cells relative to wild-type BALF cells from bleomycin-treated mice by microarray analysis.
Fig 6.
Administration of IL-13–neutralizing antibody ameliorates bleomycin–induced lung fibrosis.
(A) IL-13 protein levels in BALF. IL-13 levels in BALF from wild-type (WT) and S1pr2-/- mice on day 22 after starting saline or bleomycin (Bleo) administration were measured by ELISA. The solid and open bars indicate the values of WT and S1pr2-/- mice, respectively. In (B)-(G), the mice received intraperitoneal injection of IL-13–neutralizing antibody (αIL-13) or control IgG (IgG) (5 mg/kg, every 11 day), which was begun 1 day before starting bleomycin (Bleo) administration. The lungs and BALF were collected from mice on day 33 and analyzed. (B) Representative images at low magnification (x40) of Sirius Red–stained whole lung sections from wild-type mice given αIL-13 or IgG. Scale bar, 600 μm. (C) Left: Representative images at high magnification (x400) of Sirius Red–stained lung sections. Scale bar, 100 μm. Right: Quantification of fibrotic areas in whole lung sections. (D-G) Soluble collagen levels (D), total protein concentrations (E), total cell numbers (F), and macrophage numbers (G) in BALF. The individual values and the mean ± SEM are shown. In (A), N = 6. In (C-G), N = 4−5 from the single experiment, except the saline-administered wild-type control mice in which N was 20 from multiple experiments in (C) and 2 in (D-G). *P <0.05, **P <0.01, ****P<0.0001 (two-way ANOVA followed by the Bonferroni post-test in (A) and two-tailed Student’s t-test in (C)-(G)).
Fig 7.
STAT6 activation is suppressed in S1pr2-/- macrophages.
(A) Tyrosine phosphorylation of STAT6 and STAT1 in alveolar macrophages freshly isolated from saline–or bleomycin (Bleo)–administered wild-type (WT) and S1pr2-/- mice. Left: representative western blots. Right: quantified data. The open and solid bars indicate the values of saline–and Bleo–administered mice, respectively. (B,C) mRNA expression levels of Il4ra (B) and Il13ra1 (C) in BALF cells from WT and S1pr2-/- mice. The solid and open bars indicate the values of Bleo–and saline–administered mice, respectively. Each sample was prepared from the pooled cells obtained from 8–13 mice. Lysates of IL-13-stimulated BM–derived wild-type macrophages that had been induced to differentiate in the presence of CSF-1 were used as a positive control. The means ± SEM are shown. N = 3−6 from three or four independent experiments. **P <0.01 and ***P<0.001 (two-way ANOVA followed by the Bonferroni post-test).
Fig 8.
Pharmacological blockade of S1PR2 attenuates lung fibrosis.
The S1PR2 antagonist, S1PR2i, was orally administered into wild-type mice, which began 3 days before starting bleomycin administration. The lungs and BALF were collected from mice on day 33 and analyzed. (A) Representative images at low magnification (x40) of Sirius Red–stained whole lung sections from bleomycin–administered wild-type mice given S1PR2i or vehicle. Scale bar: 600 μm. (B) Representative images at high magnification (x400) of Sirius Red–stained lung sections. Scale bar: 100 μm. (C) Quantification of fibrotic areas in whole lung sections. (D) Soluble collagen levels in BALF. The individual values and the mean ± SEM are shown. N = 8−12 from three independent experiments, except the saline-administered wild-type control mice in which N was 20 from multiple experiments in (C) and 3 in (D). **P <0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA followed by the Bonferroni post-test).