Effect of HSP27 siRNA on bleomycin-induced pulmonary fibrosis in mice.

<p>Mice were intratracheally treated with PBS or bleomycin. On day 4, 6, 9, and 12, mice were intranasally inoculated with 5 μg of control siRNA or HSP27 siRNA using MaxSuppressor In Vivo RNA-LANCEr II. On day 14, mice were sacrificed and lungs were removed. (A) Immunoblot analysis. Protein levels of HSP27 were analyzed by immunoblotting using tissue lysates prepared from right lungs. As a loading control, β-actin was used. A representative image from six independent experiments is shown (above). Quantitative data are shown as mean ± SE (n = 6) (below). *: P<0.05 by Student’s <i>t</i>-test. (B) Masson’s trichrome staining. This was performed using tissue sections from left lungs. Representative images from three independent experiments are shown. The bars indicate 50 μm. (C) Ashcroft’s Score. Fibrosis levels were quantified by Ashcroft’s Score. Data are shown as mean ± SE (n = 7). *: P<0.05 by Student’s <i>t</i>-test. (D) Hydroxyproline content. Tissue homogenates prepared from right lungs were used to determined hydroxproline contents. Data are shown as mean ± SE (n = 7). *: P<0.05 by Student’s <i>t</i>-test. (E) Immunohistochemical staining. Tissue sections from left lungs were immunohistochemically stained for HSP27, α-SMA, and OPN. Representative images from three independent experiments are shown. The bars indicate 20 μm. (F) Airway delivery of FITC-labeled siRNA. Mice were intratracheally treated with bleomycin. On day 4, mice were intranasally inoculated with 5 μg of FITC-labeled siRNA mixed in MaxSuppressor In Vivo RNA-LANCEr II. After 1 h, lungs were removed and frozen sections were made. Tissue sections were fixed and stained for α-SMA. FITC (green) and α-SMA (red) were observed on a fluorescence microscopy. Representative images from four independent experiments are shown.</p

Upregulation of HSP27 in TGF-β1-treated lung fibroblasts.

<p>(A, B) MRC-5. Cells in monolayer were washed twice with PBS to remove FBS, cultured in Opti-MEM for 24 h, and mock-treated or treated with 1 ng/ml of TGF-β1 for the indicated lengths of time. (A) Immunoblot analysis. Protein levels of HSP27, p-HSP27, and α-SMA were determined by immunoblot analysis. For a loading control, β-actin was used. Signal intensities were quantified using Image J software and normalized by β-actin. A representative image from four independent experiments is shown in the left. Quantitative data are shown as mean ± SE (n = 4) in the right. *: P<0.05 by one-way ANOVA. (B) Quantitative PCR. Expression levels of HSP27 and α-SMA mRNAs were determined by quantitative PCR and normalized by GAPDH. Data are shown as mean ± SE (n = 4). *: P<0.05 by one-way ANOVA. (C, D) NHLF. Cells in monolayer were washed twice with PBS, cultured in Opti-MEM for 24 h, and mock-treated or treated with 2 ng/ml of TGF-β1 for indicated length of time. (C) Immunoblot analysis. This was performed as described above. For a loading control, GAPDH was used. A representative image from four independent experiments is shown in the left. Quantitative data are shown as mean ± SE (n = 4) in the right. *: P<0.05 by one-way ANOVA. (D) Quantitative PCR. This was performed as described above. Data are shown as mean ± SE (n = 4). *: P<0.05 by one-way ANOVA. (E, F) NMLF. Cells in monolayer were washed twice with PBS, cultured in Opti-MEM containing 1% FBS for 24 h, and mock-treated or treated with 4 ng/ml of TGF-β1 for the indicated length of time. (E) Immunoblot analysis. This was performed as described above. For a loading control, GAPDH was used. A representative image from six independent experiments is shown in the left. Quantitative data are shown as mean ± SE (n = 6) in the right. *: P<0.05 by one-way ANOVA. (F) Quantitative PCR. This was performed as described above. Data are shown as mean ± SE (n = 6). *: P<0.05 by one-way ANOVA.</p

Effect of HSP27 siRNA on TGF-β1-treated MRC-5.

<p>MRC-5 cells were transfected with control or HSP27 siRNA and cultured for 24 h. After washing to remove FBS, cells were placed in Opti-MEM and treated with 0.5 ng/ml of TGF-β1 for 48 h. (A) Phase contrast microscopic images. Representative results from three independent experiments are shown. The bars indicate 100 μm. (B) Cell viability assay. Dead cells were detected by staining <i>in situ</i> with 5 μg/ml of propidium iotide (PI). PI-positive cells in each field (44 mm²) were counted on a fluorescence microscope. Data are shown as mean ± SE (n = 6). *: P<0.05 by Student’s <i>t</i>-test. (C) Apoptosis assay. Apoptotic cells were detected by flow cytometry using the Annexin V and 7AAD double staining assay. The FACS plots are shown in the left with % of cells in the four gated areas. Columns in the right show % of cells in early apoptosis (Annexin V⁺ and 7AAD^‒) and late apoptotosis (Annexin V⁺ and 7AAD⁺) as mean ± SE (n = 3). *: P<0.05 by Student’s <i>t</i>-test. (D) Immunoblot assay. MRC-5 cells were transfected with control siRNA “C” or HSP27 siRNA “H” and cultured for 24 h. Then, after changing the culture medium to Opti-MEM containing 2% FBS, cells were treated with or without 0.5 ng/ml of TGF-β1 for 48 h. Protein levels of HSP27, α-SMA and fibronectin (FN) were determined by immunoblot analysis. For a loading control, α-tubulin was used. Quantitative data are shown as mean ± SE (n = 4) in the right. *: P<0.05 by Student’s <i>t</i>-test. (E) Quantitative PCR. MRC-5 cells were transfected with control or HSP27 siRNA and cultured for 24 h. Then, after changing the culture medium to Opti-MEM containing 2% FBS, cells were treated with or without 0.5 ng/ml of TGF-β1 for 24 h. Expression levels of HSP27, α-SMA, FN1, α1 type I collagen (COL1A1) and opsteopontin (OPN) mRNAs were determined by quantitative PCR and normalized by GAPDH. Data are shown as mean ± SE (n = 6). *: P<0.05 by Student’s <i>t</i>-test.</p

Upregulation of HSP27 in lung tissues of bleomycin-treated mice.

<p>Mice were intratracheally treated with PBS or bleomycin. After 14 days, mice were sacrificed and lungs were removed. (A) Immunoblot analysis. Protein levels of HSP27 and p-HSP27 were analyzed by Immunoblotting using tissue lysates prepared from right lungs. For a loading control, α-tubulin was used. Signal intensities were quantified using Image J software. A representative image from seven independent experiments is shown in the left. Quantitative data are shown as mean ± SE (n = 7) in the right. *: P<0.05 by Student’s <i>t</i>-test. (B) Immunofluorescence staining. Left lungs were fixed with 10% formaldehyde and embedded in paraffin. Tissue sections (4 μm) were double stained for HSP27 (green) and α-SMA (red), proSP-C (red), E-cadherin (E-cad, red) or OPN (red) as depicted. For nuclear staining, TO-PRO-3 (blue) was used. The bars indicate 20 μm. Representative images from three independent experiments are shown. (C) Col1a2-EGFP reporter mice. Mice were intratracheally instilled with PBS or bleomycin. After 14 days, mice were sacrificed and lungs were fixed with 4% paraformaldehyde, treated with 30% sucrose for cryoprotection, and embedded. Frozen sections (6 μm thick) were stained for HSP27 (red). Collagen Type I α2 was visualized by EGFP (green). The bars indicate 20 μm. Representative images from three independent experiments are shown.</p

Strong upregulation of HSP27 in lung tissues from IPF patients.

<p>(A) Immunohistochemical staining of HSP27 in human lung tissues. Representative images are shown (n = 5). (B) Double immunofluorescence staining of HSP27 (green) and α-SMA (red) in human lung tissues. Representative images are shown (n = 4). (C) Quantitation of HSP27 in bronchoalveolar lavage (BAL) samples. HSP27 contents in BAL samples containing 0.5% Triton X-100 were determined by ELISA. Data are shown as mean ± SE (control, n = 3; IPF, n = 6). *: P<0.05 by Student’s <i>t</i>-test.</p

Choroidal Neovascularization Is Inhibited in Splenic-Denervated or Splenectomized Mice with a Concomitant Decrease in Intraocular Macrophage

<div><p>Purpose</p><p>To determine the involvement of sympathetic activity in choroidal neovascularization (CNV) using laser-induced CNV in a mouse model.</p><p>Methods</p><p>We investigated changes in the proportions of intraocular lymphocytes, granulocytes, and three macrophage subtypes (Ly6C^hi, Ly6C^int, and Ly6C^lo) after laser injury in mice using flow cytometry, and evaluated CNV lesion size in mice lacking inflammatory cells. Further, we evaluated the lesion size in mice administered the β3 receptor antagonist, splenic-denervated and splenectomized mice. We also assessed changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenic-denervated and splenectomized mice. Lastly, lesion size was compared between splenic-denervated mice with or without adoptive transfer of macrophages following laser injury. After Ly5.1 mice spleen-derived Ly6C^hi cells were transferred into Ly5.2 mice, the proportions of intraocular Ly5.1⁺Ly6C^hi cells were compared.</p><p>Results</p><p>In WT mice, the proportion of CD4⁺ T cells recruited into the eye increased progressively from day 3 to day 7 after laser injury, whereas, intraocular CD8⁺ T cells did not change significantly. Proportions of B220⁺ cells, granulocytes, and two subtypes of intraocular macrophages (Ly6C^hi and Ly6C^lo) peaked at day 3 following laser injury. In contrast, Ly6C^int/loCD64⁺ subtype showed a significantly higher percentage at day 7 after laser injury. There were no differences in lesion size between <i>CD4</i>^{<i>–/–</i>}or <i>Rag2</i>^{<i>–/–</i>}mice and controls, whereas lesion size was significantly reduced in <i>CCR2</i>^{<i>−/−</i>} mice and clodronate liposome-treated mice. CNV lesion area was significantly reduced in mice with β3 blocker treatment, splenic-denervated and splenectomized mice compared with controls. Intraocular Ly6C^hi macrophages were also reduced by splenic denervation or splenectomy. Adoptive transfer of spleen-derived Ly6C^hi cells increased the lesion size in splenic-denervated mice. Compared with controls, intraocular donor-derived Ly6C^hi cells recruited into the eye were reduced in splenic-denervated and splenectomized mice.</p><p>Conclusions</p><p>Although lymphocytes had little effect on CNV formation, Ly6C^hi macrophages/monocytes exacerbated CNV in mice. Sympathetic activity might contribute to CNV via the recruitment of macrophages to the eye.</p></div

Changes in the proportions of circulating monocyte/macrophage subtypes in peripheral blood after laser injury.

<p>(A) Representative flow cytometry plots from WT mice. Monocyte subpopulations were gated based on Ly6C and CD43 expression to determine the proportions of classical (Ly6C^hiCD43^lo), intermediate (Ly6C^intCD43^hi) and nonclassical (Ly6C^loCD43^hi) monocytes per total leukocytes. Hi, int, and lo correspond to Ly6C^hi, Ly6C^int, and Ly6C^lo cells, respectively. (B) The proportion of circulating Ly6C^hi cells were significantly higher at day 3 after laser injury, whereas there were no changes in the proportions of Ly6C^lo and Ly6C^int cells. All experiments were performed in triplicate. *P < 0.05 versus control of the same subtype with Dunn's multiple comparison test for post-hoc analysis.</p

Change in the proportions of inflammatory cells recruited into the posterior segment of the eye in wild-type mice following laser photocoagulation.

<p>The proportion of CD4⁺ T cells increased progressively from day 3 after laser injury and greatly increased until day 7. Proportions of B220⁺ cells, granulocytes, and two subtypes of intraocular macrophages (Ly6C^hi and Ly6C^lo) peaked at day 3 after laser injury, whereas Ly6C^int/loCD64⁺ subtype showed a significantly higher percentage at 7 days after laser injury. In contrast, there were no changes in the proportion of CD8⁺ T cells after injury. All experiments were performed in triplicate. *P < 0.05 versus control of the same subtype with Dunn's multiple comparison test for post-hoc analysis.</p

Changes in the proportions of intraocular macrophages and peripheral blood monocytes in splenectomized mice after laser injury.

<p>(A) Less Ly6C^hi cells were recruited into the eye in splenectomized mice than those in controls both before laser treatment and at day 3 after laser injury, and intraocular Ly6C^int/loCD64⁺ cells were also reduced by splenectomy from before laser photocoagulation up to 7 days after laser, whereas, there were no significant changes in Ly6C^lo cells between two groups with or without laser injury. (B) In the peripheral blood, a lower proportion of Ly6C^int cells was observed before laser treatment, at day 3 and day 7 after laser injury, and Ly6C^lo cells were also reduced at day 7 after laser injury. In contrast, peripheral blood Ly6C^hi monocytes showed no differences between controls and splenectomized mice. All experiments were performed in triplicate. *P < 0.05 versus control of the same subtype with Dunn's multiple comparison test for post-hoc analysis.</p

Comparison of lesion size between wild-type (WT) and transgenic mice lacking lymphocytes at day 7 after laser injury.

<p>(A) There were no differences in mean choroidal neovascularization (CNV) area between WT and <i>CD4</i>^{<i>−/−</i>} mice or (B) between WT and <i>Rag2</i>^{<i>−/−</i>} mice. (C) Representative micrographs of CNV (white arrows) in RPE-choroid flatmounts. Scale bars, 100 μm. n = 8 for all groups.</p