Attenuation of Allergen-, IL-13-, and TGF-α-induced Lung Fibrosis after the Treatment of rIL-15 in Mice

Abstract

Endogenous IL-15 deficiency promotes lung fibrosis; therefore, we examined the effect of induced IL-15 in restricting the progression of lung fibrosis. Our objective in this work was to establish a novel therapeutic molecule for pulmonary fibrosis. Western blot, qPCR, and ELISA were performed on the lung tissues of IL-15-deficient mice, and recombinant IL-15 (rIL-15)-treated CC10-IL-13 and CC10-TGF-α mice, and allergen-challenged CC10-IL-15 mice were examined to establish the antifibrotic effect of IL-15 in lung fibrosis. We show that endogenous IL-15 deficiency induces baseline profibrotic cytokine and collagen accumulation in the lung, and pharmacological delivery of rIL-15 downregulates Aspergillus antigen-induced lung collagen, the profibrotic cytokines IL-13 and TGF-β1, and α-SMA⁺ and FSP1⁺ cells in mice. To confirm that overexpression of IL-15 diminishes pulmonary fibrosis, we generated CC10-rtTA-tetO7-IL-15 transgenic mice and challenged them with Aspergillus antigen. Aspergillus antigen-challenged, doxycycline (DOX)-treated CC10-IL-15 transgenic mice exhibited decreased collagen accumulation, profibrotic cytokine (IL-13 and TGF-β1) expression, and α-SMA⁺ and FSP1⁺ cells compared with IL-15-overexpressing mice not treated with DOX. Additionally, to establish that the antifibrotic effect of IL-15 is not limited to allergen-induced fibrosis, we showed that rIL-15 or IL-15 agonist treatment restricted pulmonary fibrosis even in CC10-IL-13 and CC10-TGF-α mice. Mechanistically, we show that T-helper cell type 17 suppressor IL-15-responsive RORγ⁺ T regulatory cells are induced in DOX-treated, allergen-challenged IL-15-overexpressing mice, which may be a novel pathway for restricting progression of pulmonary fibrosis. Taken together, our data establishes antifibrotic activity of IL-15 that might be a novel therapeutic molecule to combat the development of pulmonary fibrosis.

Keywords: IL-13; TGF-β; allergen; fibrosis; lung.

Figure 1.

Analysis of baseline tissue remodeling in endogenous IL-15 gene–deficient (IL-15^−/−) mice. (A and B) Baseline collagen deposition was examined using Masson’s trichrome staining of lung tissue sections from 20-weeks-old naive wild-type (WT) and (C and D) IL-15^−/− mice. (C) Total lung collagen was assessed using Sircol reagent, and (D) transforming growth factor β1 (TGF-β1) levels were assessed by ELISA. (E–G) Western blot analysis showed enhanced expression of TGF-β1 and α-SMA in IL-15^−/− mice (E), which was confirmed by performing GAPDH-normalized densitometry (F and G). Data are expressed as mean ± SD, n = 8 mice/group in each group, except for the Western blot analysis. Scale bars: 100 μm. SMA = smooth muscle actin.

Figure 2.

Consequences of rIL-15 pretreatment in Aspergillus-induced bronchial fibrosis in mice. (A) Representative light-microscopy photomicrographs of lung sections with Masson’s trichrome staining show perivascular and peribronchiolar collagen accumulation after 3 weeks of saline or Aspergillus challenge in mice treated with and without rIL-15. (B) Total collagen expression in saline- and rIL-15–treated, Aspergillus-challenged mice. ELISA results for profibrotic IL-13 and TGF-β1 expression in saline- and rIL-15–treated, Aspergillus-challenged mice are shown. (C and D) Immunofluorescence staining revealed α-SMA⁺ cells in Aspergillus- and rIL-15–treated, Aspergillus-challenged mice. (E) Very few eosinophils are seen in the saline-treated (for 3 wk) mice. (F) Arrows indicated FSP1⁺ cell expression in Aspergillus and rIL-15–treated Aspergillus-challenged mice. Quantitation of α-SMA⁺ and FSP1⁺ cells is shown in saline-, (G and H) Aspergillus-, and rIL-15–treated Aspergillus-challenged mice. Data are expressed as mean ± SD, n = 12 mice/group. Scale bars: 100 μm and 20 μm. ASP = Aspergillus; FSP1 = fibroblast-specific protein 1; rIL-15 = recombinant IL-15; SAL = saline.

Figure 3.

Generation of doxycycline (DOX)-regulated IL-15 transgenic mice, and analysis of tissue collagen and profibrotic cytokines in saline- and Aspergillus-exposed CC10–IL-15 bitransgenic mice. (A) Construct of (tetO) 7 CMV-IL15 transgenic mice and rtTA-CC10 transgenic mice to generate rtTA-CC10–IL-15 bitransgenic mice. (B) Induction of IL-15 levels in the lung and BAL fluid (BALF) after 3 weeks of DOX exposure to rtTA-CC10–IL-15 bitransgenic mice. (C) A representative photomicrograph of Masson’s trichrome-stained saline- and Aspergillus-challenged DOX- and no-DOX–exposed CC10–IL-15 bitransgenic mice. (D) Total lung collagen, and the profibrotic cytokines (IL-13 and TGF-β1) in saline and Aspergillus-challenged no-DOX and DOX exposed IL-15 transgenic mice are shown (E and F). Immunofluorescence staining revealed α-SMA⁺ cells in saline- and Aspergillus-challenged no-DOX– and DOX-exposed IL-15 transgenic mice. (G) Very few α-SMA⁺ cells are seen in the saline-treated (for 3 wk) mice. (H) Quantitation of α-SMA⁺ cells in saline- and Aspergillus-challenged IL-15 transgenic mice. (I) Arrows indicated FSP1⁺ cells detected in Aspergillus-challenged no-Dox– and Dox-exposed IL-15 transgenic mice. (J) Quantitation of FSP1⁺ cells in saline- and Aspergillus-challenged Dox- and no-Dox–exposed transgenic mice. Data are expressed as mean ± SD, n = 12 mice/group. Scale bars: 20 μm, 50 μm, and 100 μm. bGH = bovine growth hormone; CC10 = CC10-Clara cell 10 kD protein; CMV = cytomegalovirus (promoter); hGH = human growth hormone; rtTA = express a tet repressor-VP16 transcriptional activator fusion gene; TetO7 = tetracycline; Tg = transgenic.

Figure 4.

Pharmacological delivery of IL-15 significantly reduces IL-13–induced collagen accumulation and profibrotic cytokines in the lungs of CC10–IL-13 transgenic mice. (A) Light-microscopy photomicrographs of Masson’s trichome–stained lung sections from saline- or IL-15–treated, no-DOX– or DOX-exposed CC10–IL-13 bitransgenic mice. (B–E) Total lung collagen (B) and the profibrotic cytokines IL-13 (C), TGF-β1 (D), and α-SMA (E) in saline- and IL-15–treated no-DOX– and DOX-exposed IL-13 bitransgenic mice are shown. Data are expressed as mean ± SD, n = 12 mice/group. Scale bars: 100 μm.

Figure 5.

Pharmacological delivery of IL-15 significantly reduces TGF-α–induced collagen accumulation and profibrotic cytokines in the lungs of CC10–TGF-α transgenic mice. (A) Light-microscopy photomicrographs of Masson’s trichome–stained lung sections of saline- or IL-15–treated, no-DOX– or DOX-exposed CC10–TGF-α bitransgenic mice are shown. (B–D) Total lung collagen (B) and the profibrotic cytokine TGF-β1 (C), α-SMA (D), and the expression of lung TGF-α (E) in saline- or IL-15–treated, no-DOX– or DOX-exposed TGF-α bitransgenic mice are shown. Data are expressed as mean ± SD, n = 4 mice/group. Scale bars: 100 μm and 20 μm.

Figure 6.

Analysis of T regulatory (Treg) cells in DOX-regulated CC10–IL-15 transgenic mice after Aspergillus challenge. (A and B) Treg cells in the mediastinal lymph nodes of Aspergillus-challenged DOX- and non-DOX–exposed CC10–IL-15 overexpressing mice were examined by flow cytometry using anti-CD45-FITC, anti-CD4-PerCP, anti-RORγ-PE, anti-CD25-PE-Cy7, and anti-Foxp3-APC along with matched IgG antibodies. (C) The average percentage of CD4⁺CD25⁺Foxp3⁺ and (D) RORγ⁺CD4⁺CD25⁺Foxp3⁺ Treg cells, and their absolute number of cells from three independent experiments in mediastinal lymph nodes of mice. Data are expressed as mean ± SD.

Figure 7.

Reduction in lung collagen and the levels of profibrotic cytokines in response to pretreatment with the human IL-15 agonist ALT-803 in the lungs of Aspergillus-challenged mice. The potential of a human IL-15 agonist as a therapeutic agent was examined using an allergen-induced lung fibrosis model. (A) A highly significant reduction in collagen accumulation is shown in response to ALT-803 treatment (regimen of 5 μg on alternate days for 3 wk) in Aspergillus-challenged mice compared with saline-treated, Aspergillus-challenged mice. (B–E) Total lung collagen (B) and the profibrotic cytokines IL-13 (C), TGF-β1 (D), and α-SMA (E) in saline- and ALT-803–treated saline or Aspergillus-challenged mice are shown. (F and G) Western blot analysis showed that ALT-803 treatment downregulated Aspergillus-induced levels of profibrotic cytokines (collagen I, TGF-β1, and α-SMA) in mice (F), and this was further confirmed by GAPDH-normalized densitometry of collagen I, TGF-β1, and α-SMA (G). Data are expressed as mean ± SD, n = 12 mice/group in each group, except for the Western blot analysis. Scale bars: 100 μm.