Targeting interleukin-13 with tralokinumab attenuates lung fibrosis and epithelial damage in a humanized SCID idiopathic pulmonary fibrosis model

Abstract

The aberrant fibrotic and repair responses in the lung are major hallmarks of idiopathic pulmonary fibrosis (IPF). Numerous antifibrotic strategies have been used in the clinic with limited success, raising the possibility that an effective therapeutic strategy in this disease must inhibit fibrosis and promote appropriate lung repair mechanisms. IL-13 represents an attractive target in IPF, but its disease association and mechanism of action remains unknown. In the present study, an overexpression of IL-13 and IL-13 pathway markers was associated with IPF, particularly a rapidly progressive form of this disease. Targeting IL-13 in a humanized experimental model of pulmonary fibrosis using tralokinumab (CAT354) was found to therapeutically block aberrant lung remodeling in this model. However, targeting IL-13 was also found to promote lung repair and to restore epithelial integrity. Thus, targeting IL-13 inhibits fibrotic processes and enhances repair processes in the lung.

Figure 1.

Elevated IL-13 transcript levels in the lungs of patients with idiopathic pulmonary fibrosis (IPF). (A, B) mRNA expression measured by quantitative RT-PCR in whole lung biopsy tissue from patients with IPF (n = 8) and from nonspecific interstitial pneumonia (NSIP)-fibrotic (n = 4) and NSIP-cellular (n = 3) patients and normalized to normal lung control tissue for il-13 (A) and il-4 (B).

Figure 2.

Immunolocalization of IL-13 in interstitial lung diseases. Lung biopsy sections, taken from patients with interstitial lung disease at the time of diagnosis, were stained with anti–IL-13 to visualize the expression of IL-13. Consecutive sections were stained with IgG control (see Figure E1 in the online supplement). Pathologist-identified diseased sections from respiratory bronchiolitis interstitial lung disease (A, C), patients with IPF who experienced an acute exacerbation within 1 year of diagnosis (B and H), normal lung tissue (D and G), fibrotic NSIP (E), or IPF (F, I–K). (G–K) Representative histological sections from normal patients and patients with IPF are at ×200 magnification.

Figure 3.

Elevated IL-13 pathway activation in the lungs and circulation, more closely associated with rapidly progressing IPF. (A) Correlations in levels of mRNA expression between IL-13 pathway genes and fibrosis markers in IPF lung biopsies (n = 62). Lighter colors represent greater correlations between genes. The larger value indicates a stronger the correlation. Negative values represent a negative correlation. (B) Increased IL-13 pathway gene expression, as measured by RT-PCR, with rapidly progressing IPF and stabling progressive IPF lung biopsies in comparison to normal healthy lung tissue. (C) Increased il4r, il13rα1, gata3, and mmp7 in epithelial cell brushings from a subset of patients with IPF as measured by RT-PCR. (D) Spontaneous release of IL-13 from fibroblasts isolated from the lung biopsies of rapidly progressive patients with IPF. IL-13 was measured using specific ELISA. Data are mean ± SEM from four different patients with IPF. (E, F) Circulating CCL2/MCP-1 (E) and CCL11/eotaxin (F) were measured in the plasma of stable (n = 19) and rapid (n = 14) patients with IPF and healthy age-matched normal control subjects (n = 20). (G) Correlation of plasma IL-13 levels and the time of the first clinical event postdiagnosis in the rapid IPF patient subgroup. Correlation of lung tissue il-13rα2 transcript expression and FVC % predicted (H) and collagen 3a1 (I) determined by RT-PCR. Each data point represents an individual patient. *P < 0.05, **P < 0.01, and ***P < 0.005.

Figure 4.

IL-13 stimulation of IPF fibroblasts or epithelial cells results in an up-regulation of IL-13Rα2. (A–F) Fibroblasts from patients with IPF were stimulated with increasing concentrations of IL-13 for 24 hours. Supernatants were taken for ELISA analysis, whereas cell monolayers were lysed and assessed for changes in gene expression compared with unstimulated controls using branched DNA technology (Panomics; A) or IL-13Rα2 immunolocalized using anti–IL-13Rα2 staining and quantitated (Cellomics; B–F). Lung epithelial cells were stimulated with IL-13 for 24 hours, and il-13ra2 expression was determined using branched DNA technology (G). For fibroblast studies, data shown are mean ± SEM from four different donors (gene) and are representative of one individual donor for IL-13Ra2 surface expression (lower graph, pictures). Data are mean ± SEM. *P < 0.05 and **P < 0.01 significance in comparison to unstimulated controls.

Figure 5.

Neutralization of fibroblast-derived IL-13 inhibits lung fibrosis in an established fibroblast-driven disease setting in humanized SCID mice. (A–C) Isolated IPF fibroblasts (n = 5) or normal lung tissue fibroblasts (n = 5) were injected intravenously to SCID mice, and animals were killed at specific subsequent time points. Whole lung mRNA expression was measured by quantitative RT-PCR for il-13 (A), il113rα1 (B), and il-13rα2 (C) and normalized to lung mRNA expression levels from mice that had received normal lung tissue-derived fibroblasts. (D–N) Thirty-five days after IPF fibroblast engraftment, mice were randomized and treated with PBS control, control IgG4, or tralokinumab (anti–IL-13 mAb) every other day until analysis at Day 63. Representative mouse lung sections stained with Masson’s trichrome to depict the degree of fibrosis at Day 63 from SCID/IPF mice treated with PBS control (D), CAT251 IgG₄ isotype control (E), and tralokinumab anti–IL-13 antibody (F). Fibrosis was quantitated in all animals using a modified Ashcroft score of histological sections (G). Tralokinumab attenuated serum CC16 levels as measured by ELISA (H) and BAL caspase 3 activity (I). Data are mean ± SEM (n = 5). (J–M) Representative immunohistochemistry localizing IL-13Rα2 expression in the lungs of control SCID mice that have not received IPF fibroblast (J) or SCID/IPF mice treated with PBS control (K), isotype control (L), or tralokinumab (M). Original magnification: top panels, ×40; bottom panels, ×200. *P < 0.05, **P < 0.01, and ***P < 0.005.

Figure 6.

Enhanced epithelial markers in the lungs of SCID/IPF mice treated with tralokinumab. Whole lung mRNA expression from mice treated with isotype control or tralokinumab (anti–IL-13) was measured using quantitative RT-PCR. Levels of vimentin (A), TGF-β1 (B), surfactant protein a (C), surfactant protein b (D), surfactant protein c (E), surfactant protein d (F), and e-cadherin (G) were quantitated and normalized to lung mRNA expression levels from SCID/IPF mice that had received PBS control only. Data are mean ± SEM (n = 5). *P < 0.05.