Epithelial transglutaminase 2 is needed for T cell interleukin-17 production and subsequent pulmonary inflammation and fibrosis in bleomycin-treated mice

Abstract

Pulmonary fibrosis is a potentially life-threatening disease that may be caused by overt or asymptomatic inflammatory responses. However, the precise mechanisms by which tissue injury is translated into inflammation and consequent fibrosis remain to be established. Here, we show that in a lung injury model, bleomycin induced the secretion of IL-6 by epithelial cells in a transglutaminase 2 (TG2)-dependent manner. This response represents a key step in the differentiation of IL-17-producing T cells and subsequent inflammatory amplification in the lung. The essential role of epithelial cells, but not inflammatory cells, TG2 was confirmed in bone marrow chimeras; chimeras made in TG2-deficient recipients showed reduced inflammation and fibrosis, compared with those in wild-type mice, regardless of the bone marrow cell phenotype. Epithelial TG2 thus appears to be a critical inducer of inflammation after noninfectious pulmonary injury. We further demonstrated that fibroblast-derived TG2, acting downstream of transforming growth factor-β, is also important in the effector phase of fibrogenesis. Therefore, TG2 represents an interesting potential target for therapeutic intervention.

Figures

Figure 1.

TG2 deficiency reduces BLM-induced pulmonary inflammation and fibrosis. (A–C) Representative photographs of lungs from WT and TG2−/− mice 21 d after intratracheal instillation of BLM (1.5 mg/kg) or PBS. Sections were stained with H&E (A) or Masson-trichrome (B). Pulmonary collagen content of the lungs was determined by the Sircol assay (C). (D–F) Levels of IL-6 (D), MCP-1 (E), and MCP-3 (F) in BALF of WT and TG2−/− mice after BLM-exposure were determined by ELISA. (G–J) Inflammatory cells in BALF from WT B6 and TG2−/− mice. BLM-exposed mice were sacrificed to harvest BALF on the indicated days. Numbers of total cells (G), lymphocytes (H), macrophages (I), and neutrophils (J) in BALF were determined by flow cytometric analysis. Data represent means ± SD of three independent determinations with BALF or lung tissues, and n = 5 (A–C) or n = 3 mice/group for each time point (D–J).

Figure 2.

BLM-mediated pulmonary injury triggers a Th17 response. WT B6 and TG2−/− mice received an intratracheal instillation of BLM (1.5 mg/kg) and were sacrificed to harvest BALF on the indicated days. Cells from BALF were stimulated with PMA and ionomycin and intracellular cytokines were detected by flow cytometric analysis. (A and B) Numbers of CD4+ cells producing IL-17 (A) or IFN-γ (B) in BALF of BLM-exposed WT B6 and TG2−/− mice. (C) The percentages of CD4+ cells producing IL-17 or IFN-γ in BALF and draining lymph nodes (DLN) of WT B6 and TG2−/− mice were determined by flow cytometric analysis 10 d after BLM exposure. Dot plots are gated on CD4+ T cells. (D–H) BLM-exposed WT B6 mice were treated with IL-17RA-Fc or isotype control for 10 d (100 µg/mouse/day, i.p.). IL-17 levels (D) and the numbers of CD4+IL-17+ cells (E), total cells (F), and neutrophils (G) were determined by ELISA and flow cytometry, respectively. (H) BLM-exposed mice were treated with IL-17RA-Fc or isotype control for the times indicated. Quantitative analysis of the fibrotic area by H&E and Masson’s trichrome staining was performed 21 d after instillation of BLM. Data represent means ± SD of three independent determinations with BALF or lung tissues from n = 5 (H) or n = 3 mice/group for each time point (A–G).

Figure 3.

IL-6 blocking reduces pulmonary inflammation, Th17 responses, and fibrosis induced by BLM. (A–C) Inflammatory cells in BALF were harvested 8 d after BLM exposure. BLM-exposed WT B6 mice were treated with blocking anti–IL-6 mAb or isotype-control twice weekly, beginning 1 d before BLM treatment (100 µg/mouse, i.p.). Numbers of total cells (A), CD4+ T cells (B), and CD4+ T cells producing IL-17 (C) in BALF were determined by flow cytometry. (D) A representative photograph of lungs from isotype control– and anti–IL-6 mAb–treated WT B6 mice 21 d after BLM exposure. Sections were stained with Masson’s trichrome. (E) Quantitative analysis of the fibrotic area by H&E and Masson’s trichrome staining was performed 21 d after instillation of BLM. Data represent means ± SD of two independent determinations with BALF or lung tissues from n = 5 mice/group.

Figure 4.

BLM induces secretion of IL-6 from lung epithelium. (A) Immunofluorescence staining of IL-6 production (green) from pro-SP-C (red)–expressing type II epithelial cells in the lungs of BLM-exposed WT B6 mice. Nuclei were counterstained with DAPI (blue). (B) Lung epithelial cells expressing pro-SP-C were sort-purified (top). IL-6 levels in culture supernatants of sort-purified primary MLECs and primary MLFs from WT B6 mice as determined by ELISA. Cells (2 × 104) were treated with BLM (5 µg/ml) for the times indicated. (C) Primary MLECs from WT B6 mice were treated with BLM (5 µg/ml) in the presence of the TG2 inhibitor CyM or PBS. (D) Effect of Bay-11-7082, a chemical inhibitor for NF-κB, on BLM-induced IL-6 secretion in primary MLECs from WT B6 mice. (E) Th17 differentiation of CD4+ T cells induced by culture supernatants of primary MLECs. Lymphocytes isolated from WT B6 mice were stimulated with anti-CD3 and anti-CD28 for 3 d in the presence of culture supernatants of primary MLECs from WT and TG2−/− mice treated with PBS or BLM. As indicated, anti–IL-6 (10 µg/ml) or anti–TGF-β (10 µg/ml) antibodies were added to the culture. After restimulation with PMA and ionomycin, percentages of CD4+ T cells expressing IL-17 or IFN-γ were determined by flow cytometry. (F) IL-6 levels in the culture supernatants of lung macrophages and primary MLECs were determined by ELISA. Lung macrophages (5 × 104) and primary MLECs (2 × 104) from WT B6 and TG2−/− mice treated with PBS, LPS (1 µg/ml), or BLM (5 µg/ml) for 48 h. (B–F) Data represent means ± SD, based on three independent determinations using samples from n = 3 cell cultures. *, P ≤ 0.05; **, P ≤ 0.005. Representative data from three independent determinations are shown.

Figure 5.

Th17 cells proliferate and differentiate in the lung. (A and B) WT B6 mice received BLM intratracheally (1.5 mg/kg). BLM-exposed mice were treated with PBS or FTY720 (2 mg/kg/day, i.p.) between days 3 and 7 (analyzed on day 7), between days 3 and 8 (analyzed on day 8), and between days 5 and 8 (analyzed on day 8). Mice were sacrificed and BALF and draining lymph nodes (DLN) harvested on the days noted above. The percentages of CD4+ cells producing IL-17 or IFN-γ in draining lymph nodes (DLN; A) and BALF (B) were determined by flow cytometric analysis. (C) BLM-exposed WT B6 mice were treated with PBS or FTY720 (2 mg/kg/day, i.p.) at day 5, 6, or 7 after BLM-exposure. Numbers and percentages of CD4+ cells producing IL-17 in BALF and DLN were determined by flow cytometric analysis 8 d after BLM exposure. (D and E) WT B6 (Thy1.2) mice received BLM intratracheally (1.5 mg/kg). On the next day, BLM-exposed mice were adoptively transferred with CFSE-labeled Thy1.1+ CD4+ T cells (5 × 106 cells). To deplete endogenous T cells, anti-Thy1.2 antibody (30-H12; 300 µg/mouse) was administered i.p. to recipient mice on days −1 and 2 of BLM treatment. (D) Mice were sacrificed to harvest leukocytes from lungs on the days indicated. The percentages of donor Thy1.1+ CD4+ cells producing IL-17 were determined by flow cytometric analysis. (E) The percentages of donor Thy1.1+ CD4+ cells producing IL-17 in lungs and draining lymph nodes (DLN) of recipient mice were determined by flow cytometric analysis 8 d after PBS or BLM exposure. (F and G) WT B6 (Thy1.2) mice were adoptively transferred with CFSE-labeled naive phenotype Thy1.1+ CD4+ T cells (5 × 106 cells) 1 d after BLM exposure. Some mice received FTY720 10 h before naive phenotype Thy1.1+ CD4+ T cell transfer. Percentages (F) and numbers (G) of donor Thy1.1+ CD4+ cells producing IL-17 in lungs and DLN of recipient mice were determined by flow cytometric analysis 8 d after PBS or BLM exposure. (C and G) Data represent means ± SD of three independent determinations with BALF or lung tissues from n = 5 mice/group.

Figure 6.

Chimeric mice with TG2−/− recipients exhibit diminished pulmonary inflammation and fibrosis in response to BLM. BM chimeras were prepared by irradiation of WT B6 or TG2−/− mice, followed by T cell–depleted BM cell reconstitution (BMWT→WT, BMTG2−/−→WT, BMWT→TG2−/−, and BMTG2−/−→TG2−/−). (A) The percentage of CD4+IL-17+ cells in BALF of the chimeras was determined by flow cytometric analysis 10 d after BLM exposure. (B) IL-6 levels in BALF from chimeras 3 d after BLM exposure was determined by ELISA. (C) Representative photographs of lungs from chimeras are shown. Lung tissues were prepared 21 d after BLM instillation and stained with Masson’s trichrome. (D and E) Quantitative analysis of fibrosis was performed using Masson’s trichrome staining (D) and pulmonary collagen content (Sircol assay; E) 21 d after BLM exposure. Data represent means ± SD of three independent determinations with BALF or lung tissues from n = 5 mice/group.

Figure 7.

Inhibition of TG2 by CyM during either the early or late phase effectively reduces lung fibrosis induced by BLM. (A) Representative photographs of lungs from WT mice 21 d after exposure to BLM. BLM-exposed mice were treated with PBS or CyM (40 mg/kg/day, i.p.) during the early phase (days 0–10), late phase (days 11–21), or for all 21 d. Sections were stained with Masson’s trichrome. (B and C) Quantitative analysis of fibrotic area (B) and pulmonary collagen content (C) 21 d after BLM exposure. Data represent means ± SD of three independent determinations with lung tissues from n = 5 mice/group for each time point.