Immune Netw.  2015 Jun;15(3):142-149. 10.4110/in.2015.15.3.142.

Osteopontin Potentiates Pulmonary Inflammation and Fibrosis by Modulating IL-17/IFN-gamma-secreting T-cell Ratios in Bleomycin-treated Mice

• Oh K
• Seo MW
• Kim YW
• Lee DS

• KMID: 2168040
• DOI: http://doi.org/10.4110/in.2015.15.3.142

Abstract

Lung fibrosis is a life-threatening disease caused by overt or insidious inflammatory responses. However, the mechanism of tissue injury-induced inflammation and subsequent fibrogenesis remains unclear. Recently, we and other groups reported that Th17 responses play a role in amplification of the inflammatory phase in a murine model induced by bleomycin (BLM). Osteopontin (OPN) is a cytokine and extracellular-matrix-associated signaling molecule. However, whether tissue injury causes inflammation and consequent fibrosis through OPN should be determined. In this study, we observed that BLM-induced lung inflammation and subsequent fibrosis was ameliorated in OPN-deficient mice. OPN was expressed ubiquitously in the lung parenchymal and bone-marrow-derived components and OPN from both components contributed to pathogenesis following BLM intratracheal instillation. Th17 differentiation of CD4+ alphabeta T cells and IL-17-producing gammadelta T cells was significantly reduced in OPN-deficient mice compared to WT mice. In addition, Th1 differentiation of CD4+ alphabeta T cells and the percentage of IFN-gamma-producing gammadelta T cells increased. T helper cell differentiation in vitro revealed that OPN was preferentially upregulated in CD4+ T cells under Th17 differentiation conditions. OPN expressed in both parenchymal and bone marrow cell components and contributed to BLM-induced lung inflammation and fibrosis by affecting the ratio of pathogenic IL-17/protective IFN-gamma T cells.

Keyword

Osteopontin; Pulmonary fibrosis; alphabeta T cells; IL-17; Th17 differentiation

MeSH Terms

Animals
Bleomycin
Bone Marrow Cells
Fibrosis*
Inflammation
Interleukin-17
Lung
Mice*
Osteopontin*
Pneumonia*
Pulmonary Fibrosis
T-Lymphocytes*
T-Lymphocytes, Helper-Inducer
Bleomycin
Interleukin-17
Osteopontin

Figure

• 

  Figure 1 OPN deficiency reduces BLM-induced pulmonary inflammation and fibrosis. (A) Representative photographs of lungs from WT and OPN-/- mice 21 days after intratracheal instillation of BLM (1.5 mg/kg). Sections were stained with H&E. (B) OPN expression levels in diverse tissues from WT B6 mice were analyzed using RT-PCR. (C) BM chimeras were prepared by irradiation of WT or OPN-/- mice, followed by T-cell-depleted BM cell reconstitution (BMWT→WT, BMWT→ OPN-/-, BMOPN-/-→WT, and BMOPN-/-→OPN-/-). Two months after BM cell reconstitution, pulmonary fibrosis was induced with BLM. Representative photographs of lungs 21 days after intratracheal instillation with BLM (1.5 mg/kg).

• 

  Figure 2 Lymphocytes of spleens, lymph nodes, and liver from WT and OPN-/- mice were harvested and stained with antibodies for αβTCR, γδTCR, CD4, CD8, CD11c, and B220 molecules. Cell populations were detected using flow cytometric analysis.

• 

  Figure 3 WT and OPN-/- mice that received an intratracheal instillation of BLM (1.5 mg/kg) were sacrificed to harvest BALF on 10 days. Cells from BALF were stimulated with PMA, and ionomycin, surface markers and intracellular cytokines were detected using flow cytometric analysis. (A) The percentages of CD4+ or γδTCR+ cells in BALF were detected using flow cytometric analysis. (B) The percentage of CD4+ or γδTCR+ cells producing IL-17 or IFN-γ in BALF of BLM-exposed WT and OPN-/- mice. (C) The number of CD4+ T cells in BALF. (D) The number of γδ T cells in BALF.

• 

  Figure 4 (A) Naïve CD4+ T cells, CD8+ T cells, CD11c+ dendritic cells, and B220+ B cells from lymph nodes and spleens were purified. OPN mRNA expression was determined using RT-PCR. (B) Purified CD4+ or CD8+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies for 24 and 48 h. (C) Purified CD4+ T cells were stimulated with anti-CD3 and anti-CD28 antibodies under the Th0 (no additive), Th1 (10 ng/ml IL-12, 10 µg/ml anti-IL-4), Th2 (10 ng/ml IL-4, 10 µg/ml anti-IL-12, 10 µg/ml anti-IFN-γ), and Th17 (10 ng/ml IL-6, 5 ng/ml TGF-β, 10 µg/ml anti-IL-4, 10 µg/ml anti-IFN-γ) cell generation conditions. Representative data from three independent determinations are shown.

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