Elsevier

Immunobiology

Volume 218, Issue 2, February 2013, Pages 245-254
Immunobiology

Differential role of regulatory T cells in early and late stages of pulmonary fibrosis

https://doi.org/10.1016/j.imbio.2012.05.020Get rights and content

Abstract

Regulatory T cells (Tregs) are a specific subset of T lymphocytes that regulate the function of other subsets of lymphocytes. Contradictory results have been reported regarding the role of Tregs in lung fibrosis. We wished to clarify the role of Tregs in the early and late stages of bleomycin-induced lung fibrosis in mice by depleting them with anti-CD25+ antibody (PC61). Mice treated with PC61 in early stages had significantly decreased number of CD4+CD25+ T cells compared to mice treated with the isotype control. The number of inflammatory cells, the concentrations of collagen, TGFβ1, the content of collagen and hydroxyproline in lung tissue were significantly reduced in PC61-treated mice compared to mice treated with the isotype control group. Pathological examination of the lung also disclosed reduced fibrotic changes and decreased fibrosis score in the PC61 group compared to control group. By contrast, mice treated with PC61 in late stages of the disease showed more infiltration of inflammatory cells and higher fibrotic score and hydroxyproline content in the lungs than mice treated with the isotype control. Our results suggest that Tregs play a detrimental role in early stages but protective role in late stages of pulmonary fibrosis in mice.

Introduction

Pulmonary fibrosis is the result of an acute and/or chronic lung injury characterized by excessive and aberrant deposition of connective tissue matrix in the alveolar and interstitial compartments of the lung (Wilson and Wynn 2009). The initial insult may be triggered by oxidants, drugs, viruses or, more frequently, by unknown agents (Wilson and Wynn 2009). Lung injury is followed by an abnormal accumulation and proliferation of mesenchymal cells, which secrete abundant extracellular matrix proteins (e.g., collagen, elastin, proteoglycans, fibronectin, laminin and tenascins) within the alveoli and interstitium leading to thickening of alveolar walls, impairment of gas exchange and increase in the stiffness of the lung ultimately causing scarring of the lungs (Wilson and Wynn 2009). The disease in humans is associated with a group of disorders of the lower respiratory tract known as the interstitial lung diseases (Wilson and Wynn 2009). Of these, the most frequent entity is the subgroup, idiopathic pulmonary fibrosis (IPF). The etiology of IPF is unknown and curative therapy is not currently available (King et al. 2011). The prognosis of IPF patients is poor, their median survival being only 3 years from the time of diagnosis (Hoo and Whyte, 2011, King et al., 2011, Nathan et al., 2011).

Following lung injury, there is an accumulation of inflammatory mediators and immune cells including macrophages, eosinophils and T cells that take part in the pathogenesis of pulmonary fibrosis (Wynn 2008). The subset of tissue-infiltrating T cells can modify the process of fibrosis. T helper (Th)2 cells that secrete interleukin (IL)-4, IL-13, IL-5 are predominantly associated with profibrotic inflammatory responses, whereas Th1 cells exert antifibrotic activity by secreting interferon (IFN)γ and IL-2 (Wynn 2008). Deficiency of a population of CD25+CD4+ T cells, known as regulatory T cells (Tregs), has also been implicated in advanced stages of the disease. In sarcoidosis, Tregs were unable to inhibit the release of proinflammatory cytokines, and in IPF, the reduction in the number and suppressing activity of Tregs was correlated with clinical parameters of disease severity (Kotsianidis et al., 2009, Rappl et al., 2011). However, the role of Tregs in early stages of IPF remains unclear.

Investigation of the disease in humans is difficult because already at diagnosis the fibrotic process is generally in advanced stages; thus, animal models of fibrosis are currently being used to clarify potential mechanistic pathways. Bleomycin (BLM)-induced lung fibrosis is the best characterized and a widely used animal model for evaluating the therapeutic efficacy of experimental agents (Moore and Hogaboam 2008). Administration of BLM by intravenous, intratracheal, intraperitoneal or subcutaneous routes induces acute pan-alveolitis during the first week, the inflammatory response reaches a peak on the 7th day after beginning drug infusion (Moore and Hogaboam, 2008, Yasui et al., 2001); this early phase is followed by a late process characterized by enhanced proliferation of myofibroblasts and secretion/deposition of extracellular matrix proteins in the lungs (Moore and Hogaboam 2008). Similar to the disease in humans, more activation of Th2 and Th17 cells compared to Th1 cells is critical for the induction of fibrosis by BLM in mice (Luzina et al. 2008); however, contradictory results have been reported on the number and functional activity of Tregs in the BLM-induced lung fibrosis (Du et al., 2009, Trujillo et al., 2010). A previous study has shown significant increase in the number of CD4+CD25+FoxP3+ T cells, whereas another study reported almost undetectable level of this cell population in the lungs from C57BL/6 mice with lung fibrosis induced by BLM.

The currently study was undertaken to clarify the role of Tregs in lung fibrogenesis induced in mice by BLM during different phases of the disease. The mice were depleted of Tregs by treatment with anti-CD25+ antibody during the early (3 days before BLM), intermediate (mid, 3 days before and 7 days after BLM) and late (9 and 16 days after BLM) phases of the disease and the development of lung fibrosis was compared.

Section snippets

Animals and disease model

Pathogen free 8- to 10-wk-old, female, C57BL/6 mice, weighing 20–23 g were purchased from Nihon SLC (Hamamatsu, Japan). Lung injury was induced by constant subcutaneous infusion of BLM through osmotic minipumps (Alzet, model 2001; Durect Corp., Cupertino, CA), as originally described by Harrison and Lazo (Harrison and Lazo 1987). BLM (Nihon Kayaku; Tokyo, Japan) was dissolved in sterile saline and loaded into 7-day minipumps. In control animals the osmotic minipumps were loaded with sterile

Depletion of CD4+CD25+ T cells in early and late stages of lung fibrosis

To examine if PC61 could deplete CD4+CD25+ T cells, the number of these cells was determined in lung tissue from animals that had lung fibrosis induced by BLM. The number of CD4+CD25+ T cells was significantly decreased on days 3, 7 and 16, and the number of CD4+CD25+FoxP3+ T cells was significantly decreased on day 3 in mice treated with PC61 3 days (BLM/early-PC61) before BLM infusion compared to those treated with the isotype control antibody (BLM/control) (Supplemental Fig. S1 and Fig. 1).

Discussion

The results of this study showed that depletion of the subpopulation of CD4+CD25+ regulatory T cells in the early stages of BLM-induced lung injury is associated with reduced fibrosis but that depletion later leads to enhanced lung fibrosis.

Conclusion

The results of this study suggest that Tregs play a detrimental role in the early stages but a protective role in late stages of BLM-induced pulmonary fibrosis, suggesting that therapeutic strategies might need to be different in the different phases of the disease.

Acknowledgment

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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