Regulatory T cells reduce acute lung injury fibroproliferation by decreasing fibrocyte recruitment

Abstract

Acute lung injury (ALI) causes significant morbidity and mortality. Fibroproliferation in ALI results in worse outcomes, but the mechanisms governing fibroproliferation remain poorly understood. Regulatory T cells (Tregs) are important in lung injury resolution. Their role in fibroproliferation is unknown. We sought to identify the role of Tregs in ALI fibroproliferation, using a murine model of lung injury. Wild-type (WT) and lymphocyte-deficient Rag-1(-/-) mice received intratracheal LPS. Fibroproliferation was characterized by histology and the measurement of lung collagen. Lung fibrocytes were measured by flow cytometry. To dissect the role of Tregs in fibroproliferation, Rag-1(-/-) mice received CD4(+)CD25(+) (Tregs) or CD4(+)CD25(-) Tcells (non-Tregs) at the time of LPS injury. To define the role of the chemokine (C-X-C motif) ligand 12 (CXCL12)-CXCR4 pathway in ALI fibroproliferation, Rag-1(-/-) mice were treated with the CXCR4 antagonist AMD3100 to block fibrocyte recruitment. WT and Rag-1(-/-) mice demonstrated significant collagen deposition on Day 3 after LPS. WT mice exhibited the clearance of collagen, but Rag-1(-/-) mice developed persistent fibrosis. This fibrosis was mediated by the sustained epithelial expression of CXCL12 (or stromal cell-derived factor 1 [SDF-1]) that led to increased fibrocyte recruitment. The adoptive transfer of Tregs resolved fibroproliferation by decreasing CXCL12 expression and subsequent fibrocyte recruitment. Blockade of the CXCL12-CXCR4 axis with AMD3100 also decreased lung fibrocytes and fibroproliferation. These results indicate a central role for Tregs in the resolution of ALI fibroproliferation by reducing fibrocyte recruitment along the CXCL12-CXCR4 axis. A dissection of the role of Tregs in ALI fibroproliferation may inform the design of new therapeutic tools for patients with ALI.

Figure 1.

Adoptive transfer of T regulatory cells (Tregs; CD4⁺CD25⁺ cells) but not CD4⁺CD25⁻ cells into Rag-1^−/− mice restored normal resolution of lung injury. (A) Survival curves after intratracheal LPS for all groups (*P = 0.021 by survival log-rank, and ^#P = 0.024 by χ² test, n ≥ 14 in all groups). (B) Weight loss presented as percentage of baseline weight after intratracheal LPS for all groups (*P < 0.001, and ^#P < 0.001, n ≥ 14, all groups). (C) Bronchoalveolar lavage (BAL) cell counts on Day 3 and Day 7 after intratracheal LPS or intratracheal water for all groups (*P = 0.012 and ^#P < 0.05, n > 4, all groups). (D) BAL protein measured by the method of Lowry on Day 3 and Day 7 after intratracheal LPS or intratracheal water for all groups (*P = 0.001 and ^#P = 0.006, n ≥ 4 all groups). AT, adoptive transfer; WT, wild-type.

Figure 2.

Adoptive transfer of Tregs reduced fibroproliferation in Rag-1^−/− mice after LPS injury. (A) Lung sections were stained with Masson’s trichrome to highlight collagen deposition on Day 3 and Day 7 after intratracheal LPS or intratracheal water in WT and Rag-1^−/− mice (n ≥ 3 in each group; images are of representative examples, ×200 magnification). (B) Lung sections were stained with Masson’s trichrome on Day 7 after intratracheal LPS or intratracheal water in Rag-1^−/− mice, Rag-1^−/− mice + Tregs, and Rag-1^−/− mice + non-Tregs (images are of representative examples, ×200 magnification, n ≥ 3 in each group). (C) Lung collagen was measured by Sircol assay in all groups on Day 3 and Day 7 after intratracheal LPS (*P = 0.009 and ^#P = 0.047, n ≥ 4, all groups). (D) BAL collagen was measured by Sircol assay in all groups on Day 3, Day 7, and Day 10 after intratracheal LPS (*P = 0.002, ^†P = 0.003, ^‡P = 0.005, and ^#P = 0.005, n ≥ 3, all groups). (E) Lung collagen was positively correlated with the natural log of BAL collagen (r = 0.73, P < 0.001, using Pearson product moment correlation).

Figure 3.

Fibrocytes were initially increased after LPS injury, and were reduced in the presence of Tregs. (A) Lung fibrocyte number was determined using flow cytometry to identify CD45⁺Col-1⁺CXCR4⁺ cells in lung single-cell suspensions in all groups on Day 3 and Day 7 after intratracheal LPS (*P = 0.007 and ^#P = 0.017, n ≥ 6, all groups). (B) BAL fibrocyte number was determined using flow cytometry to identify CD45⁺Col-1⁺CXCR4⁺ cells in BAL single-cell suspensions on Day 3 and Day 7 after intratracheal LPS (*P < 0.05 and ^#P < 0.05, n ≥ 4, all groups).

Figure 4.

Epithelial cells were an important source of CXCL12 after LPS-induced lung injury. (A) Lung sections were stained for CXCL12 by immunohistochemistry on Day 3 and Day 7 after intratracheal LPS. Epithelial cells (arrows) appeared to be the predominant source of CXCL12 staining (images are of representative examples, ×200 magnification, n ≥ 2 mice in each group; inset, ×400 magnification). (B) Single-cell suspensions were prepared from the lungs of WT and Rag-1^−/− mice on Day 7 after intratracheal LPS or sterile water. Cells were stained with biotinylated anti-CD31 and anti-CD45, followed by V450-conjugated streptavidin as well as allophycocyanin-conjugated (APC)–Cy7–conjugated anti-CD326. Cells were fixed, permeabilized, and stained with APC-conjugated anti-CXCL12. Cells were gated for CD31 and CD45 positivity, and CXCL12 expression was examined by mean fluorescence intensity (MFI) (*P < 0.001, n = 16 mice). (C) CD31⁻CD45⁻ cells were gated for CD326 (epithelial cell adhesion molecule [EpCAM]) expression to identify epithelial cells. CXCL12 expression in CD326⁺ and CD326⁻ cells was examined by MFI (*P < 0.001, n = 16 mice).

Figure 5.

Adoptive transfer of Tregs reduced lung CXCL12 concentrations. CXCL12 concentrations were measured in whole-lung homogenates by ELISA. (A) CXCL12 concentrations were positively correlated with the natural log of lung fibrocyte numbers (r = 0.91, R² = 0.84, P < 0.001, using linear regression on log-transformed data; n = 9 total mice where CXCL12 concentrations and lung fibrocyte numbers were available from the same time point: 5 Rag-1^−/− AT non-Tregs, 3 Rag-1^−/− AT-Tregs, and 1 WT Day 7 LPS). (B) CXCL12 concentrations were determined by ELISA on whole-lung homogenates on Day 3 and day 7 after intratracheal LPS (*P < 0.001, ^#P < 0.001, ^†P = 0.009, ^¥P = 0.01, and ^‡P = 0.036, n ≥ 4, all groups).

Figure 6.

Blockade of the CXCR4 receptor with AMD3100 reduced fibroproliferation in Rag-1^−/− mice by reducing fibrocyte recruitment to the lung after LPS injury. (A) Weight loss presented as percentage of baseline weight after intratracheal LPS for Rag-1^−/− mice that received intraperitoneal AMD3100 or intraperitoneal PBS. (B) Lung fibrocyte number was determined using flow cytometry to identify CD45⁺Col-1⁺CXCR4⁺ cells in lung single-cell suspensions in both groups on Day 7 after intratracheal LPS (*P = 0.001; n ≥ 7, all groups). (C) Lung sections were stained with Masson’s trichrome to highlight collagen deposition on Day 7 after intratracheal LPS in both groups (images are of representative examples; magnification, ×200). (D) Lung collagen was measured by Sircol assay in both groups on Day 7 after intratracheal LPS (*P = 0.012; n ≥ 5, all groups). (E) CXCL12 concentrations were measured in whole-lung homogenates by ELISA in both groups on Day 7 after intratracheal LPS (n ≥ 5, all groups).