Foxp3+ regulatory T cells promote lung epithelial proliferation

Abstract

Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality each year. There is a paucity of information regarding the mechanisms necessary for ARDS resolution. Foxp3(+) regulatory T cells (Foxp3(+) T(reg) cells) have been shown to be an important determinant of resolution in an experimental model of lung injury. We demonstrate that intratracheal delivery of endotoxin (lipopolysaccharide) elicits alveolar epithelial damage from which the epithelium undergoes proliferation and repair. Epithelial proliferation coincided with an increase in Foxp3(+) T(reg) cells in the lung during the course of resolution. To dissect the role that Foxp3(+) T(reg) cells exert on epithelial proliferation, we depleted Foxp3(+) T(reg) cells, which led to decreased alveolar epithelial proliferation and delayed lung injury recovery. Furthermore, antibody-mediated blockade of CD103, an integrin, which binds to epithelial expressed E-cadherin decreased Foxp3(+) T(reg) numbers and decreased rates of epithelial proliferation after injury. In a non-inflammatory model of regenerative alveologenesis, left lung pneumonectomy, we found that Foxp3(+) T(reg) cells enhanced epithelial proliferation. Moreover, Foxp3(+) T(reg) cells co-cultured with primary type II alveolar cells (AT2) directly increased AT2 cell proliferation in a CD103-dependent manner. These studies provide evidence of a new and integral role for Foxp3(+) T(reg) cells in repair of the lung epithelium.

Figure 1. Lung epithelial cells are more proliferative after LPS-induced ALI

Epithelial proliferation levels peak during ALI resolution. WT mice (n = 8–14 per group per time point) were challenged with IT LPS. (A) Representative lung sections were stained with H&E to reveal morphologic changes at time points after IT LPS. Original magnification, x40. (B–D) BAL total protein (B), BAL total cell counts (C), and BAL soluble RAGE measured by ELISA (D) were determined in WT mice controls or after treatment with LPS. *p < 0.001 versus control by Mann Whitney. (E–G) Total lung cell count (E), Total CD326⁺ or E-cadherin⁺ cell counts (F), and percentage of proliferating (ki-67⁺) CD326⁺ or E-cadherin⁺ cells (G) were determined by flow cytometry in WT mice controls or after treatment with IT LPS. #p < 0.05 versus control by Mann Whitney. (H) Alveolar Treg (CD4⁺CD25⁺Foxp3⁺) numbers at baseline and after injury in WT mice. (I) Total Foxp3⁺ T_reg numbers (GFP⁺ cells) in single cell suspensions of Foxp3^gfp mice at baseline and 7 days after treatment with IT water or LPS. (n = 7 per group). ^p < 0.01 versus control by Mann Whitney. Figure p values determined by Mann Whitney.

Figure 2. Epithelial proliferation during ALI resolution is impaired in T_reg-depleted mice

T_reg-depleted mice have decreased levels of epithelial proliferation. (A) Foxp3^gfp or Foxp3^DTR mice (n = 5–9 per group) were challenged with IT LPS (day 0) and/or diphtheria toxin (50 μg/kg or 10 μg/kg) administered at day -2, -1, 1, 3, and 5 and harvested at day 7 post LPS. (B) Representative lung sections were stained with H&E to reveal morphologic changes 7 days after IT LPS ± DT. Original magnification, x40. (C–F) Total lung cell count (C), Total CD326⁺ cell numbers (D), proliferating epithelial cell (CD326⁺ki-67⁺) numbers (E) and percentage of proliferating CD326⁺ cells compared to total CD326⁺ cells (F) were determined by flow cytometry. p values determined by Mann Whitney.

Figure 3. T_reg number and epithelial proliferation after ALI is abrogated with antibody blockade of CD103

CD103 blockade decreased lung Foxp3⁺ T_reg number and subsequent epithelial proliferation after ALI. (A) WT mice (n = 10–12 per group per time point) were challenged with IT LPS and the number of Foxp3⁺ T_reg cells in the lung cell suspensions were identified by flow cytometry along with the percentage of Foxp3⁺ cells that co-expressed CD103 (n = 7–9 per group per time point). *p < 0.05; ^p < 0.005 versus control by Mann Whitney. (B–F) WT mice (n = 9 per group) were challenged with IT LPS along with administration of either an anti-CD103 antibody (150 μg) or isotype control (150 μg rat IgG) at days 0, 1, 3, and 5 post IT LPS (B). The number of Foxp3⁺ cells in the lung cell suspensions were determined by flow cytometry (C) along with the total number (D) and percentage (E) of proliferating CD326⁺ cells (CD326⁺ki-67⁺ cells) cells. (F) Total lung Foxp3⁺ cell number positively correlated with CD326⁺ki-67⁺ epithelial cell number (Pearson r = 0.8730, R² = 0.7621, p < 0.0001 using linear regression). #p = 0.0939; *p < 0.05 by Mann Whitney.

Figure 4. Adoptive transfer of T_regs augments epithelial proliferation after ALI in Rag-1^−/− mice

AT of WT T_regs augments epithelial proliferation after ALI in Rag-1^−/− mice. Rag-1^−/− mice were challenged with IT LPS and 1 hour afterward received 1 × 10⁶ WT CD4⁺CD25⁺, WT CD4⁺CD25⁻ or CD103^−/−CD4⁺CD25⁺ cells and lungs harvested at day 7 post LPS administration. (A) Percentage of proliferating CD326⁺ki-67⁺ epithelial cells at day 7 post LPS in WT mice or Rag-1^−/− mice after infusion of designated lymphocytes subsets (n = 6–12 per group). (B) Total number of Foxp3⁺ cells in the lungs of WT mice or Rag-1^−/− mice after infusion of designated lymphocytes subsets (n = 4–7 per group). p values determined by student t-test.

Figure 5. T_regs augment epithelial proliferation in mice after PNX

WT, Rag-1^−/−, Foxp3^gfp, and Foxp3^DTR mice underwent left lung pneumonectomy. Foxp3^gfp and Foxp3^DTR mice undergoing PNX were also administered DT at the time of PNX and at days 2, 4, and 6 post PNX. (A) Representative lung sections were stained with H&E to reveal morphologic changes 7 days post procedure in WT sham, WT PNX, and Foxp3^DTR mice (PNX and DT). Original magnification, x40 and 100x. (B–C) WT mice (n = 5 per group) alveolar (B) and lung (C) Foxp3⁺ T_reg number at baseline and at 7 days post sham or PNX. *p < 0.05 versus control/sham by 1 way ANOVA. (D–F) Total lung cell count (D), CD326⁺ cell numbers (E) and percentage of proliferating epithelial cells (CD326⁺ki-67⁺) number (F) were determined by flow cytometry 7 days after sham procedure or PNX (n = 6–13 per group). p values determined by Mann Whitney.

Figure 6. T_regs enhance proliferation of AT2 cells in vitro

Primary AT2 cells were isolated by sorting GFP⁺ cells from SP-C^GFP mice and co-cultured with lymphocytes subsets (WT CD4⁺CD25⁺, WT CD4⁺CD25⁻) at a lymphocyte: AT2 ratio of 1:5, n ≥ 5 for each condition. Fold increase (compared to media alone) in proliferation (CD326⁺ki-67⁺) was determined after 24 hours of co-culture of AT2 cells with either CD4⁺CD25⁻, CD4⁺CD25⁺, CD4⁺CD25⁺ plus addition of CD103 blocking antibody, or CD4⁺CD25⁺ separated from AT2 cells through a Transwell insert. p values determined by Mann Whitney.