Bronchoalveolar Tregs are associated with duration of mechanical ventilation in acute respiratory distress syndrome

Abstract

Background: Foxp3⁺ regulatory T cells (Tregs) play essential roles in immune homeostasis and repair of damaged lung tissue. We hypothesized that patients whose lung injury resolves quickly, as measured by time to liberation from mechanical ventilation, have a higher percentage of Tregs amongst CD4⁺ T cells in either airway, bronchoalveolar lavage (BAL) or peripheral blood samples.

Methods: We prospectively enrolled patients with ARDS requiring mechanical ventilation and collected serial samples, the first within 72 h of ARDS diagnosis (day 0) and the second 48-96 h later (day 3). We analyzed immune cell populations and cytokines in BAL, tracheal aspirates and peripheral blood, as well as cytokines in plasma, obtained at the time of bronchoscopy. The study cohort was divided into fast resolvers (FR; n = 8) and slow resolvers (SR; n = 5), based on the median number of days until first extubation for all participants (n = 13). The primary measure was the percentage of CD4⁺ T cells that were Tregs.

Results: The BAL of FR contained more Tregs than SR. This finding did not extend to Tregs in tracheal aspirates or blood. BAL Tregs expressed more of the full-length FOXP3 than a splice variant missing exon 2 compared to Tregs in simultaneously obtained peripheral blood.

Conclusion: Tregs are present in the bronchoalveolar space during ARDS. A greater percentage of CD4⁺ cells were Tregs in the BAL of FR than SR. Tregs may play a role in the resolution of ARDS, and enhancing their numbers or functions may be a therapeutic target.

Keywords: Acute respiratory distress syndrome; Regulatory T cells; Resolution.

Fig. 1

Treg percentage during ARDS in the bronchoalveolar lavage (BAL), tracheal aspirate, and peripheral blood. Percentages of CD4⁺ cells that are FOXP3⁺ were quantified in cells obtained from BAL, tracheal aspirate, or peripheral blood. a The mean percentages of CD4⁺ cells that are FOXP3⁺ in two BALs performed during bronchoscopy at two time points (n = 13). Each participant is identified by a unique symbol. Each is categorized as a slow or fast resolver, based on the median number of days to extubation. Slow resolvers are open symbols with a dashed line connecting points, while fast resolvers are filled in solid black with a solid line connecting points. b The percentage of CD4⁺ cells that are FOXP3⁺ for each lavage (two for each participant) at Day 0. Participants are categorized as slow or fast resolvers. c The average percentage of FOXP3⁺ CD4⁺ cells of the two BALs during bronchoscopy performed on Day 0. Participants are categorized as slow or fast resolvers (SR, n = 5; FR, n = 8). d The highest percentage of FOXP3⁺ CD4⁺ cells obtained at either time point is shown for slow and fast resolvers (SR, n = 5; FR, n = 8). e The change in average percentages of FOXP3⁺ CD4⁺ cells between Day 0 and Day 3; participants are categorized as slow or fast resolvers (SR n = 5; FR n = 5). Of note, the lower FR number is because 3 FR were extubated before the second bronchoscopy time point. f The percentages of CD4⁺ cells that are FOXP3⁺ in tracheal aspirates obtained just prior to bronchoscopy at Day 0 and 3 (Day 0, n = 12; Day 3, n = 11). g The percentage of FOXP3⁺ CD4⁺ cells in tracheal aspirates at Day 0. Participants are categorized as slow or fast resolvers (SR, n = 5; FR, n = 7). h The highest percentage of FOXP3⁺ CD4⁺ cells in tracheal aspirates obtained at either Day 0 or Day 3; participants categorized as slow or fast resolvers (SR, n = 5; FR, n = 8). i The change in average percentages of FOXP3⁺ CD4⁺ cells in tracheal aspirates between Day 0 and Day 3; participants grouped as slow or fast resolvers (SR, n = 5; FR, n = 5). j The percentages of CD4⁺ cells that are FOXP3⁺ in peripheral blood obtained at the time of bronchoscopy at two time points (Day 0, n = 12; Day 3, n = 9). k The percentage of FOXP3⁺ CD4⁺ cells in peripheral blood at Day 0. Participants are categorized as slow or fast resolvers (SR, n = 5; FR, n = 7). l The highest percentage of FOXP3⁺ CD4⁺ cells in peripheral blood at either time point, participants categorized as slow or fast resolvers (SR, n = 5; FR, n = 8). m The change in average percentages of FOXP3⁺ CD4⁺ cells in peripheral blood between Day 0 to Day 3, participants categorized as slow or fast resolvers (SR, n = 4; FR, n = 4). Data are expressed as the mean ± standard error of the mean. *P < 0.05, ***P < 0.001

Fig. 2

FOXP3 mRNA splicing differs in Tregs from the BAL compared to the peripheral blood. a Schematic of two antibody clones that distinguish two of the most common FOXP3 isoforms. Both antibodies (clones PCH101 and 150D) bind to the full-length protein, whereas clone 150D does not bind to the splice variant missing region of FOXP3 coded by exon 2, part of the repressor domain of FOXP3. The regions where two FOXP3 monoclonal antibodies, 150D and PCH101, bind to the FOXP3 protein are illustrated. b Flow cytometric plots and gating schemes used to identify the full length FOXP3 and the splice variant of FOXP3 are illustrated for Tregs in the peripheral blood or BAL using a set of stored blood and BAL cell samples. Gating and dot plot results are representative of at least three independent experiments. c Percentage of full length FOXP3 isoform⁺ cells to total FOXP3⁺ Tregs isolated from either the peripheral blood or BAL samples (Day 0: n = 9 lavages, blood n = 10 samples; Day 3: n = 5 lavages, blood n = 7 samples). Data were analyzed using a two way repeated measure ANOVA, with both days and type as repeated factors. The blood results are significantly different from the BAL results on both day 0 and day 3 (P = 0.0245). The data are not different between days. d, e Immunofluorescence of BAL (d) or peripheral blood (e) cytospins prepared from a participant and stained for DNA, CD4⁺, FOXP3⁺ clone 150D, and FOXP3⁺ clone PCH101. Arrows indicate Tregs. White bar: 20 µM. f RNA-Seq data examining two FOXP3 splice variants in a previously reported data set of Tregs isolated from human breast tissue or peripheral blood mononuclear cells (PBMC) [19]. Data expressed as the mean ± SEM. P values for the RNA-Seq data determined by paired and independent t-tests. *P < 0.05

Fig. 3

Immune cell subsets identified in the bronchoalveolar lavage (BAL), tracheal aspirate, or peripheral blood by flow cytometry. Box-and-Whisker plots showing median, minimum, and maximum with overlay of individual data points (dots) of immune cells, expressed as a percentage of CD45⁺ cells at day 0 and/or day 3 after enrollment. a–d BAL immune cells expressed as a percentage of CD45⁺ cells at day 0 or day 3 after enrollment. a Percentages are shown for all participants at both time points. b Fast and slow responders are compared combining data from both days. c Fast and slow responders are compared at Day 0. d Fast and slow responders are compared at Day 3. e–h The tracheal aspirate immune cells expressed as a percentage of CD45⁺ cells at day 0 or day 3 after enrollment. Percentages are shown for participants at both time points (e), grouped as fast or slow resolvers (f), and categorized as slow or fast resolvers at Day 0 (g) or Day 3 (h). i–l The peripheral blood immune cells expressed as a percentage of CD45⁺ cells at day 0 or day 3 after enrollment. Percentages are shown for participants at both time points (i), grouped as fast or slow resolvers (j) and categorized as slow or fast resolvers at Day 0 (k) or Day 3 (l). The flow cytometric plots and gating scheme used for the identification of immune cells with gating were adapted from [23]. Data were analyzed per type of cell, without correction of multiple comparisons. For each cell type, repeated measure ANOVA was used to compare day 0 and 3, fast and slow resolvers, and interaction

Fig. 4

Percentage of CD3⁺ cells that are either CD4⁺ or CD8⁺ in the bronchoalveolar lavage (BAL) or peripheral blood from participants at either Day 0 or Day 3. Box-and-Whisker plots showing median, minimum, and maximum with overlay of individual data points (dots) of the percentage of CD3⁺ cells that are either CD4⁺ or CD8⁺ on day 0 or day 3 after enrollment. a, b Percentage of CD3⁺ lymphocytes that are either CD4⁺ or CD8⁺ in BAL, shown as the average of two lavages performed during bronchoscopy at Day 0 (a) and Day 3 (b). The participants are categorized as slow (SR) or fast resolvers (FR) at either Day 0 (SR n = 5; FR n = 8) or Day 3 (SR n = 4; FR n = 4). c, d Percentage of CD3⁺ lymphocytes that are either CD4⁺ or CD8⁺ in the peripheral blood, shown for all samples at Day 0 (c) and Day 3 (d). The participants are categorized as slow or fast resolvers at either Day 0 (SR n = 5; FR n = 7) or Day 3 (SR n = 4; FR n = 5). No significant differences were identified. Data were analyzed per cell type, without correction for multiple comparisons. For each cell type, repeated measure ANOVA was used to compare day 0 and 3, fast and slow resolvers, and interaction

Fig. 5

Immune mediators in bronchoalveolar lavage (BAL) or peripheral plasma from participants at either Day 0 or Day 3. Box-and-Whisker plots showing median, minimum, and maximum with overlay of individual data points (dots) of selected concentrations of mediators from Tables 3 and 4 measured in BAL or plasma at Day 0 or Day 3 after enrollment. In each figure, the data are shown for all samples, as well as when grouped as slow resolvers (SR) and fast resolvers (FR). a–d BAL concentrations of sCD163, IL-11, IL-26, and osteopontin. Day 0: n = 12, Day 3: n = 8; Combined days: SR = 9, FR = 11; Day 0: SR = 5, FR = 7; Day 3: SR = 4, FR = 5. e, f Plasma cytokine concentrations of TNFSF13 and TNFSF13B. Day 0: n = 12, Day 3 n = 10; Combined days: SR = 10, FR = 12; Day 0: SR = 5, FR = 7; Day 3: SR = 5, FR = 5. For each cell type, repeated measure ANOVA was used to compare day 0 and 3, fast and slow resolvers, and interactions. *P < 0.05