Transcriptional analysis of Foxp3+ Tregs and functions of two identified molecules during resolution of ALI

Abstract

Recovery from acute lung injury (ALI) is an active process. Foxp3+ Tregs contribute to recovery from ALI through modulating immune responses and enhancing alveolar epithelial proliferation and tissue repair. The current study investigates Treg transcriptional profiles during resolution of ALI in mice. Tregs from either lung or splenic tissue were isolated from uninjured mice or mice recovering from ALI and then examined for differential gene expression between these conditions. In mice with ALI, Tregs isolated from the lungs had hundreds of differentially expressed transcripts compared with those from the spleen, indicating that organ specificity and microenvironment are critical in Treg function. These regulated transcripts suggest which intracellular signaling pathways modulate Treg behavior. Interestingly, several transcripts having no prior recognized function in Tregs were differentially expressed by lung Tregs during resolution. Further investigation into 2 identified transcripts, Mmp12 and Sik1, revealed that Treg-specific expression of each plays a role in Treg-promoted ALI resolution. This study provides potentially novel information describing the signals that may expand resident Tregs, recruit or retain them to the lung during ALI, and modulate their function. The results provide insight into both tissue- and immune microenvironment-specific transcriptional differences through which Tregs direct their effects.

Keywords: Cellular immune response; Immunology; Pulmonology; T cells; Transcription.

Figure 1. Foxp3⁺ Tregs increase in the lung during resolution of ALI.

Male Foxp3^EGFP mice were challenged with LPS (3 mg/kg) intratracheally. (A) Body weight relative to baseline measured daily before and after injury (n = 15–47 per time point). (B–D) Total lung cell count in the lung digest (B), total cell counts in the bronchoalveolar lavage (BAL) (C), and BAL total protein concentration (D) were determined in Foxp3^EGP mice, either unchallenged or after treatment with LPS (n = 10–22 per time point; combined from 2–3 separate experiments). (E–F) The number of GFP⁺ cells in the BAL (E) or lung digests (F) of unchallenged mice and mice 3, 7, or 10 days after LPS (n = 10–16 per condition). (G) The percentage of total CD4⁺ cells that were Foxp3⁺, determined in unchallenged mice or mice 3, 7, or 10 days after LPS (n = 10–16 per condition). Data are expressed as the mean ± SEM where applicable. P values were determined by 1-way ANOVA with Tukey’s multiple comparison test between conditions. **P < 0.01 ***P < 0.001, ****P < 0.0001.

Figure 2. The impact of both an inflammatory stimulus and location on the Treg transcriptome.

Tregs were isolated from the lungs of unchallenged Foxp3^EGFP mice or from the lungs and spleens from Foxp3^EGFP mice 7 days after LPS (3 mg/kg i.t.) challenge. (A) Body weight relative to baseline measured daily before and after injury (n = 19 females; 39 males; 58 total). Data are expressed as the mean ± SEM. (B) Principal component analysis (PCA) of mRNA expression demonstrating the contribution of the top 3 principal components to the variance in the 3 Treg sets. Each symbol represents 1 sort of pooled Tregs from mice (15–27 mice/experiment); 3 arrays/set. (C) Venn diagram showing the overlap of significantly differentially expressed transcripts between sets (adjusted P < 0.05, fold change > 2). Area of the circles reflects the relative number of differentially expressed transcripts for each comparison. Numbers in parentheses indicate the total from the respective pairwise comparison. These transcripts are listed in Supplemental Tables 1–3. (D) Unsupervised hierarchical clustering with Euclidean distance metric of 1,599 differentially expressed transcripts identified in all 3 comparisons. The dendrogram identifies clustering across transcripts (top) or across experimental samples (right). The heatmap depicts the log₂ normalized intensity, and each row represents Treg RNA from a pooled set of mice. As described in the Methods, transcripts between sorted Treg sets were identified by filtering at adjusted P < 0.05 and fold change > 2 (bright blue for lower expression levels and bright red for high expression levels). (E) Differentially expressed genes (DEGs) between resolving lung Tregs and unchallenged lung Tregs. Unsupervised hierarchical clustering of the unique 78 DEGs between lung Treg samples. The heatmap depicts the log₂ normalized intensity across samples. Each column represents Treg RNA from pooled Tregs, and each row represents a DEG. The dendrogram identifies clustering across experimental samples (top) or DEGs (right). (F) Select list of transcripts with a >2-fold change between resolving lung Tregs and unchallenged lung Treg comparisons. (G) Select list of transcripts with a >2-fold change between resolving lung Tregs and resolving splenic Treg comparisons.

Figure 3. Protein expression of receptors and other surface molecules on Foxp3⁺ Tregs in the lung and spleen and their numbers in lung during ALI resolution compared with unchallenged mice.

Foxp3^EGFP mice were challenged with LPS (3 mg/kg i.t.) and compared with unchallenged Foxp3^EGFP mice (labeled as “LPS–” mice in figures). (A) Percentages of CD4⁺ cells were compared with the total number of lymphocytes in the lymphocyte gate determined by flow cytometry in lung or spleen single cell suspensions of unchallenged (control) mice or at 7 days after LPS (n = 6–8 per condition). (B) The percentage of total CD4⁺ cells that expressed Foxp3 was determined (n = 6–14 per set). (C) Median fluorescence intensity (MFI) of GFP expression driven by the endogenous Foxp3 promoter was determined in the lung and spleen in unchallenged mice or at 7 days after LPS (n = 6–8 per condition). (D–F) Percentages of CD103⁺Foxp3⁺ cells (D), IL18R⁺Foxp3⁺ cells (E), and ST2⁺Foxp3⁺ cells (F) as a percentage of total Foxp3⁺ cells (n = 6–14 per condition). (G–I) Numbers of CD103⁺Foxp3⁺ cells (G), IL18R⁺Foxp3⁺ cells (H), and ST2⁺Foxp3⁺ cells (I) determined in the lung between conditions (n = 6–8 per condition). Data are expressed as the mean ± SEM. Mann Whitney rank sum test determined P values.

Figure 4. Treg-depleted mice have increased immune cell numbers during resolution of acute lung injury.

(A) Foxp3^EGFP or Foxp3^DTR mice (n = 9–15 per group, combined from 2 separate experiments) were challenged with intraperitoneal diphtheria toxin (DT; 50 μg/kg or 10 μg/kg) administered at days –2, –1, 1, 3, and 5 along with intratracheal LPS was administered at day 0. Mice were examined at 7 days after LPS for immune cell populations. (B) Body weight relative to baseline determined after injury. (C) Representative H&E lung sections reveal increased cellularity 7 days after LPS and DT in Treg-depleted mice. Gray scale bar: 1 mM. Black scale bar: 500 μM. Maroon scale bar: 250 μM. (D) Total lung cell count from single cell lung suspension obtained at day 7 after DT and LPS treatment demonstrated increase cellularity in Treg-depleted mice. (E) Total CD4⁺Foxp3⁺ lymphocyte numbers determined and which confirm Treg depletion. (F–J) Changes in myeloid cell subsets as total numbers in single cell suspensions determined using a published flow cytometric panel and gating approach (30). Treg-depleted mice had significant increases in alveolar macrophages, neutrophils, and interstitial macrophages. (K–Q) Changes in lymphocyte subsets as a total numbers in single cell suspensions determined and demonstrated increased numbers of CD3⁺, CD4⁺, and CD8⁺, γδ⁺, NK, and NKT lymphocytes. (N–O) Numbers of CD19⁺ and γδ⁺ lymphocytes were also found at lower numbers in Treg-depleted mice. Data are expressed as the mean ± SEM. P values calculated by Mann Whitney rank sum test. *P < 0.05, **P < 0.01 ***P < 0.001, ****P < 0.0001.

Figure 5. Treg-depleted mice repleted with Tregs lacking MMP12 expression have more neutrophils during lung resolution.

(A) Mmp12 transcript concentrations determined by digital droplet RT-PCR analysis. CD4⁺GFP⁺ cells (Tregs) sorted from the lungs of Foxp3^EGFP mice either from control lungs or lungs resolving from ALI (n = 2 per group, representative of 3 separate experiments). P value by 2-tailed Student t test. (B–L) Foxp3^DTR (DTR) mice were given diphtheria toxin (DT) to deplete endogenous Tregs as previously described (16), followed by LPS. One hour after LPS challenge, the DTR mice received 1 × 10⁶ CD4⁺CD25⁺ cells (Tregs) from either Foxp3^EGFP or Mmp12^–/– mice. n = 12–14 per group, combined from 2 separate experiments. Samples are combined from 2 independent experiments (B). Adoptive transfer of Mmp12^–/– Tregs have similar weight recovery (C), BAL cell counts (D), and BAL total protein (E) after LPS-induced ALI compared with Foxp3^EGFP Tregs (Mmp12^+/+) 7 days after LPS administration. Total lung cell count and the number of Tregs between the adoptive transfer conditions did not differ (F and G). Changes in myeloid cell subsets as total numbers in single cell suspensions determined using a published flow cytometric panel and gating approach (30) (H–L). Foxp3^DTR mice depleted of endogenous Tregs and then repleted with Mmp12^–/– Tregs at the time of injury had greater neutrophil cell numbers and a trend for increased interstitial macrophages numbers compared with Foxp3^DTR mice repleted with Mmp12^+/+ Tregs 7 days after LPS administration (I–J). Data are expressed as the mean ± SEM. P values calculated by Mann Whitney rank sum test.

Figure 6. Treg-depleted mice repleted with Tregs lacking SIK1 expression have more Tregs and increased Treg CD103 expression during lung resolution.

Foxp3^DTR (DTR) mice were given diphtheria toxin (DT) to deplete endogenous Tregs as previously described (16), followed by LPS. One hour after LPS challenge, the DTR mice received 1 × 10⁶ CD4⁺CD25⁺ cells (Tregs) from either Foxp3^EGFP or Sik1^–/– mice. Samples are combined from 4 independent experiments (n = 22–35 per group). (A) Weight recovery after LPS-induced ALI is similar after adoptive transfer of Sik1^–/– Tregs compared with Foxp3^EGFP Tregs (Sik1^+/+) 7 days after LPS administration. (B) Total lung cell count did not differ between the adoptive transfer conditions. (C–E) Foxp3^DTR mice depleted of endogenous Tregs and then repleted with Sik1^–/– Tregs at the time of injury had greater numbers of Foxp3⁺ cells (C), decreased Foxp3⁺ expression as measured by median fluorescence intensity (MFI) in Foxp3⁺ cells (D), and a greater percentage of Foxp3⁺ cells coexpressing CD103 (E). (F–G) The increased percentage of Foxp3⁺ cells expressing CD103⁺ correlated with a greater number of Foxp3⁺CD103⁺ Tregs (F) and a higher expression of CD103 in Tregs as measured by MFI (G). (H) The percentage of Foxp3⁺ cell that coexpressed IL18R was also increased in Foxp3^DTR mice repleted with Sik1^–/– Tregs compared with Sik1^+/+ Tregs. (I–M) Changes in myeloid cell subsets as total numbers in single cell suspensions determined using a published flow cytometric panel and gating approach (30). Foxp3^DTR mice depleted of endogenous Tregs and then repleted with Sik1^–/– Tregs at the time of injury had less Ly6C^– monocyte/macrophage numbers compared with Foxp3^DTR mice repleted with Sik1^+/+ Tregs. Data are expressed as the mean ± SEM. P values calculated by Mann Whitney rank sum test.