Transcriptional analysis of Foxp3+ Tregs and functions of two identified molecules during resolution of ALI
Jason R. Mock, … , Hong Dang, Claire M. Doerschuk
Jason R. Mock, … , Hong Dang, Claire M. Doerschuk
Published February 12, 2019
Citation Information: JCI Insight. 2019;4(6):e124958. https://doi.org/10.1172/jci.insight.124958.
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Research Article Immunology Pulmonology

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.

Authors

Jason R. Mock, Catherine F. Dial, Miriya K. Tune, Dustin L. Norton, Jessica R. Martin, John C. Gomez, Robert S. Hagan, Hong Dang, Claire M. Doerschuk

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Figure 5

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

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Treg-depleted mice repleted with Tregs lacking MMP12 expression have mor...
(A) Mmp12 transcript concentrations determined by digital droplet RT-PCR analysis. CD4+GFP+ cells (Tregs) sorted from the lungs of Foxp3EGFP 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) Foxp3DTR (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 Foxp3EGFP 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 Foxp3EGFP 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). Foxp3DTR 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 Foxp3DTR 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.