Abstract Rationale Foxp3+ regulatory T cells (Tregs) play key roles in resolving lung injury through multiple mechanisms. Our prior work showed that Tregs isolated from the lung during LPS-induced acute lung injury (ALI) display distinct gene expression profiles compared with naïve pulmonary Tregs. Among the differentially expressed genes, Mmp12, encoding a matrix metalloproteinase, was increased more than twenty-fold in resolving pulmonary Tregs. Methods Using the Cre-lox system, we generated a new strain of mice with a deletion in Mmp12 specifically in Tregs (Mmp12ΔTreg). Two established models of ALI were utilized (1) intratracheal lipopolysaccharide (LPS) and (2) a H1N1 mouse-adapted influenza strain (PR8) to examine the role of Mmp12-specific Treg expression on resolving lung injury. Lungs were collected at a resolving time point (day 7 LPS; day 15 PR8) from Mmp12ΔTreg mice or control Foxp3YFP-Cre mice (WT). Immune cell kinetics were assessed using multicolor flow cytometry. N-TAILS proteomics was performed on the bronchoalveolar lavage (BAL) in the LPS model and lung lysates in the influenza model to evaluate differences in the neo-N-termini of proteins formed by proteases between the two strains. Additionally, splenic Tregs from Mmp12ΔTreg or WT mice were isolated for bulk RNA sequencing. Results There were few transcriptional differences in gene expression between the splenic WT and Mmp12ΔTreg Tregs. Mmp12ΔTreg mice demonstrated greater concentrations of two pro-inflammatory soluble mediators, TNF-α and IFN-γ, at peak injury in the LPS-induced ALI model compared to WT controls. Flow cytometric analysis demonstrated that Mmp12ΔTreg mice had higher numbers of neutrophils and B cells in LPS-induced ALI. Similarly, in the influenza model, Mmp12ΔTreg mice had higher neutrophil counts at an early time point of resolution. N-TAILS proteomic analysis of BAL supernatant from day 7 post-LPS demonstrated enrichment of several neo-N-termini in WT but not Mmp12ΔTreg mice, suggesting an effect of Treg-expressed Mmp12 proteolysis. In lung lysates at influenza resolution, a majority of the protein fragments enriched in either the WT or Mmp12ΔTreg mice are involved in immune function. Conclusion The effect of Mmp12-deficient Tregs results in increased inflammation in the immune microenvironment in both models of ALI. Our next steps will focus on validating that Treg-expressed Mmp12-mediated proteolysis is critical for pathways that promote lung injury resolution. This abstract is funded by: 1F31HL180024-01
Bose et al. (Fri,) studied this question.