B18-02 Th17 Immunity Is Implicated in Accelerating Vascular Remodeling After Foxp3 Ablation

Abstract Rationale Despite advances in Group 1 Pulmonary Arterial Hypertension (PAH) treatment, gaps remain in understanding the pathobiology of PAH associated with autoimmune conditions like Connective-Tissue Disease. Methods The chronic inflammatory model of pulmonary hypertension (CIPH model, PMCID: PMC7578108) develops occlusive neointimal lesions and right ventricular systolic pressure (RVSP) elevation after six weeks of daily antigen (HDM) exposure. Foxp3IRES-DTR-GFP mice (Foxp3Dtr) enable selective deletion of Foxp3 expressing cells, including anti-inflammatory T regulatory cells (Tregs), after intraperitoneal diphtheria toxin administration. Pairs of male and female Foxp3Dtr mice were used in the CIPH model. At the start of neointima formation either PBS or diphtheria toxin were administered every other day for 3 doses. At four weeks and six days, mice were euthanized and lung tissue was analyzed using immunohistochemistry, RNA in situ hybridization, and single cell analysis. Single-cell Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) was conducted and included amplified mRNA immune response analysis, whole transcriptome and T-Cell Receptor (TCR) sequencing, and epitope identification with barcoded antibodies. Finally, Balb/c mice in the CIPH model were treated with either (1) IL-17 neutralizing antibody, (2) Interferon-γ neutralizing antibody, or (3) control IgG antibody. After six weeks, mice underwent RVSP measurement and histological assessment. Result CIPH Foxp3Dtr mice who received diphtheria toxin had significantly accelerated neointima development versus those who received PBS (Figure 1A-C). Ablation of Foxp3 Tregs was verified by GFP expression (Figure 1D). CITE-Seq analysis revealed significant changes in monocyte, macrophage, B-, and T-Cell populations after Treg ablation. Focusing on T Cells, Treg ablation increased Th1 and Th17 populations and decreased Th2 populations. T-Cell clonal analysis by paired, single cell TCR sequencing found significant clonal expansion of Interferon-γ and IL17 expressing clones after Treg ablation. RNA in situ hybridization verified concentrated perivascular Il17a expressing T-cells. In the CIPH model, vessel occlusion by neointima was significantly reduced by Il17 blockade (11% vs. 62% in control, p 0.001) and partially reduced by Interferon-γ blockade (27% vs. 62% in control, p 0.001). Notably, RVSP was significantly reduced by Il17 blockade (22.4 vs. 29 mmHg in control, p = 0.021) but not Interferon-γ blockade (26.2 vs. 29.0 mmHg in control). Conclusions Deletion of Foxp3+ Tregs accelerates neointima development, increases Th1 and Th17 populations, and decreases Th2 populations. Anti-Il17 antibody therapy inhibits experimental pulmonary hypertension development. These data could provide a rationale for anti-IL17 therapies in autoimmunity-associated PAH. This abstract is funded by: Parker B Francis Fellowship, Pulmonary Hypertension Association, National Institutes of Health