Central Role of Dendritic Cells in Pulmonary Arterial Hypertension in Human and Mice

Abstract

The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is not fully understood, but evidence is accumulating that immune dysfunction plays a significant role. We previously reported that 31-week-old Tnfaip3^DNGR1-KO mice develop pulmonary hypertension (PH) symptoms. These mice harbor a targeted deletion of the TNFα-induced protein-3 (Tnfaip3) gene, encoding the NF-κB regulatory protein A20, specifically in type I conventional dendritic cells (cDC1s). Here, we studied the involvement of dendritic cells (DCs) in PH in more detail. We found various immune cells, including DCs, in the hearts of Tnfaip3^DNGR1-KO mice, particularly in the right ventricle (RV). Secondly, in young Tnfaip3^DNGR1-KO mice, innate immune activation through airway exposure to toll-like receptor ligands essentially did not result in elevated RV pressures, although we did observe significant RV hypertrophy. Thirdly, PH symptoms in Tnfaip3^DNGR1-KO mice were not enhanced by concomitant mutation of bone morphogenetic protein receptor type 2 (Bmpr2), which is the most affected gene in PAH patients. Finally, in human IPAH lung tissue we found co-localization of DCs and CD8+ T cells, representing the main cell type activated by cDC1s. Taken together, these findings support a unique role of cDC1s in PAH pathogenesis, independent of general immune activation or a mutation in the Bmpr2 gene.

Keywords: BMPR2; Tnfaip3; Toll-like receptor; dendritic cells; inflammation; pulmonary arterial hypertension.

Figure 1

Increased myocardial infiltration of dendritic cells (DCs) in the right ventricle (RV) of Tnfaip3^DNGR1-KO mice. (a) Flow cytometry analysis for CD45+ cells (alive CD45⁺ cells), DCs (CD3-/CD19-, MHC-II+CD11c+), T cells (CD3+) and B cells (CD19+) in separately measured right and left ventricle cell suspensions from hearts of Tnfaip3^DNGR1-WT/KO mice; (b) mRNA expression (relative to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene) of DC markers CD11c and BATF3, and the T cell subset markers CD4 and CD8 in 24- and 31-week-old Tnfaip3^DNGR1-KO and wild-type (WT) control mice; (c) whole mount analysis of right ventricle staining for YFP (green) and MHCII (red) in age 14-, 24- or 31-week-old Tnfaip3^DNGR1-KO mice. Results are presented as mean values + standard deviations of 4–6 mice per group. * p < 0.05, ** p < 0.01; (d) Elastin van Gieson (EvG)-stained whole heart section histology of Tnfaip3^DNGR1-WT and Tnfaip3^DNGR1-KO mice for representative sections. Scale in left panels is 2.5 mm and in right panels 100 µm. Arrows indicate lymphocytes.

Figure 2

TLR4 activation leads to RV hypertrophy and cDC1 phenotype changes in Tnfaip3^DNGR1-KO mice. (a) Scheme of intratracheal (i.t.) administration of CpG, lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C) in WT and Tnfaip3^DNGR1-KO mice on day 0 and 7, and analysis on day 21; (b) right ventricular systolic pressure (RVSP) in toll-like receptor (TLR)-exposed WT and Tnfaip3^DNGR1-KO mice, determined by right heart catheterization; (c) hypertrophy of RV measured by Fulton index (right ventricle/ left ventricle + septum); (d) assessment of the indicated cell types in LPS-exposed WT and Tnfaip3^DNGR1-KO mice. Proportion of the CD64⁺GR1⁻ macrophage/monocyte population from total alive cells is indicated below the pie charts; (e,f) quantification of total DCs (e) and the CD103+ cDC1/CD11b+ cDC2 subset ratio (f) in the lungs of the indicated mice, as determined by flow cytometry; (g) quantification of surface CD86, CD40 and PD-L1 expression on CD103+ cDC1s (left) and CD11b+ cDC2s (right) in the indicated mouse groups. Flow cytometry analyses are shown as histogram overlays of CD86 expression (top), as dot plots with CD64/CD40 profiles (middle) and dot plots with CD64/PD-L1 profiles (bottom) of gated cDC1s. Results are presented as mean values + standard deviation of 3–10 mice (b,c) or 3–6 (e–g) mice per group. MFI = median fluorescence intensity. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Concomitant Bmpr2 mutation does not alter the pulmonary hypertension (PH) phenotype in Tnfaip3^DNGR1-KO mice. (a) Right ventricular systolic pressure (RVSP), determined by right heart catheterization and hypertrophy of RV measured by Fulton index (right ventricle/ left ventricle + septum) of the indicated mouse groups; (b) quantification of lung CD103+CD11c+ cDC1 frequencies and CD86 expression (MFI = median fluorescence intensity) by flow cytometry; (c–f) quantification of CD64+GR1- monocyte/macrophage population (c), CD3+ T cells (d), memory CD44+CD62L- CD8+ T cells (e) or CD19+ B cells (f) determined by flow cytometry in the lungs of the indicated mouse groups. Results are presented as mean + standard deviation of 10–31 mice (a) or 4–12 mice (b–e) per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

DCs and T cells are more often in close proximity around vessels and in parenchyma in Tnfaip3^DNGR1-KO mice. (a) DC (CD11c, red) and T cell (CD3, blue) staining on lung cryosections of 31-week-old WT mice of which an area around a vessel (top) and parenchyma (bottom) of the same mouse is depicted. Magnification of selected areas (right); (b) DC (CD11c, red) and T cell (CD3, blue) staining on lung cryosections of 31-week-old Tnfaip3^DNGR1-KO mice of which an area around a vessel (top) and parenchyma (bottom) of the same mouse is depicted. Magnification of selected areas (right); (c) DC (CD11c, red) and CD8+ T cells (CD8, blue) of WT (top) and Tnfaip3^DNGR1-KO (bottom) mice around a vessel. Magnification of selected area (right). Data shown are representative for 4–6 mice per group. Scale in left panels is 50 µm and of right panels 25 µm. Blue arrows indicate T-cells, and red arrows indicate DCs in the larger magnification (right panels).

Figure 5

DCs are in close proximity to CD8+ T cells around vessels and in parenchyma in lungs of idiopathic pulmonary arterial hypertension (IPAH) patients. (a,b) 4-plex chromogenic multiplex staining of DCs (CD206+, yellow), macrophages (CD68+, purple with or without CD206), T cells (CD8, DAB) and α-SMA (green) around a vessel and intraparenchymal (a) or in parenchyma (b) and the magnification of the indicated area (right) in the same healthy tissue from a smoking patient who was diagnosed with adenocarcinoma; (c,d) determination of DCs (CD206+CD68-), CD8+ T cells (CD8+), macrophages (CD68+) and vessels (α-SMA) around a fully occluded vessel (c) or partly occluded vessel (d) and the magnification of the indicated area (right) of the same IPAH patient with moderate remodeling; (e,f) determination of DCs, CD8+ T cells, macrophages and vessels around a fully occluded vessel (e) or partly occluded vessel (f) and the magnification of the indicated area (right) of the same a IPAH patient with extensive remodeling and immune cell infiltration. Data shown are representative for 6 IPAH patients. Sporadic CD206+CD68- cells with a morphology suggesting neutrophil identity were not regarded as DCs. Scale in left panels is 100 µm and of right panels 50 µm. Arrows indicate DC and CD8 co-localization