Predominance of M2 macrophages in organized thrombi in chronic thromboembolic pulmonary hypertension patients.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a debilitating disease characterized by thrombotic occlusion of pulmonary arteries and vasculopathy, leading to increased pulmonary vascular resistance and progressive right-sided heart failure. Thrombotic lesions in CTEPH contain CD68+ macrophages, and increasing evidence supports their role in disease pathogenesis. Macrophages are classically divided into pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which are involved in wound healing and tissue repair. Currently, the phenotype of macrophages and their localization within thrombotic lesions of CTEPH are largely unknown. In our study, we subclassified thrombotic lesions of CTEPH patients into developing fresh thrombi (FT) and organized thrombi (OT), based on the degree of fibrosis and remodeling. We used multiplex immunofluorescence histology to identify immune cell infiltrates in thrombotic lesions of CPTEH patients. Utilizing software-assisted cell detection and quantification, increased proportions of macrophages were observed in immune cell infiltrates of OT lesions, compared with FT. Strikingly, the proportions with a CD206+INOS- M2 phenotype were significantly higher in OT than in FT, which mainly contained unpolarized macrophages. Taken together, we observed a shift from unpolarized macrophages in FT toward an expanded population of M2 macrophages in OT, indicating a dynamic role of macrophages during CTEPH pathogenesis.

Kynurenine metabolites predict survival in pulmonary arterial hypertension: A role for IL-6/IL-6Rα.

Activation of the kynurenine pathway (KP) has been reported in patients with pulmonary arterial hypertension (PAH) undergoing PAH therapy. We aimed to determine KP-metabolism in treatment-naïve PAH patients, investigate its prognostic values, evaluate the effect of PAH therapy on KP-metabolites and identify cytokines responsible for altered KP-metabolism. KP-metabolite levels were determined in plasma from PAH patients (median follow-up 42 months) and in rats with monocrotaline- and Sugen/hypoxia-induced PH. Blood sampling of PAH patients was performed at the time of diagnosis, six months and one year after PAH therapy. KP activation with lower tryptophan, higher kynurenine (Kyn), 3-hydroxykynurenine (3-HK), quinolinic acid (QA), kynurenic acid (KA), and anthranilic acid was observed in treatment-naïve PAH patients compared with controls. A similar KP-metabolite profile was observed in monocrotaline, but not Sugen/hypoxia-induced PAH. Human lung primary cells (microvascular endothelial cells, pulmonary artery smooth muscle cells, and fibroblasts) were exposed to different cytokines in vitro. Following exposure to interleukin-6 (IL-6)/IL-6 receptor α (IL-6Rα) complex, all cell types exhibit a similar KP-metabolite profile as observed in PAH patients. PAH therapy partially normalized this profile in survivors after one year. Increased KP-metabolites correlated with higher pulmonary vascular resistance, shorter six-minute walking distance, and worse functional class. High levels of Kyn, 3-HK, QA, and KA measured at the latest time-point were associated with worse long-term survival. KP-metabolism was activated in treatment-naïve PAH patients, likely mediated through IL-6/IL-6Rα signaling. KP-metabolites predict response to PAH therapy and survival of PAH patients.

Evidence for a Role of CCR6+ T Cells in Chronic Thromboembolic Pulmonary Hypertension.

Introduction: Previous studies have shown an increase of T cells and chemokines in vascular lesions of patients with chronic thromboembolic pulmonary hypertension (CTEPH). However, detailed characterization of these T cells is still lacking, nor have treatment effects been evaluated. Methods: We included 41 treatment-naive CTEPH patients at diagnosis, 22 patients at 1-year follow-up, and 17 healthy controls (HCs). Peripheral blood T cells were characterized by flow cytometry for subset distribution, cytokine expression and activation marker profile. We used multiplex immunofluorescence to identify CCR6+ T cells in endarterectomy tissue from 25 patients. Results: At diagnosis, proportions of CCR6+ CD4+ T cells were increased in CTEPH patients compared with HCs. Patients displayed a significantly reduced production capacity of several cytokines including TNFα, IFNγ, GM-CSF and IL-4 in CD4+ T cells, and TNFα and IFNγ in CD8+ T cells. CD4+ and CD8+ T cells showed increased expression of the immune checkpoint protein CTLA4. Multivariate analysis separated CTEPH patients from HCs, based on CCR6 and CTLA4 expression. At 1-year follow-up, proportions of CCR6+CD4+ T cells were further increased, IFNγ and IL-17 production capacity of CD4+ T cells was restored. In nearly all vascular lesions we found substantial numbers of CCR6+ T cells. Conclusion: The observed increase of CCR6+ T cells and modulation of the IFNγ and IL-17 production capacity of circulating CD4+ T cells at diagnosis and 1-year follow-up - together with the presence of CCR6+ T cells in vascular lesions - support the involvement of the Th17-associated CCR6+ T cell subset in CTEPH.

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

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 Tnfaip3DNGR1-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 Tnfaip3DNGR1-KO mice, particularly in the right ventricle (RV). Secondly, in young Tnfaip3DNGR1-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 Tnfaip3DNGR1-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.

DNGR1-Cre-mediated Deletion of Tnfaip3/A20 in Conventional Dendritic Cells Induces Pulmonary Hypertension in Mice.

Chronic perivascular inflammation is a prominent feature in the lungs of idiopathic pulmonary arterial hypertension. Although the proportions of conventional dendritic cells (cDCs) and plasmacytoid DCs are increased in idiopathic pulmonary arterial hypertension lungs, it remains unknown whether activated cDCs play a pathogenic role. The Tnfaip3 gene encodes the ubiquitin-binding protein A20, which is a negative regulator of NF-κB, critically involved in DC activation. Targeting of Tnfaip3/A20 in cDCs was achieved by Clec9a (DNGR1)-Cre-mediated excision of the Tnfaip3 gene in Tnfaip3DNGR1-KO mice. Mice were evaluated for signs of pulmonary hypertension (PH) using right heart catheterization, echocardiography, and measurement of the Fulton index. Inflammation was assessed by immunohistochemistry and flow cytometry. Pulmonary cDCs and monocyte-derived DCs from 31-week-old Tnfaip3DNGR1-KO mice showed modulated expression of cell surface activation markers compared with Tnfaip3DNGR1-WT mice. Tnfaip3DNGR1-KO mice developed elevated right ventricular systolic pressure and right ventricular hypertrophy. The lungs of these mice displayed increased vascular remodeling and perivascular and peribronchial immune cell infiltration resembling tertiary lymphoid organs. Proportions of activated T cells and expression of IL-1ß, IL-6, and IL-10 were enhanced in the lungs of Tnfaip3DNGR1-KO mice. Autoreactive IgA and IgG1 was detected in BAL and autoreactive IgA recognizing pulmonary endothelial antigens was present in the serum of Tnfaip3DNGR1-KO mice. All signs of PH were ameliorated in Tnfaip3DNGR1-KO mice by anti-IL-6 antibody treatment. These results indicate that activation of the NF-κB pathway in DCs, through deletion of A20/Tnfaip3, leads to experimental PH with accompanied pulmonary inflammation in an IL-6-dependent fashion.

DNGR1-mediated deletion of A20/Tnfaip3 in dendritic cells alters T and B-cell homeostasis and promotes autoimmune liver pathology.

Dendritic cells (DCs) are central regulators of tolerance versus immunity. The outcome depends amongst others on DC subset and activation status. Whereas CD11b+ type 2 conventional DCs (cDC2s) initiate proinflammatory helper T (Th)-cell responses, CD103+ cDC1s are crucial for regulatory T-cell (Treg) induction and CD8+ T-cell activation. DC activation is controlled by the transcription factor NF-κB. Ablation of A20/Tnfaip3, a critical regulator of NF-κB activation, in DCs leads to constitutive DC activation and development of systemic autoimmunity. We hypothesized that the activation status of cDCs controls the development of autoimmunity. To target cDCs, DNGR1(Clec9a)-cre-mediated excision of A20/Tnfaip3 was used through generation of Tnfaip3fl/flxClec9a+/cre (Tnfaip3DNGR1-KO) mice. Immune cell activation was evaluated at 31-weeks of age. We found that DNGR1-cre-mediated deletion of A20/Tnfaip3 resulted in liver pathology characterized by inflammatory infiltrates adjacent to the portal triads. Both cDC subsets as well as monocyte-derived DCs (moDCs) in Tnfaip3DNGR1-KO livers harbored an activated phenotype. Specifically, the costimulatory molecule CD40 in liver cDCs and moDCs was regulated by A20/Tnfaip3 expression. Livers from Tnfaip3DNGR1-KO mice had augmented proportions of Th1, Th17, Treg, and follicular Th (Tfh)-cells compared to control mice, accompanied by an increase in IgA-producing plasma cells. Serum IgA from Tnfaip3DNGR1-KO mice recognized self-proteins, specifically cytoplasmic proteins in liver periportal regions. These data show that enhanced activation of cDCs and moDCs, due to A20/Tnfaip3 ablation, promotes the development of organ-specific autoimmunity but not systemic autoimmunity. This model could be useful to examine the pathobiological processes contributing to autoimmune liver diseases.

The prognostic value of various biomarkers in adults with pulmonary hypertension; a multi-biomarker approach.

BACKGROUND: This study aimed to investigate the prognostic value of six different biomarkers in patients with pulmonary hypertension (PH) and to explore whether a multi-biomarker approach can contribute to a better risk stratification. METHODS: In this prospective study, patients with PH were included at the day of the diagnostic right heart catheterization between May 2012 and October 2016. Venous blood sampling included; NT-proBNP, high sensitive troponin-T, high sensitive CRP, galectin-3, red blood cell distribution width and eGFR. Associations between biomarker levels and the primary endpoint (death or lung transplantation) and secondary endpoint (death, lung transplantation or heart failure) were assessed with Cox regression, adjusted for age and sex. Additionally, adjustment for the REVEAL risk score was performed. RESULTS: In total, 106 patients were included (median age 58.7 [IQR 47.0-69.2] years, 64% women, 51% pulmonary arterial hypertension). After a median follow-up duration of 23.9 [IQR 15.1-40.0] months, respectively 29 and 37 patients reached the primary and secondary endpoint. All six biomarkers, except eGFR, were significantly associated with the endpoints. A multi-biomarker approach including the number of elevated biomarkers per patient, demonstrated that patients were at higher risk of adverse events as more biomarker levels were elevated (HR for each extra elevated biomarker; 1.33, 95% CI 1.07-1.64, P = .01). However, a single as well as a combination of multiple biomarkers, did not yield prognostic value independent of the REVEAL risk score. CONCLUSIONS: Various biomarkers are associated with the event-free survival in adults with PH. However, risk stratification exclusively based on single or a combination of biomarkers seems not superior to existing risk scores.