WNT3a and WNT5a Transported by Exosomes Activate WNT Signaling Pathways in Human Cardiac Fibroblasts.

WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20⁻150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3ß (GSK3ß), induced nuclear translocation of ß-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/ß-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate ß-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.

The AP-1 transcription factor Fosl-2 drives cardiac fibrosis and arrhythmias under immunofibrotic conditions.

Fibrotic changes in the myocardium and cardiac arrhythmias represent fatal complications in systemic sclerosis (SSc), however the underlying mechanisms remain elusive. Mice overexpressing transcription factor Fosl-2 (Fosl-2tg) represent animal model of SSc. Fosl-2tg mice showed interstitial cardiac fibrosis, disorganized connexin-43/40 in intercalated discs and deregulated expression of genes controlling conduction system, and developed higher heart rate (HR), prolonged QT intervals, arrhythmias with prevalence of premature ventricular contractions, ventricular tachycardias, II-degree atrio-ventricular blocks and reduced HR variability. Following stimulation with isoproterenol Fosl-2tg mice showed impaired HR response. In contrast to Fosl-2tg, immunodeficient Rag2-/-Fosl-2tg mice were protected from enhanced myocardial fibrosis and ECG abnormalities. Transcriptomics analysis demonstrated that Fosl-2-overexpression was responsible for profibrotic signature of cardiac fibroblasts, whereas inflammatory component in Fosl-2tg mice activated their fibrotic and arrhythmogenic phenotype. In human cardiac fibroblasts FOSL-2-overexpression enhanced myofibroblast signature under proinflammatory or profibrotic stimuli. These results demonstrate that under immunofibrotic conditions transcription factor Fosl-2 exaggerates myocardial fibrosis, arrhythmias and aberrant response to stress.

Regulation of Monocyte Adhesion and Type I Interferon Signaling by CD52 in Patients With Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is characterized by dysregulation of type I interferon (IFN) signaling. CD52 is known for its immunosuppressive functions in T cells. This study was undertaken to investigate the role of CD52 in monocyte adhesion and type I IFN signaling in patients with SSc. METHODS: Transcriptome profiles of circulating CD14+ monocytes from patients with limited cutaneous SSc (lcSSc), patients with diffuse cutaneous SSc (dcSSs), and healthy controls were analyzed by RNA sequencing. Levels of CD52, CD11b/integrin αΜ, and CD18/integrin ß2 in whole blood were assessed by flow cytometry. CD52 expression was analyzed in relation to disease phenotype (early, lcSSc, dcSSc) and autoantibody profiles. The impact of overexpression, knockdown, and antibody blocking of CD52 was analyzed by gene and protein expression assays and functional assays. RESULTS: Pathway enrichment analysis indicated an increase in adhesion- and type I IFN-related genes in monocytes from SSc patients. These cells displayed up-regulated expression of CD11b/CD18, reduced expression of CD52, and enhanced adhesion to intercellular adhesion molecule 1 and endothelial cells. Changes in CD52 expression were consistent with the SSc subtypes, as well as with immunosuppressive treatments, autoantibody profiles, and monocyte adhesion properties in patients with SSc. Overexpression of CD52 led to decreased levels of CD18 and monocyte adhesion, while knockdown of CD52 increased monocyte adhesion. Experiments with the humanized anti-CD52 monoclonal antibody alemtuzumab in blood samples from healthy controls increased monocyte adhesion and CD11b/CD18 expression, and enhanced type I IFN responses. Monocytic CD52 expression was up-regulated by interleukin-4 (IL-4)/IL-13 via the STAT6 pathway, and was down-regulated by lipopolysaccharide and IFNs α, ß, and γ in a JAK1 and histone deacetylase IIa (HDAC IIa)-dependent manner. CONCLUSION: Down-regulation of the antiadhesion CD52 antigen in CD14+ monocytes represents a novel mechanism in the pathogenesis of SSc. Targeting of the IFN-HDAC-CD52 axis in monocytes might represent a new therapeutic option for patients with early SSc.

Elevated Fibronectin Levels in Profibrotic CD14+ Monocytes and CD14+ Macrophages in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by overproduction of extracellular matrix (ECM) and multiorgan fibrosis. Animal studies pointed to bone marrow-derived cells as a potential source of pathological ECM-producing cells in immunofibrotic disorders. So far, involvement of monocytes and macrophages in the fibrogenesis of SSc remains poorly understood. Methods and Results: Immunohistochemistry analysis showed accumulation of CD14+ monocytes in the collagen-rich areas, as well as increased amount of alpha smooth muscle actin (αSMA)-positive fibroblasts, CD68+ and mannose-R+ macrophages in the heart and lungs of SSc patients. The full genome transcriptomics analyses of CD14+ blood monocytes revealed dysregulation in cytoskeleton rearrangement, ECM remodeling, including elevated FN1 (gene encoding fibronectin) expression and TGF-ß signalling pathway in SSc patients. In addition, single cell RNA sequencing analysis of tissue-resident CD14+ pulmonary macrophages demonstrated activated profibrotic signature with the elevated FN1 expression in SSc patients with interstitial lung disease. Peripheral blood CD14+ monocytes obtained from either healthy subjects or SSc patients exposed to profibrotic treatment with profibrotic cytokines TGF-ß, IL-4, IL-10, and IL-13 increased production of type I collagen, fibronectin, and αSMA. In addition, CD14+ monocytes co-cultured with dermal fibroblasts obtained from SSc patients or healthy individuals acquired a spindle shape and further enhanced production of profibrotic markers. Pharmacological blockade of the TGF-ß signalling pathway with SD208 (TGF-ß receptor type I inhibitor), SIS3 (Smad3 inhibitor) or (5Z)-7-oxozeaenol (TGF-ß-activated kinase 1 inhibitor) ameliorated fibronectin levels and type I collagen secretion. Conclusions: Our findings identified activated profibrotic signature with elevated production of profibrotic fibronectin in CD14+ monocytes and CD14+ pulmonary macrophages in SSc and highlighted the capability of CD14+ monocytes to acquire a profibrotic phenotype. Taking together, tissue-infiltrating CD14+ monocytes/macrophages can be considered as ECM producers in SSc pathogenesis.

Involvement of the myeloid cell compartment in fibrogenesis and systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune fibrotic disease of unknown aetiology that is characterized by vascular changes in the skin and visceral organs. Autologous haematopoietic stem cell transplantation can improve skin and organ fibrosis in patients with progressive disease and a high risk of organ failure, indicating that cells originating in the bone marrow are important contributors to the pathogenesis of SSc. Animal studies also indicate a pivotal function of myeloid cells in the development of fibrosis leading to changes in the tissue architecture and dysfunction in multiple organs such as the heart, lungs, liver and kidney. In this Review, we summarize current knowledge about the function of myeloid cells in fibrogenesis that occurs in patients with SSc. Targeted therapies currently in clinical studies for SSc might affect myeloid cell-related pathways. Therefore, myeloid cells might be used as cellular biomarkers of disease through the application of high-dimensional techniques such as mass cytometry and single-cell RNA sequencing.

Love, madness and social order: love melancholy in France and England in the late sixteenth and early seventeenth centuries.

The concept of "illness's social course" can be approached from two stand-points. We can trace both the way the social world shapes the course of an illness and the way an illness' symptoms shape the social world. The purpose of this study is to locate the specific illness of love melancholy in a specific historical and social context, namely that of France and England in the late sixteenth and early seventeenth centuries, in order to explain the intense discussion on the disorder during that period. This attempt is done with respect to the two dimensions of the concept of "illness' social course" and in the light of constructivist commentary on psychological disorders, which regards them as local stress idioms shaped by a specific social and cultural context.