Complement-containing small extracellular vesicles from adventitial fibroblasts induce proinflammatory and metabolic reprogramming in macrophages

Abstract

Pulmonary hypertension (PH) is a severe cardiopulmonary disease characterized by complement-dependent, fibroblast-induced perivascular accumulation and proinflammatory activation of macrophages. We hypothesized that, in PH, nanoscale-sized small extracellular vesicles (sEVs), released by perivascular/adventitial fibroblasts, are critical mediators of complement-dependent pro-inflammatory activation of macrophages. Pulmonary adventitial fibroblasts were isolated from calves with severe PH (PH-Fibs) and age-matched controls (CO-Fibs). PH-Fibs exhibited increased secretion of sEVs, compared to CO-Fibs, and sEV biological activity was tested on mouse and bovine bone marrow-derived macrophages (BMDMs) and showed similar responses. PH-Fib-sEVs induced augmented expression of pro-inflammatory cytokines/chemokines and metabolic genes in BMDMs, compared to CO-Fib-sEVs.Pharmacological blockade of exosome release from PH-Fibs resulted in significant attenuation of pro-inflammatory activation of BMDMs. "Bottom-up" proteomic analyses revealed significant enrichment of complement and Coagulation cascades in PH-Fib-sEVs, including augmented expression of complement component C3. We therefore examined whether PH-Fib-sEVs-mediated pro-inflammatory activation of BMDMs was complement C3-dependent. Treatment of PH-Fibs with siC3-RNA significantly attenuated the capacity of PH-Fib-sEVs for pro-inflammatory activation of BMDMs. PH-Fib-sEVs mediated pro-glycolytic alterations and complement-dependent activation of macrophages toward a pro-inflammatory phenotype, as confirmed by metabolomic studies. Thus, fibroblast-released sEVs can serve as critical mediators of complementinduced perivascular/microenvironmental inflammation in PH.

Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury

“…Future work will need to address whether the protective effects of lungderived C3 in severe bacterial pneumonia is due to the biosynthesized C3 that is stored in cells, or is secreted by neighboring lung epithelial cells and is internalized (16,21). Second, there are sources of C3 in the lungs other than lung epithelial cells, such as monocyte/macrophages, fibroblasts and mesenchymal stem cells (15,(49)(50)(51). Future studies should determine if delivering C3 to the lung via these other sources is sufficient to mitigating lung injury in a paracrine manner.…”

Coagulation potential and the integrated omics of extracellular vesicles from COVID-19 positive patient plasma

“…All extracts were analyzed twice (20 µL injection each) by ultra-high-performance liquid chromatography using a UHPLC Vanquish coupled with a Q Exactive mass spectrometer (both from ThermoFisher Scientific Inc., San Jose, CA, USA) using both negative and positive polarity modes. For each method, the UHPLC utilized an Acquity HSS column at a flow rate increasing from 0.3 to 0.4 mL/min or 0.325 to 0.4 mL/min for 17 or 15 min for lipidomics, or at a flow rate of 450 µL/min on a Kinetex C18 column (150 × 2.1 mm, 1.7 μm, Phenomenex, Torrance, CA, USA) using 5-min gradients in positive and negative ion polarity modes for metabolomics, respectively, as described previously 42,43 .…”

Retention of functional mitochondria in mature red blood cells from patients with sickle cell disease

“…Repeated transition from low to high oxygen saturation in peripheral tissues and lung capillaries can trigger reverse electron flow in the electron transport chain 22,23 and produce a phenotype similar to reperfusion injury, comparable to that in the re-oxygenation response to ischaemic 22,23 or haemorrhagic hypoxia. 24,25 Carboxylic acids generated by this mechanism can also trigger immune-metabolic cascades, 26 thereby promoting neutrophil infiltration in the lung and triggering acute lung injury, 27 or, in the chronic setting, producing pulmonary vascular remodelling, macrophage activation 28,29 in the pulmonary adventitia, and, ultimately, pulmonary hypertension; 30,31 the latter occurs with an incidence of 6%-10% in SCD based on pulmonary artery catheterization. 32 Herein, we examined mitochondria retention in mature RBCs from patients with SCD and evaluated the functional capabilities of these mitochondria.…”