Aberrant lipid metabolism in macrophages is associated with granuloma formation in sarcoidosis
Rationale: Chronic sarcoidosis is a complex granulomatous disease with limited treatment options that can progress over time. Understanding the molecular pathways contributing to disease would aid in new therapeutic development.  Objectives: To understand if macrophages from non-resolving chronic sarcoidosis patients are predisposed to macrophage aggregation and granuloma formation, and if modulation of the underlying molecular pathways influence sarcoidosis granuloma formation.  Methods: Macrophages were cultivated in vitro from isolated peripheral blood CD14+ monocytes and evaluated for spontaneous aggregation. Transcriptomics analyses, phenotypic and drug inhibitory experiments were performed on these monocyte-derived macrophages. Human skin biopsies from sarcoidosis patients and a myeloid Tsc2-specific sarcoidosis mouse model were analyzed for validatory experiments. Measurements and Main Results: Monocyte-derived macrophages from chronic sarcoidosis patients spontaneously formed extensive granulomas in vitro compared to healthy controls. Transcriptomic analyses separated healthy and sarcoidosis macrophages and identified an enrichment in lipid metabolic processes. In vitro patient granulomas, sarcoidosis mouse model granulomas, and those directly analyzed from lesional patient skin expressed an aberrant lipid metabolism profile and contained increased neutral lipids. Conversely, a combination of statins and cholesterol-reducing agents reduced granuloma formation both in vitro and in vivo in a sarcoidosis mouse model.  Conclusions: Together, our findings show that altered lipid metabolism in sarcoidosis macrophages is associated with its predisposition to granuloma formation and suggest cholesterol-reducing therapies as a treatment option in patients. Overall design: CD14-isolated monocytes from 5 chronic sarcoidosis patients and 4 healthy controls were differentiated with 500 IU/mL rhGM-CSF for 3 days. Up to 1.2 x106 CD14+ monocytes or monocyte-derived macrophages from patients and healthy controls were carefully washed, harvested and lysed in 700 ul Qiazol (Qiagen) and stored at -80°C until RNA isolation. CD14+ cells were lysed on the day of sample collection, whereas macrophages were harvested after 3 days of differentiation with GM-CSF. RNA was isolated from the aforementioned samples using the miRNeasy Kit (Qiagen) and concentration and purity assessed using a Nanodrop 2000c Spectrophotometer (Thermo Scientific). RNA quality of the samples was further assessed on the Agilent Bioanalyzer RNA 6000 Nano Electropherogram, and library preparation performed on samples with RIN quality between 8-10. 100ng of total RNA was used for library preparation using the NEBNext Ultra II directional RNA library preparation kit with poly-A tail enrichment. Thereafter, stranded mRNA 75 bp single read RNA sequencing was performed on the Illumina NextSeq 500 sequencer.