Altered lipid homeostasis and autophagy precipitate diffuse alveolar hemorrhage in murine lupus

Abstract

Abnormal autophagy regulation is implicated in lupus and other autoimmune diseases. We investigated autophagy in the murine pristane-induced lupus model. Pristane causes monocyte/macrophage-mediated endoplasmic reticulum (ER) stress in lung endothelial cells and diffuse alveolar hemorrhage (DAH) indistinguishable from DAH in lupus patients. Enlarged macrophages with abundant lipid droplets containing neutral lipid and exhibiting increased autophagosome staining were observed in the lung and peritoneal macrophages after pristane treatment. Cellular overload of neutral lipid can lead to selective autophagy (lipophagy) of lipid droplets and transport to lysosomes. The autophagy inducer rapamycin decreased neutral lipid staining but aggravated DAH, while an autophagy inhibitor (3-methyladenine) blocked the onset of DAH. Pristane-induced autophagy in macrophages was confirmed by acridine orange assay and LC3 western blot. Pristane also enlarged lysosomal volume and enhanced cathepsin S, D, and K expression while decreasing lysosomal acid lipase activity. If the capacity to degrade neutral lipid into free cholesterol and fatty acids is overwhelmed, lysosomes enlarge and can release cathepsins into the cytoplasm promoting cell death. Increasing lysosomal cholesterol content by blocking the Niemann-Pick C disease protein NPC1 protects against lysosome-dependent cell death. Treatment with NPC1 inhibitors U18666A or cepharanthine, which stabilize lysosomes, normalized lysosomal volume, reversed ER stress, and prevented DAH in pristane-treated mice. We conclude that pristane disrupts lipid homeostasis, promoting autophagy, lysosomal dysfunction, ER stress, and cell death leading to DAH. NPC1 inhibition reverses these abnormalities, preventing DAH. The findings shed light on the role of autophagy and lysosomal dysfunction in the pathogenesis of lupusAbbreviations 25HC, 25-hydroxycholesterol; 3MA, 3-methyladenine; ACAT, acyl-coenzyme A: cholesterol acetyltransferase; AO, acridine orange; B6, C57BL/6 mice; BODIPY, 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; Ch25h, cholesterol 25-hydroxylase; DAH, diffuse alveolar hemorrhage; ER, endoplasmic reticulum; IFN, interferon; LAL, lysosomal acid lipase; LAMP-1, lysosomal-associated membrane protein 1; LDCD, lysosome-dependent cell death; LDL, low density lipoprotein; LMP, lysosomal membrane permeabilization; LPS, lipopolysaccharide; LXR, liver X receptor; Mφ, macrophage; MFI, mean fluorescence intensity; MO, mineral oil; mTORC, mechanistic target of rapamycin complex 1; NPC1, Niemann-Pick C disease protein 1; PI3K, phosphoinositide 3-kinase; PECs, peritoneal exudate cells; qPCR, quantitative real-time PCR; SLE, systemic lupus erythematosus; TFEB, transcription factor EB; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; UPR, unfolded protein response; PECs, peritoneal exudate cells.

Keywords

• Cholesterol
• Endoplasmic reticulum stress
• Lipid droplets
• Lysosomes
• Macrophages
• Lung