Aging disturbed lysosomes to modulate efferocytosis-induced SPP1-expressing macrophage and inhibit pulmonary fibrosis reversal

Background: Reversibility of pulmonary fibrosis decreased with aging. Macrophages (Mφ) play a key role in fibrosis regression. Published scRNA-seq results suggest Mφ expressing high level SPP1 (SPP1^hi-Mφ) accumulated in fibrotic lungs and contributed to fibrosis, indicating persistent existence of SPP1^hi-Mφ may prevent fibrosis regression. However, how aging modulates SPP1^hi-Mφ to inhibit pulmonary fibrosis reversal is unknown. Methods and Results: We isolated bone marrow derived Mφ (BMDM) from 6-week-old and 8-month-old mice (6w-BMDM and 8m-BMDM). And induce SPP1^hi-Mφ by efferocytosis. We found SPP1 was elevated at 6 hours. 12 hours later, it was decreased, coupled with declined TGFβ secretion and increased extracellular MMP13 in 6w-BMDM, suggesting pro-fibrotic SPP1^hi-Mφ switched to pro-fibrolytic SPP1^lo-Mφ. While SPP1 expression maintained at a high level in 8m-BMDM. Consistently, 3 weeks after bleomycin treatment, SPP1^hi-Mφ accumulated in lungs. 6 weeks later, its number decreased in lungs of 6-week-old mice, but not in 8-month-old mice. Furthermore, we found efferocytosis led to lysosomal iron overload. In 6w-BMDM, iron content returned to normal level spontaneously while remained in 8m-BMDM. Chelating iron promoted the transfer of SPP1^hi-Mφ to SPP1^lo-Mφ. In addition, lysosomal acidity decreased with aging. Bafilomycin, which disturbed lysosomal acidity, blocked the switch of SPP1^hi-Mφ to SPP1^lo-Mφ and lysosomal iron homeostasis recovery in 6w-BMDM. Conclusions: Aging inhibited lysosomal iron homeostasis recovery to prevent the switch of pro-fibrotic SPP1^hi-Mφ to pro-fibrolytic SPP1^lo-Mφ, which may contribute to decreased reversibility of lung fibrosis.