Tissue fibrosis is a major cause of mortality that results from the deposition of matrix proteins by an activated mesenchyme. Macrophages accumulate in fibrosis, but the role of specific subgroups in supporting fibrogenesis has not been investigated in vivo. Here we used single-cell RNA sequencing (scRNA-seq) to characterize the heterogeneity of macrophages in bleomycin-induced lung fibrosis in mice. A novel computational framework for the annotation of scRNA-seq by reference to bulk transcriptomes (SingleR) enabled the subclustering of macrophages and revealed a disease-associated subgroup with a transitional gene expression profile intermediate between monocyte-derived and alveolar macrophages. These CX3CR1
+
SiglecF
+
transitional macrophages localized to the fibrotic niche and had a profibrotic effect
in vivo
. Human orthologues of genes expressed by the transitional macrophages were upregulated in samples from patients with idiopathic pulmonary fibrosis. Thus, we have identified a pathological subgroup of transitional macrophages that are required for the fibrotic response to injury.
Pathologic features of idiopathic pulmonary fibrosis (IPF) include genetic predisposition, activation of the unfolded protein response, telomere attrition, and cellular senescence. The mechanisms leading to alveolar epithelial cell (AEC) senescence are poorly understood. MicroRNAs (miRNAs) have been reported as regulators of cellular senescence. Senescence markers including p16, p21, p53, and senescence-associated β-galactosidase (SA-βgal) activity were measured in type II AECs from IPF lungs and unused donor lungs. miRNAs were quantified in type II AECs using gene expression arrays and quantitative RT-PCR. Molecular markers of senescence (p16, p21, and p53) were elevated in IPF type II AECs. SA-βgal activity was detected in a greater percentage in type II AECs isolated from IPF patients (23.1%) compared to patients with other interstitial lung diseases (1.2%) or normal controls (0.8%). The relative levels of senescence-associated miRNAs miR-34a, miR-34b, and miR-34c, but not miR-20a, miR-29c, or miR-let-7f were significantly higher in type II AECs from IPF patients. Overexpression of miR-34a, miR-34b, or miR-34c in lung epithelial cells was associated with higher SA-βgal activity (27.8%, 35.1%, and 38.2%, respectively) relative to control treated cells (8.8%). Targets of miR-34 miRNAs, including E2F1, c-Myc, and cyclin E2, were lower in IPF type II AECs. These results show that markers of senescence are uniquely elevated in IPF type II AECs and suggest that the miR-34 family of miRNAs regulate senescence in IPF type II AECs.
SUMMARY
The environmentally widespread polysaccharide chitin is degraded and recycled by ubiquitous bacterial and fungal chitinases. Although vertebrates express active chitinases from evolutionarily conserved loci, their role in mammalian physiology is unclear. We show that distinct lung epithelial cells secrete acidic mammalian chitinase (AMCase), which is required for airway chitinase activity. AMCase-deficient mice exhibit premature morbidity and mortality, concomitant with accumulation of environmentally-derived chitin polymers in the airways and expression of pro-fibrotic cytokines. Over time, these mice develop spontaneous pulmonary fibrosis, which is ameliorated by restoration of lung chitinase activity by genetic or therapeutic approaches. AMCase-deficient epithelial cells express fibrosis-associated gene sets linked with cell stress pathways. Mice with lung fibrosis due to telomere dysfunction and humans with interstitial lung disease also accumulate excess chitin polymers in their airways. These data suggest that altered chitin clearance could exacerbate fibrogenic pathways in the setting of lung diseases characterized by epithelial cell dysfunction.
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