Rationale: Idiopathic pulmonary fibrosis (IPF) involves the accumulation of α-smooth muscle actin–expressing myofibroblasts arising from interactions with soluble mediators such as transforming growth factor-β1 (TGF-β1) and mechanical influences such as local tissue stiffness. Whereas IPF fibroblasts are enriched for aerobic glycolysis and innate immune receptor activation, innate immune ligands related to mitochondrial injury, such as extracellular mitochondrial DNA (mtDNA), have not been identified in IPF. Objectives: We aimed to define an association between mtDNA and fibroblast responses in IPF. Methods: We evaluated the response of normal human lung fibroblasts (NHLFs) to stimulation with mtDNA and determined whether the glycolytic reprogramming that occurs in response to TGF-β1 stimulation and direct contact with stiff substrates, and spontaneously in IPF fibroblasts, is associated with excessive levels of mtDNA. We measured mtDNA concentrations in bronchoalveolar lavage (BAL) from subjects with and without IPF, as well as in plasma samples from two longitudinal IPF cohorts and demographically matched control subjects. Measurements and Main Results: Exposure to mtDNA augments α-smooth muscle actin expression in NHLFs. The metabolic changes in NHLFs that are induced by interactions with TGF-β1 or stiff hydrogels are accompanied by the accumulation of extracellular mtDNA. These findings replicate the spontaneous phenotype of IPF fibroblasts. mtDNA concentrations are increased in IPF BAL and plasma, and in the latter compartment, they display robust associations with disease progression and reduced event-free survival. Conclusions: These findings demonstrate a previously unrecognized and highly novel connection between metabolic reprogramming, mtDNA, fibroblast activation, and clinical outcomes that provides new insight into IPF.

Background and objective: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis and variable clinical course. Although matrix metalloproteinase‐7 (MMP‐7) is emerging as an important IPF biomarker, reproducibility across studies is unclear. We aimed to determine whether a previously reported prognostic threshold for MMP‐7 was predictive of mortality in an independent cohort of IPF patients.Methods: MMP‐7 concentrations obtained from heparinized plasma samples were determined by ELISA in 97 patients with IPF and 41 healthy controls. The association of the previously published heparin plasma MMP‐7 threshold of 12.1 ng/mL with all‐cause mortality or transplant‐free survival (TFS) was determined, either as an independent biomarker or as part of the modified personal clinical and molecular mortality index (m‐PCMI).Results: MMP‐7 plasma concentrations were significantly higher in IPF patients compared to healthy controls (14.40 ± 6.55 ng/mL vs 6.03 ± 2.51 ng/mL, P < 0.001). The plasma MMP‐7 threshold of 12.1 ng/mL was significantly associated with both all‐cause mortality and TFS (unadjusted Cox proportional hazard ratio (HR) = 25.85 and 15.49, 95% CI: 10.91–61.23 and 5.41–44.34, respectively, P < 0.001). MMP‐7 concentrations, split by 12.1 ng/mL, were significantly (P < 0.05) predictive of mortality and TFS after adjusting for age, gender, smoking and baseline pulmonary function parameters, in a multivariate Cox proportional hazards model. MMP‐7 concentrations were negatively correlated with diffusing lung capacity of carbon monoxide (DL_CO) (r = −0.21, P = 0.02), and positively with a mortality risk scoring system (GAP) that combines age, gender, forced vital capacity (FVC) and DL_CO (r = 0.32, P = 0.001).Conclusion: This study confirms that MMP‐7 concentrations could be used to accurately predict outcomes across cohorts and centres, when similar collection protocols are applied.

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5′ single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung–blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

The chronic progressive decline in lung function observed in idiopathic pulmonary fibrosis (IPF) appears to result from persistent nonresolving injury to the epithelium, impaired restitution of the epithelial barrier in the lung, and enhanced fibroblast activation. Thus, understanding these key mechanisms and pathways modulating both is essential to greater understanding of IPF pathogenesis. We examined the association of VEGF with the IPF disease state and preclinical models in vivo and in vitro. Tissue and circulating levels of VEGF were significantly reduced in patients with IPF, particularly in those with a rapidly progressive phenotype, compared with healthy controls. Lung-specific overexpression of VEGF significantly protected mice following intratracheal bleomycin challenge, with a decrease in fibrosis and bleomycin-induced cell death observed in the VEGF transgenic mice. In vitro, apoptotic endothelial cell–derived mediators enhanced epithelial cell injury and reduced epithelial wound closure. This process was rescued by VEGF pretreatment of the endothelial cells via a mechanism involving thrombospondin-1 (TSP1). Taken together, these data indicate beneficial roles for VEGF during lung fibrosis via modulating epithelial homeostasis through a previously unrecognized mechanism involving the endothelium.

Fibrosis is a macrophage-driven process of uncontrolled extracellular matrix accumulation. Neuronal guidance proteins such as netrin-1 promote inflammatory scarring. We found that macrophage-derived netrin-1 stimulates fibrosis through its neuronal guidance functions. In mice, fibrosis due to inhaled bleomycin engendered netrin-1–expressing macrophages and fibroblasts, remodeled adrenergic nerves, and augmented noradrenaline. Cell-specific knockout mice showed that collagen accumulation, fibrotic histology, and nerve-associated endpoints required netrin-1 of macrophage but not fibroblast origin. Adrenergic denervation; haploinsufficiency of netrin-1’s receptor, deleted in colorectal carcinoma; and therapeutic α₁ adrenoreceptor antagonism improved collagen content and histology. An idiopathic pulmonary fibrosis (IPF) lung microarray data set showed increased netrin-1 expression. IPF lung tissues were enriched for netrin-1⁺ macrophages and noradrenaline. A longitudinal IPF cohort showed improved survival in patients prescribed α₁ adrenoreceptor blockade. This work showed that macrophages stimulate lung fibrosis via netrin-1–driven adrenergic processes and introduced α₁ blockers as a potentially new fibrotic therapy.

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of sterol regulatory element binding protein ( SREBP ) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immunometabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in bronchoalveolar lavage (BAL) cells isolated from patients with IPF compared with healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti–miR-33 peptide nucleic acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. These studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a potentially novel therapeutic approach to treat this disease.