No abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease, but the mechanisms driving progression remain incompletely defined. We previously reported that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs), which serve as a cell of origin for IPF fibroblasts. Proliferating IPF MPCs are located at the periphery of fibroblastic foci in an active cellular front at the interface between the myofibroblast-rich focus core and adjacent normal alveolar structures. Among a large set of genes that distinguish IPF MPCs from their control counterparts, we identified IL-8 as a candidate mediator of IPF MPC fibrogenicity and driver of fibrotic progression. IPF MPCs and their progeny displayed increased steady-state levels of IL-8 and its cognate receptor CXCR1 and secreted more IL-8 than did controls. IL-8 functioned in an autocrine manner promoting IPF MPC self-renewal and the proliferation and motility of IPF MPC progeny. Secreted IL-8 also functioned in a paracrine manner stimulating macrophage migration. Analysis of IPF lung tissue demonstrated codistribution of IPF MPCs with activated macrophages in the active cellular front of the fibroblastic focus. These findings indicate that IPF MPC-derived IL-8 is capable of expanding the mesenchymal cell population and recruiting activated macrophages cells to actively evolving fibrotic lesions.

Rationale The phenotypes of vascular smooth-muscle cells (vSMCs) comprise a continuum bounded by predominantly contractile and synthetic cells. Some evidence suggests that contractile vSMCs can assume a more synthetic phenotype in response to ischemic injury, but the mechanisms that activate this phenotypic switch are poorly understood. Objective To determine whether lactate, which increases in response to regional ischemia, may promote the synthetic phenotype in vSMCs. Methods and Results Experiments were performed with vSMCs that had been differentiated from human induced pluripotent stem cells and then cultured in glucose-free, lactate-enriched (L+) medium or in standard (L−) medium. Compared to the L− medium, the L+ medium was associated with significant increases in synthetic vSMC marker expression, proliferation, and migration, and with significant declines in contractile and apoptotic activity. Furthermore, these changes were accompanied by increases in the expression of monocarboxylic acid transporters (MCT) and were generally attenuated both by the blockade of MCT activity and by transfection with iRNA for N-myc downstream regulated gene (NDRG). Proteomics, biomarker, and pathway analyses suggested that the L+ medium tended to upregulate the expression of synthetic vSMC markers, the production of extracellular proteins that participate in tissue construction or repair, and the activity of pathways that regulate cell proliferation and migration. Observations in hypoxia-cultured vSMCs were similar to those in L+ cultured vSMCs, and assessments in a swine myocardial infarction model suggested that measurements of lactate levels, lactate dehydrogenase levels, vSMC proliferation, and MCT and NDRG expression were greater in the ischemic zone than in nonischemic tissues. Conclusions These results demonstrate for the first time that vSMCs assume a more synthetic phenotype in a microenvironment that is rich in lactate. Thus, mechanisms that link glucose metabolism to vSMC phenotypic switching could play a role in the pathogenesis and treatment of cardiovascular disease.

Our data identify suppression of fibroblast Dicer1 expression in the myofibroblast-rich IPF fibroblastic focus core as a central step in the mechanism by which the ECM sustains fibrosis progression in IPF.

No abstract

Idiopathic pulmonary fibrosis is a progressive fibrotic lung disease. We previously identified fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that serve as drivers of progressive fibrosis. Recent single cell RNA sequencing work revealed that IPF MPCs with the highest transcriptomic network entropy differ the most from control MPCs; and that increased CD44 was a marker of these IPF MPCs. We hypothesize that IPF MPCs with high CD44 (CD44 hi ) expression will display enhanced fibrogenicity. We demonstrate that CD44 expressing MPCs are present at the periphery of the IPF fibroblastic focus, placing them in regions of active fibrogenesis. In a humanized mouse xenograft model, CD44 hi IPF MPCs are more fibrogenic than CD44 lo IPF MPCs and knock-down of CD44 diminishes their fibrogenicity. CD44 hi IPF MPCs display increased expression of pluripotency markers and enhanced self-renewal compared to CD44 lo IPF MPCs; properties potentiated by IL-8. The mechanism involves the accumulation of CD44 within the nucleus where it associates with the chromatin modulator protein BRG1 and the Zeb1 transcription factor. This CD44/BRG1/Zeb1 nuclear protein complex targets the Sox2 gene promoting its upregulation and self-renewal. Our data implicate CD44 interaction with the epigenetic modulator protein BRG1 in conveying IPF MPCs with cell-autonomous fibrogenicity.

In Idiopathic Pulmonary Fibrosis (IPF), there is unrelenting scarring of the lung mediated by pathological mesenchymal progenitor cells (MPCs) that manifest autonomous fibrogenicity in xenograft models. To determine where along their differentiation trajectory IPF MPCs acquire fibrogenic properties, we analyzed the transcriptome of 335 MPCs isolated from the lungs of 3 control and 3 IPF patients at the single-cell level. Using transcriptional entropy as a metric for differentiated state, we found that the least differentiated IPF MPCs displayed the largest differences in their transcriptional profile compared to control MPCs. To validate entropy as a surrogate for differentiated state functionally, we identified increased CD44 as a characteristic of the most entropic IPF MPCs. Using FACS to stratify IPF MPCs based on CD44 expression, we determined that CD44hi IPF MPCs manifested an increased capacity for anchorage-independent colony formation compared to CD44lo IPF MPCs. To validate our analysis morphologically, we used two differentially expressed genes distinguishing IPF MPCs from control (CD44, cell surface; and MARCKS, intracellular). In IPF lung tissue, pathological MPCs resided in the highly cellular perimeter region of the fibroblastic focus. Our data support the concept that IPF fibroblasts acquire a cell-autonomous pathological phenotype early in their differentiation trajectory.

The arginine decarboxylase pathway, which converts arginine to agmatine, is present in both humans and most bacterial pathogens. In humans agmatine is a neurotransmitter with affinities towards α2-adrenoreceptors, serotonin receptors, and may inhibit nitric oxide synthase. In bacteria agmatine serves as a precursor to polyamine synthesis and was recently shown to enhance biofilm development in some strains of the respiratory pathogen Pseudomonas aeruginosa. We determined agmatine is at the center of a competing metabolism in the human lung during airways infections and is influenced by the metabolic phenotypes of the infecting pathogens. Ultra performance liquid chromatography with mass spectrometry detection was used to measure agmatine in human sputum samples from patients with cystic fibrosis, spent supernatant from clinical sputum isolates, and from bronchoalvelolar lavage fluid from mice infected with P. aeruginosa agmatine mutants. Agmatine in human sputum peaks during illness, decreased with treatment and is positively correlated with inflammatory cytokines. Analysis of the agmatine metabolic phenotype in clinical sputum isolates revealed most deplete agmatine when grown in its presence; however a minority appeared to generate large amounts of agmatine presumably driving sputum agmatine to high levels. Agmatine exposure to inflammatory cells and in mice demonstrated its role as a direct immune activator with effects on TNF-α production, likely through NF-κB activation. P. aeruginosa mutants for agmatine detection and metabolism were constructed and show the real-time evolution of host-derived agmatine in the airways during acute lung infection. These experiments also demonstrated pathogen agmatine production can upregulate the inflammatory response. As some clinical isolates have adapted to hypersecrete agmatine, these combined data would suggest agmatine is a novel target for immune modulation in the host-pathogen dynamic.