Efforts to decipher chronic lung disease and to reconstitute functional lung tissue through regenerative medicine have been hampered by an incomplete understanding of cell-cell interactions governing ...tissue homeostasis. Because the structure of mammalian lungs is highly conserved at the histologic level, we hypothesized that there are evolutionarily conserved homeostatic mechanisms that keep the fine architecture of the lung in balance. We have leveraged single-cell RNA sequencing techniques to identify conserved patterns of cell-cell cross-talk in adult mammalian lungs, analyzing mouse, rat, pig, and human pulmonary tissues. Specific stereotyped functional roles for each cell type in the distal lung are observed, with alveolar type I cells having a major role in the regulation of tissue homeostasis. This paper provides a systems-level portrait of signaling between alveolar cell populations. These methods may be applicable to other organs, providing a roadmap for identifying key pathways governing pathophysiology and informing regenerative efforts.
Precision medicine is defined by the National Institute of Health's Precision Medicine Initiative Working Group as an approach to disease treatment that takes into account individual variability in ...genes, environment, and lifestyle. There has been increased interest in applying the concept of precision medicine to idiopathic pulmonary fibrosis, in particular to search for genetic and molecular biomarker-based profiles (so called endotypes) that identify mechanistically distinct disease subgroups. The relevance of precision medicine to idiopathic pulmonary fibrosis is yet to be established, but we believe that it holds great promise to provide targeted and highly effective therapies to patients. In this manuscript, we describe the field's nascent efforts in genetic/molecular endotype identification and how environmental and behavioral subgroups may also be relevant to disease management.
Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B ...have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF.
First, we did a three-stage genome-wide association study (GWAS): stage one was a discovery GWAS; and stages two and three were independent case-control studies. DNA samples from European-American patients with IPF meeting standard criteria were obtained from several US centres for each stage. Data for European-American control individuals for stage one were gathered from the database of genotypes and phenotypes; additional control individuals were recruited at the University of Pittsburgh to increase the number. For controls in stages two and three, we gathered data for additional sex-matched European-American control individuals who had been recruited in another study. DNA samples from patients and from control individuals were genotyped to identify SNPs associated with IPF. SNPs identified in stage one were carried forward to stage two, and those that achieved genome-wide significance (p<5 × 10(-8)) in a meta-analysis were carried forward to stage three. Three case series with follow-up data were selected from stages one and two of the GWAS using samples with follow-up data. Mortality analyses were done in these case series to assess the SNPs associated with IPF that had achieved genome-wide significance in the meta-analysis of stages one and two. Finally, we obtained gene-expression profiling data for lungs of patients with IPF from the Lung Genomics Research Consortium and analysed correlation with SNP genotypes.
In stage one of the GWAS (542 patients with IPF, 542 control individuals matched one-by-one to cases by genetic ancestry estimates), we identified 20 loci. Six SNPs reached genome-wide significance in stage two (544 patients, 687 control individuals): three TOLLIP SNPs (rs111521887, rs5743894, rs5743890) and one MUC5B SNP (rs35705950) at 11p15.5; one MDGA2 SNP (rs7144383) at 14q21.3; and one SPPL2C SNP (rs17690703) at 17q21.31. Stage three (324 patients, 702 control individuals) confirmed the associations for all these SNPs, except for rs7144383. Linkage disequilibrium between the MUC5B SNP (rs35705950) and TOLLIP SNPs (rs111521887 r(2)=0·07, rs5743894 r(2)=0·16, and rs5743890 r(2)=0·01) was low. 683 patients from the GWAS were included in the mortality analysis. Individuals who developed IPF despite having the protective TOLLIP minor allele of rs5743890 carried an increased mortality risk (meta-analysis with fixed-effect model: hazard ratio 1·72 95% CI 1·24-2·38; p=0·0012). TOLLIP expression was decreased by 20% in individuals carrying the minor allele of rs5743890 (p=0·097), 40% in those with the minor allele of rs111521887 (p=3·0 × 10(-4)), and 50% in those with the minor allele of rs5743894 (p=2·93 × 10(-5)) compared with homozygous carriers of common alleles for these SNPs.
Novel variants in TOLLIP and SPPL2C are associated with IPF susceptibility. One novel variant of TOLLIP, rs5743890, is also associated with mortality. These associations and the reduced expression of TOLLIP in patients with IPF who carry TOLLIP SNPs emphasise the importance of this gene in the disease.
National Institutes of Health; National Heart, Lung, and Blood Institute; Pulmonary Fibrosis Foundation; Coalition for Pulmonary Fibrosis; and Instituto de Salud Carlos III.
We hypothesized B cells are involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a progressive, restrictive lung disease that is refractory to glucocorticoids and other nonspecific ...therapies, and almost invariably lethal. Accordingly, we sought to identify clinically associated B cell-related abnormalities in these patients. Phenotypes of circulating B cells were characterized by flow cytometry. Intrapulmonary processes were evaluated by immunohistochemistry. Plasma B lymphocyte stimulating factor (BLyS) was assayed by ELISA. Circulating B cells of IPF subjects were more Ag differentiated, with greater plasmablast proportions (3.1 ± 0.8%) than in normal controls (1.3 ± 0.3%) (p < 0.03), and the extent of this differentiation correlated with IPF patient lung volumes (r = 0.44, p < 0.03). CD20(+) B cell aggregates, diffuse parenchymal and perivascular immune complexes, and complement depositions were all prevalent in IPF lungs, but much less prominent or absent in normal lungs. Plasma concentrations of BLyS, an obligate factor for B cell survival and differentiation, were significantly greater (p < 0.0001) in 110 IPF (2.05 ± 0.05 ng/ml) than among 53 normal (1.40 ± 0.04 ng/ml) and 90 chronic obstructive pulmonary disease subjects (1.59 ± 0.05 ng/ml). BLyS levels were uniquely correlated among IPF patients with pulmonary artery pressures (r = 0.58, p < 0.0001). The 25% of IPF subjects with the greatest BLyS values also had diminished 1-y survival (46 ± 11%), compared with those with lesser BLyS concentrations (81 ± 5%) (hazard ratio = 4.0, 95% confidence interval = 1.8-8.7, p = 0.0002). Abnormalities of B cells and BLyS are common in IPF patients, and highly associated with disease manifestations and patient outcomes. These findings have implications regarding IPF pathogenesis and illuminate the potential for novel treatment regimens that specifically target B cells in patients with this lung disease.
Previously we described a reliable method based on immunodepletion for isolating mesenchymal stem cells (MSCs) from murine bone marrow and showed that, after intracranial transplantation, the cells ...migrated throughout forebrain and cerebellum and adopted neural cell fates. Here we systemically administered MSCs purified by immunodepletion from male bleomycin (BLM)-resistant BALB/c mice into female BLM-sensitive C57BL/6 recipients and quantified engraftment levels in lung by real-time PCR. Male DNA accounted for 2.21 × 10-5% of the total lung DNA in control-treated mice but was increased 23-fold (P = 0.05) in animals exposed to BLM before MSC transplantation. Fluorescence in situ hybridization revealed that engrafted male cells were localized to areas of BLM-induced injury and exhibited an epithelium-like morphology. Moreover, purification of type II epithelial cells from the lungs of transplant recipients resulted in a 3-fold enrichment of male, donor-derived cells as compared with whole lung tissue. MSC administration immediately after exposure to BLM also significantly reduced the degree of BLM-induced inflammation and collagen deposition within lung tissue. Collectively, these studies demonstrate that murine MSCs home to lung in response to injury, adopt an epithelium-like phenotype, and reduce inflammation and collagen deposition in lung tissue of mice challenged with BLM.
Many of the interstitial lung diseases represent a diagnostic and therapeutic challenge because their clinical and even histologic features are often nonspecific. Likewise, the transcriptional ...signatures of most of them are unknown.
To compare the gene expression patterns from patients with idiopathic pulmonary fibrosis (IPF) hypersensitivity pneumonitis (HP), and nonspecific interstitial pneumonia (NSIP) using custom oligonucleotide microarrays.
We profiled lung biopsies from 15 patients with IPF, 12 with HP, and eight with NSIP. Labeled complementary ribonucleic acid was hybridized to a custom Affymetrix oligonucleotide DNA microarray using standard Affymetrix protocols. The custom array, Hu03, contained 59,619 probe sets representing an estimated 46,000 gene clusters.
We identified statistically significant gene expression signatures that characterize HP and IPF. The HP gene expression signature was enriched for genes that are functionally associated with inflammation, T-cell activation, and immune responses, whereas the IPF signature was characterized by the expression of tissue remodeling, epithelial, and myofibroblast genes. We then compared these gene expression signatures to classify NSIP, a histologic pattern that is often difficult to differentiate consistently from HP and IPF. Two cases exhibited an IPF-like gene expression, another one could be more properly classified as HP, whereas others did not resemble HP or IPF, suggesting that they may represent idiopathic NSIP.
Our results underscore the value of gene expression signatures to classify the interstitial lung diseases and to understand pathogenic mechanisms, and suggest new ways to improve the diagnosis and treatment of patients with these diseases.
Most cell types in multicellular organisms can perform multiple functions. However, not all functions can be optimally performed simultaneously by the same cells. Functions incompatible at the level ...of individual cells can be performed at the cell population level, where cells divide labor and specialize in different functions. Division of labor can arise due to instruction by tissue environment or through self-organization. Here, we develop a computational framework to investigate the contribution of these mechanisms to division of labor within a cell-type population. By optimizing collective cellular task performance under trade-offs, we find that distinguishable expression patterns can emerge from cell-cell interactions versus instructive signals. We propose a method to construct ligand-receptor networks between specialist cells and use it to infer division-of-labor mechanisms from single-cell RNA sequencing (RNA-seq) and spatial transcriptomics data of stromal, epithelial, and immune cells. Our framework can be used to characterize the complexity of cell interactions within tissues.
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•Division of labor theory predicts distinct, mechanism-dependent spatial patterns•Spatial proximity of specialist cells suggests involvement of cell interactions•Archetype crosstalk networks reveal patterns of interactions between specialist cells•Enterocytes and fibroblasts exemplify distinct division of labor strategies
Two of the mechanisms that can drive division of labor in tissues are external signaling gradients and cell-cell interactions. Theory for optimal task allocation explains how to distinguish between the two strategies in single-cell and spatial omics data. This is demonstrated for colon fibroblasts, intestinal enterocytes, lung fibroblasts, and macrophages.
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a ...peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression.
We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins-MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A-that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval CI 92.7%-100%) and specificity of 98.1% (95% CI 89.9%-100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%-100%) and specificity of 87.2% (95% CI 72.6%-95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DLCO%).
Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.
Danger signals, or damage-associated molecular patterns (DAMPs), instigate mitochondrial innate immune responses wherein mitochondrial antiviral signaling protein (MAVS) functions as a key platform ...molecule to mediate them. The role of MAVS in the pathogenesis of idiopathic pulmonary fibrosis (IPF), however, has not yet been identified. Whether MAVS signalling can be modulated by currently existing drugs has also not been explored.We used an established model of pulmonary fibrosis to demonstrate that MAVS is a critical mediator of multiple DAMP signalling pathways and the consequent lung fibrosis after bleomycin-induced injury
After bleomycin injury, MAVS expression was mainly observed in macrophages. Multimeric MAVS aggregation, a key event of MAVS signalling activation, was significantly increased and persisted in bleomycin-injured lungs. A proapoptotic BH3 mimetic, ABT-263, attenuated the expression of MAVS and its signalling and, consequently, the development of experimental pulmonary fibrosis. In contrast, the therapeutic effects of nintedanib and pirfenidone, two drugs approved for IPF treatment, were not related to the modulation of MAVS or its signalling. Multimeric MAVS aggregation was significantly increased in lungs from IPF patients as well.MAVS may play an important role in the development of pulmonary fibrosis, and targeting MAVS with BH3 mimetics may provide a novel and much needed therapeutic strategy for IPF.
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