The histone chaperone NASP maintains H3-H4 reservoirs in the early Drosophila embryo.

Histones are essential for chromatin packaging, and histone supply must be tightly regulated as excess histones are toxic. To drive the rapid cell cycles of the early embryo, however, excess histones are maternally deposited. Therefore, soluble histones must be buffered by histone chaperones, but the chaperone necessary to stabilize soluble H3-H4 pools in the Drosophila embryo has yet to be identified. Here, we show that CG8223, the Drosophila homolog of NASP, is a H3-H4-specific chaperone in the early embryo. We demonstrate that, while a NASP null mutant is viable in Drosophila, NASP is a maternal effect gene. Embryos laid by NASP mutant mothers have a reduced rate of hatching and show defects in early embryogenesis. Critically, soluble H3-H4 pools are degraded in embryos laid by NASP mutant mothers. Our work identifies NASP as the critical H3-H4 histone chaperone in the Drosophila embryo.

BRWD3 promotes KDM5 degradation to maintain H3K4 methylation levels.

Histone modifications are critical for regulating chromatin structure and gene expression. Dysregulation of histone modifications likely contributes to disease states and cancer. Depletion of the chromatin-binding protein BRWD3 (Bromodomain and WD repeat-containing protein 3), a known substrate-specificity factor of the Cul4-DDB1 E3 ubiquitin ligase complex, results in increased H3K4me1 (H3 lysine 4 monomethylation) levels. The underlying mechanism linking BRWD3 and H3K4 methylation, however, has yet to be defined. Here, we show that depleting BRWD3 not only causes an increase in H3K4me1 levels but also causes a decrease in H3K4me3 (H3 lysine 4 trimethylation) levels, indicating that BRWD3 influences H3K4 methylation more broadly. Using immunoprecipitation coupled to quantitative mass spectrometry, we identified an interaction between BRWD3 and the H3K4-specific lysine demethylase 5 (KDM5/Lid), an enzyme that removes tri- and dimethyl marks from H3K4. Moreover, analysis of ChIP-seq (chromatin immunoprecipitation sequencing) data revealed that BRWD3 and KDM5 are significantly colocalized throughout the genome and H3K4me3 are highly enriched at BRWD3 binding sites. We show that BRWD3 promotes K48-linked polyubiquitination and degradation of KDM5 and that KDM5 degradation is dependent on both BRWD3 and Cul4. Critically, depleting KDM5 fully restores altered H3K4me3 levels and partially restores H3K4me1 levels upon BRWD3 depletion. Together, our results demonstrate that BRWD3 regulates KDM5 activity to balance H3K4 methylation levels.

Quantifying the proportion of different cell types in the human cortex using DNA methylation profiles.

BACKGROUND: Due to interindividual variation in the cellular composition of the human cortex, it is essential that covariates that capture these differences are included in epigenome-wide association studies using bulk tissue. As experimentally derived cell counts are often unavailable, computational solutions have been adopted to estimate the proportion of different cell types using DNA methylation data. Here, we validate and profile the use of an expanded reference DNA methylation dataset incorporating two neuronal and three glial cell subtypes for quantifying the cellular composition of the human cortex. RESULTS: We tested eight reference panels containing different combinations of neuronal- and glial cell types and characterised their performance in deconvoluting cell proportions from computationally reconstructed or empirically derived human cortex DNA methylation data. Our analyses demonstrate that while these novel brain deconvolution models produce accurate estimates of cellular proportions from profiles generated on postnatal human cortex samples, they are not appropriate for the use in prenatal cortex or cerebellum tissue samples. Applying our models to an extensive collection of empirical datasets, we show that glial cells are twice as abundant as neuronal cells in the human cortex and identify significant associations between increased Alzheimer's disease neuropathology and the proportion of specific cell types including a decrease in NeuNNeg/SOX10Neg nuclei and an increase of NeuNNeg/SOX10Pos nuclei. CONCLUSIONS: Our novel deconvolution models produce accurate estimates for cell proportions in the human cortex. These models are available as a resource to the community enabling the control of cellular heterogeneity in epigenetic studies of brain disorders performed on bulk cortex tissue.

Expression of SARS-CoV-2 Nonstructural Proteins 3 and 4 Can Tune the Unfolded Protein Response in Cell Culture.

Coronaviruses (CoV), including SARS-CoV-2, modulate host proteostasis through the activation of stress-responsive signaling pathways such as the Unfolded Protein Response (UPR), which remedies misfolded protein accumulation by attenuating translation and increasing protein folding capacity. While CoV nonstructural proteins (nsps) are essential for infection, little is known about the role of nsps in modulating the UPR. We characterized the impact of overexpression of SARS-CoV-2 nsp4, a key driver of replication, on the UPR in cell culture using quantitative proteomics to sensitively detect pathway-wide upregulation of effector proteins. We find that nsp4 preferentially activates the ATF6 and PERK branches of the UPR. Previously, we found that an N-terminal truncation of nsp3 (nsp3.1) can suppress pharmacological ATF6 activation. To determine how nsp3.1 and nsp4 tune the UPR, their coexpression demonstrated that nsp3.1 suppresses nsp4-mediated PERK, but not ATF6 activation. Reanalysis of SARS-CoV-2 infection proteomics data revealed time-dependent activation of PERK targets early in infection, which subsequently fades. This temporal regulation suggests a role for nsp3 and nsp4 in tuning the PERK pathway to attenuate host translation beneficial for viral replication while avoiding later apoptotic signaling caused by chronic activation. This work furthers our understanding of CoV-host proteostasis interactions and highlights the power of proteomic methods for systems-level analysis of the UPR.

Small-molecule endoplasmic reticulum proteostasis regulator acts as a broad-spectrum inhibitor of dengue and Zika virus infections.

Flaviviruses, including dengue and Zika, are widespread human pathogens; however, no broadly active therapeutics exist to fight infection. Recently, remodeling of endoplasmic reticulum (ER) proteostasis by pharmacologic regulators, such as compound 147, was shown to correct pathologic ER imbalances associated with protein misfolding diseases. Here, we establish an additional activity of compound 147 as an effective host-centered antiviral agent against flaviviruses. Compound 147 reduces infection by attenuating the infectivity of secreted virions without causing toxicity in host cells. Compound 147 is a preferential activator of the ATF6 pathway of the ER unfolded protein response, which requires targeting of cysteine residues primarily on protein disulfide isomerases (PDIs). We find that the antiviral activity of 147 is independent of ATF6 induction but does require modification of reactive thiols on protein targets. Targeting PDIs and additional non-PDI targets using RNAi and other small-molecule inhibitors was unable to recapitulate the antiviral effects, suggesting a unique polypharmacology may mediate the activity. Importantly, 147 can impair infection of multiple strains of dengue and Zika virus, indicating that it is suitable as a broad-spectrum antiviral agent.

Comparative Host Interactomes of the SARS-CoV-2 Nonstructural Protein 3 and Human Coronavirus Homologs.

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold-like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, nonstructural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) data set for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance, nuclear protein import for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

Characterization of Suboptimal Responses to Fetoscopic Endoluminal Tracheal Occlusion in Congenital Diaphragmatic Hernia.

INTRODUCTION: The aim of the study was to characterize the changes in fetal lung volume following fetoscopic endoluminal tracheal occlusion (FETO) that are associated with infant survival and need for extracorporeal membrane oxygenation (ECMO) in congenital diaphragmatic hernia (CDH). METHODS: Fetuses with CDH who underwent FETO at a single institution were included. CDH cases were reclassified by MRI metrics [observed-to-expected total lung volume (O/E TLV) and percent liver herniation]. The percent changes of MRI metrics after FETO were calculated. ROC-derived cutoffs of these changes were derived to predict infant survival to discharge. Regression analyses were done to determine the association between these cutoffs with infant survival and ECMO need, adjusted for site of CDH, gestational age at delivery, fetal sex, and CDH severity. RESULTS: Thirty CDH cases were included. ROC analysis demonstrated that post-FETO increases in O/E TLV had an area under the curve of 0.74 (p = 0.035) for the prediction of survival to hospital discharge; a cutoff of less than 10% was selected. Fetuses with a <10% post-FETO increase in O/E TLV had lower survival to hospital discharge [44.8% vs. 91.7%; p = 0.018] and higher ECMO use [61.1% vs. 16.7%; p = 0.026] compared to those with an O/E TLV increase ≥10%. Similar results were observed when the analyses were restricted to left-sided CDH cases. A post-FETO <10% increase in O/E TLV was independently associated with lower survival at hospital discharge (aOR: 0.073, 95% CI: 0.008-0.689; p = 0.022) and at 12 months of age (aOR: 0.091, 95% CI: 0.01-0.825; p = 0.036) as well as with higher ECMO use (aOR: 7.88, 95% CI: 1.31-47.04; p = 0.024). CONCLUSION: Fetuses with less than 10% increase in O/E TLV following the FETO procedure are at increased risk for requiring ECMO and for death in the postnatal period when adjusted for gestational age at delivery, CDH severity, and other confounders.

Structure and Function of the α-Hydroxylation Bimodule of the Mupirocin Polyketide Synthase.

Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA-A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly.

Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity.

The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and preemptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants because of their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared with TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile, and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.

Mindfulness-Based Analgesia or Placebo Effect? The Development and Evaluation of a Sham Mindfulness Intervention for Acute Experimental Pain.

OBJECTIVE: Meta-analyses indicate that mindfulness meditation is efficacious for chronic and acute pain, but most available studies lack active control comparisons. This raises the possibility that placebo-related processes may account, at least in part, for mindfulness effects. The objective of this study was to develop a closely matched sham mindfulness condition to establish whether placebo effects contribute to mindfulness-based interventions for pain. METHODS: We developed and validated a closely matched sham mindfulness intervention then compared it with 6 × 20-minute sessions of focused-attention mindfulness and a no-treatment condition in 93 healthy volunteers undergoing acute experimental heat pain. RESULTS: The sham mindfulness intervention produced equivalent credibility ratings and expectations of improvement as the mindfulness intervention but did not influence mindfulness-related processes. In contrast, mindfulness increased "observing" relative to no treatment but not sham. Mindfulness (F(1,88) = 7.06, p = .009, ηp2 = 0.07) and sham (F(1,88) = 6.47, p = .012, ηp2 = 0.07) moderately increased pain tolerance relative to no treatment, with no difference between mindfulness and sham (F(1,88) = 0.01, p = .92, ηp2 < 0.001). No differences were found for pain threshold. Similarly, neither mindfulness nor sham reduced pain intensity or unpleasantness relative to no treatment, although mindfulness reduced pain unpleasantness relative to sham (F(1,88) = 5.03, p = .027, ηp2 = 0.05). CONCLUSIONS: These results suggest that placebo effects contribute to changes in pain tolerance after mindfulness training, with limited evidence of specific effects of mindfulness training on pain unpleasantness relative to sham, but not no treatment. To disentangle the specific analgesic effects of mindfulness from placebo-related processes, future research should prioritize developing and incorporating closely matched sham conditions.Trial Registration: Australian New Zealand Clinical Trials Registry (ACTRN12618001175268).

COVID-19 Crisis Reduces Free Tropospheric Ozone Across the Northern Hemisphere.

Throughout spring and summer 2020, ozone stations in the northern extratropics recorded unusually low ozone in the free troposphere. From April to August, and from 1 to 8 kilometers altitude, ozone was on average 7% (≈4 nmol/mol) below the 2000-2020 climatological mean. Such low ozone, over several months, and at so many stations, has not been observed in any previous year since at least 2000. Atmospheric composition analyses from the Copernicus Atmosphere Monitoring Service and simulations from the NASA GMI model indicate that the large 2020 springtime ozone depletion in the Arctic stratosphere contributed less than one-quarter of the observed tropospheric anomaly. The observed anomaly is consistent with recent chemistry-climate model simulations, which assume emissions reductions similar to those caused by the COVID-19 crisis. COVID-19 related emissions reductions appear to be the major cause for the observed reduced free tropospheric ozone in 2020.

Recalibrating the epigenetic clock: implications for assessing biological age in the human cortex.

Human DNA methylation data have been used to develop biomarkers of ageing, referred to as 'epigenetic clocks', which have been widely used to identify differences between chronological age and biological age in health and disease including neurodegeneration, dementia and other brain phenotypes. Existing DNA methylation clocks have been shown to be highly accurate in blood but are less precise when used in older samples or in tissue types not included in training the model, including brain. We aimed to develop a novel epigenetic clock that performs optimally in human cortex tissue and has the potential to identify phenotypes associated with biological ageing in the brain. We generated an extensive dataset of human cortex DNA methylation data spanning the life course (n = 1397, ages = 1 to 108 years). This dataset was split into 'training' and 'testing' samples (training: n = 1047; testing: n = 350). DNA methylation age estimators were derived using a transformed version of chronological age on DNA methylation at specific sites using elastic net regression, a supervised machine learning method. The cortical clock was subsequently validated in a novel independent human cortex dataset (n = 1221, ages = 41 to 104 years) and tested for specificity in a large whole blood dataset (n = 1175, ages = 28 to 98 years). We identified a set of 347 DNA methylation sites that, in combination, optimally predict age in the human cortex. The sum of DNA methylation levels at these sites weighted by their regression coefficients provide the cortical DNA methylation clock age estimate. The novel clock dramatically outperformed previously reported clocks in additional cortical datasets. Our findings suggest that previous associations between predicted DNA methylation age and neurodegenerative phenotypes might represent false positives resulting from clocks not robustly calibrated to the tissue being tested and for phenotypes that become manifest in older ages. The age distribution and tissue type of samples included in training datasets need to be considered when building and applying epigenetic clock algorithms to human epidemiological or disease cohorts.

Mechanism of Ganglioside Receptor Recognition by Botulinum Neurotoxin Serotype E.

The botulinum neurotoxins are potent molecules that are not only responsible for the lethal paralytic disease botulism, but have also been harnessed for therapeutic uses in the treatment of an increasing number of chronic neurological and neuromuscular disorders, in addition to cosmetic applications. The toxins act at the cholinergic nerve terminals thanks to an efficient and specific mechanism of cell recognition which is based on a dual receptor system that involves gangliosides and protein receptors. Binding to surface-anchored gangliosides is the first essential step in this process. Here, we determined the X-ray crystal structure of the binding domain of BoNT/E, a toxin of clinical interest, in complex with its GD1a oligosaccharide receptor. Beyond confirmation of the conserved ganglioside binding site, we identified key interacting residues that are unique to BoNT/E and a significant rearrangement of loop 1228-1237 upon carbohydrate binding. These observations were also supported by thermodynamic measurements of the binding reaction and assessment of ganglioside selectivity by immobilised-receptor binding assays. These results provide a structural basis to understand the specificity of BoNT/E for complex gangliosides.

An Esterase-like Lyase Catalyzes Acetate Elimination in Spirotetronate/Spirotetramate Biosynthesis.

Spirotetronate and spirotetramate natural products include a multitude of compounds with potent antimicrobial and antitumor activities. Their biosynthesis incorporates many unusual biocatalytic steps, including regio- and stereo-specific modifications, cyclizations promoted by Diels-Alderases, and acetylation-elimination reactions. Here we focus on the acetate elimination catalyzed by AbyA5, implicated in the formation of the key Diels-Alder substrate to give the spirocyclic system of the antibiotic abyssomicin C. Using synthetic substrate analogues, it is shown that AbyA5 catalyzes stereospecific acetate elimination, establishing the (R)-tetronate acetate as a biosynthetic intermediate. The X-ray crystal structure of AbyA5, the first of an acetate-eliminating enzyme, reveals a deviant acetyl esterase fold. Molecular dynamics simulations and enzyme assays show the use of a His-Ser dyad to catalyze either elimination or hydrolysis, via disparate mechanisms, under substrate control.

High resolution crystal structures of Clostridium botulinum neurotoxin A3 and A4 binding domains.

Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening condition, botulism. However, while they have the potential to cause serious harm, they are increasingly being utilised for therapeutic applications. BoNTs comprise of seven distinct serotypes termed BoNT/A through BoNT/G, with the most widely characterised being sub-serotype BoNT/A1. Each BoNT consists of three structurally distinct domains, a binding domain (HC), a translocation domain (HN), and a proteolytic domain (LC). The HC domain is responsible for the highly specific targeting of the neurotoxin to neuronal cell membranes. Here, we present two high-resolution structures of the binding domain of subtype BoNT/A3 (HC/A3) and BoNT/A4 (HC/A4) at 1.6 Šand 1.34 Šresolution, respectively. The structures of both proteins share a high degree of similarity to other known BoNT HC domains whilst containing some subtle differences, and are of benefit to research into therapeutic neurotoxins with novel characteristics.

HIF1A up-regulates the ADORA2B receptor on alternatively activated macrophages and contributes to pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a deadly chronic lung disease. Extracellular accumulation of adenosine and subsequent activation of the ADORA2B receptor play important roles in regulating inflammation and fibrosis in IPF. Additionally, alternatively activated macrophages (AAMs) expressing ADORA2B have been implicated in mediating adenosine's effects in IPF. Although hypoxic conditions are present in IPF, hypoxia's role as a direct modulator of macrophage phenotype and identification of factors that regulate ADORA2B expression on AAMs in IPF is not well understood. In this study, an experimental mouse model of pulmonary fibrosis and lung samples from patients with IPF were used to examine the effects and interactions of macrophage differentiation and hypoxia on fibrosis. We demonstrate that hypoxia-inducible factor 1-α (HIF1A) inhibition in late stages of bleomycin-induced injury attenuates pulmonary fibrosis in association, with reductions in ADORA2B expression in AAMs. Additionally, ADORA2B deletion or pharmacological antagonism along with HIF1A inhibition disrupts AAM differentiation and subsequent IL-6 production in cultured macrophages. These findings suggest that hypoxia, through HIF1A, contributes to the development and progression of pulmonary fibrosis through its regulation of ADORA2B expression on AAMs, cell differentiation, and production of profibrotic mediators. These studies support a potential role for HIF1A or ADORA2B antagonists in the treatment of IPF.-Philip, K., Mills, T. W., Davies, J., Chen, N.-Y., Karmouty-Quintana, H., Luo, F., Molina, J. G., Amione-Guerra, J., Sinha, N., Guha, A., Eltzschig, H. K., Blackburn, M. R. HIF1A up-regulates the ADORA2B receptor on alternatively activated macrophages and contributes to pulmonary fibrosis.

Adverse life outcomes associated with adolescent psychotic experiences and depressive symptoms.

PURPOSE: To investigate whether psychotic experiences and depressive symptoms at ages 12 and 18 years are associated with adverse life outcomes across a range of functional domains between 16 and 20 years of age. METHODS: Data were gathered from ALSPAC, a UK birth cohort. Individuals were assessed with the semi-structured Psychosis-Like Symptoms Interview and the Short Mood and Feeling Questionnaire at ages 12 and 18 years. Logistic regression was used to explore associations with outcomes in education, occupation, social functioning, substance use (alcohol, cannabis, smoking, and other drugs), and illegal behaviour between the ages of 16 and 20 years. All associations were adjusted for socio-demographic and childhood confounders and for comorbid psychotic experiences or depressive symptoms. RESULTS: Psychotic experiences and depression at age 12 were associated with poorer educational, occupational, and social outcomes between the ages of 16 and 20; these withstood adjustment for confounding. Depressive symptoms at age 12 were also associated with harmful drinking. Psychotic experiences and depression at age 18 were additionally associated with other forms of substance use and illegal behaviour. Comorbidity had little impact at age 12, but was associated with significantly worse educational, social, and substance use outcomes at age 18. CONCLUSIONS: Adolescent psychotic experiences and depression represent a risk marker for a number of later adverse outcomes, most consistently with education and employment, but also social impairment, harmful drinking, and substance use. This highlights the importance of recognizing adolescent psychopathology, so that support can be provided to try and minimize adverse outcomes.

Metal- and Oxidant-Free Alkenyl C-H/Aromatic C-H Cross-Coupling Using Electrochemically Generated Iodosulfonium Ions.

A three-step transformation consisting of 1) addition of electrochemically generated iodosulfonium ions to vinylarenes to give (1-aryl-2-iodoethoxy)sulfonium ions, 2) nucleophilic substitution by subsequently added aromatic compounds to give 1,1-diaryl-2-iodoethane, and 3) elimination of HI with a base to give 1,1-diarylethenes was developed. The transformation serves as a powerful metal- and chemical-oxidant-free method for alkenyl C-H/aromatic C-H cross-coupling.

Extracellular adenosine levels are associated with the progression and exacerbation of pulmonary fibrosis.

Idiopathic pulmonary fibrosis is a devastating lung disease with limited treatment options. The signaling molecule adenosine is produced in response to injury and serves a protective role in early stages of injury and is detrimental during chronic stages of disease such as seen in lung conditions such as pulmonary fibrosis. Understanding the association of extracellular adenosine levels and the progression of pulmonary fibrosis is critical for designing adenosine based approaches to treat pulmonary fibrosis. The goal of this study was to use various models of experimental lung fibrosis to understand when adenosine levels are elevated during pulmonary fibrosis and whether these elevations were associated with disease progression and severity. To accomplish this, extracellular adenosine levels, defined as adenosine levels found in bronchioalveolar lavage fluid, were determined in mouse models of resolvable and progressive pulmonary fibrosis. We found that relative bronchioalveolar lavage fluid adenosine levels are progressively elevated in association with pulmonary fibrosis and that adenosine levels diminish in association with the resolution of lung fibrosis. In addition, treatment of these models with dipyridamole, an inhibitor of nucleoside transporters that potentiates extracellular adenosine levels, demonstrated that the resolution of lung fibrosis is blocked by the failure of adenosine levels to subside. Furthermore, exacerbating adenosine levels led to worse fibrosis in a progressive fibrosis model. Increased adenosine levels were associated with elevation of IL-6 and IL-17, which are important inflammatory cytokines in pulmonary fibrosis. These results demonstrate that extracellular adenosine levels are closely associated with the progression of experimental pulmonary fibrosis and that this signaling pathway may mediate fibrosis by regulating IL-6 and IL-17 production.