University of Alabama at Birmingham researchers have found a novel, previously unreported pathogenic entity that is a fundamental link between chronic inflammation and tissue destruction in the lungs of patients with chronic obstructive pulmonary disease, or COPD. COPD is the fourth-leading cause of death in the world.
This pathogenic entity — exosomes from activated polymorphonuclear leukocytes, or PMNs — caused COPD damage when the small, subcellular particles, collected from purified PMNs, were instilled into the lungs of healthy mice. Remarkably, the UAB researchers also collected exosomes from the lung fluids of human patients with COPD and the lung fluids of neonatal ICU babies with the lung disease bronchopulmonary dysplasia; when those human-derived exosomes were instilled into the lungs of healthy mice, they also caused COPD lung damage. Damage was primarily from PMN-derived exosomes from the human lungs.
“This report seems to provide the first evidence of the capability of a defined non-infectious subcellular entity to recapitulate disease phenotype when transferred from human to mouse,” said J. Edwin Blalock, Ph.D., professor of pulmonary, allergy and critical care medicine in the UAB Department of Medicine. “I think this could be a very profound discovery. A lot of what we have found here will apply in other tissues, depending on the disease.”
Other diseases marked by immune cell inflammation and tissue destruction include heart attacks, metastatic cancer, and chronic kidney disease. The activated PMN exosomes may also contribute to lung damage in other lung diseases that have excessive PMN-driven inflammation, such as cystic fibrosis. The study is reported in the journal Cell.
“These findings highlight a novel role of the innate immune response in chronic lung diseases and could be used for the development of new diagnostics and therapeutics for COPD and possibly cystic fibrosis,” said James Kiley, Ph.D., director of the Division of Lung Diseases at the National Heart, Lung, and Blood Institute, part of the National Institutes of Health.
COPD, a smoking-associated disease, is marked by PMN-driven inflammation in the lungs. Damage to the lung tissue leads to airway obstruction, shortness of breath, and respiratory failure. PMN immune cells, also known as neutrophils, are part of the body’s white blood cell defense against infections and tissue damage. They comprise 60 percent of the body’s white blood cells, or about 2.5 billion PMNs in each pint of blood. PMNs are voracious eaters of microbes or damaged human cells after activation by a signal of infection.
All cells shed exosomes. These tiny extracellular membrane-bound vesicles can be mediators of cell-to-cell communication, and they can ferry a diverse cargo of proteins, lipids, and nucleic acids from cell to cell. The UAB research focused on a recently found third role for exosomes — the ability to harbor protease enzymes.
Activated PMNs are known to release neutrophil elastase, or NE, a protease that can degrade type I collagen and elastin. The collagen and elastin proteins help form the extracellular matrix that glues cells together. In the lungs, the extracellular matrix and lung cells are sheets of tissue that help form the tiny alveoli, where the lung exchanges oxygen and carbon dioxide. In COPD, the damaged alveoli enlarge, reducing oxygen exchange and forcing the heart to pump harder to push blood through the lungs.
NE and other proteases from PMNs can attack microbes. Healthy lungs are protected by anti-proteases that can inhibit the proteases. Normally, NE is inhibited by a robust barrier of alpha1-antitrypsin in the lung.
Blalock and fellow researchers investigated whether NE might exist in an exosomal form and whether such exosomes might bypass alpha1-antitrypsin inhibition to contribute to inflammatory lung disease.
They found that exosomes from quiescent PMNs did not cause COPD when transferred to healthy mice. In contrast, exosomes from activated PMNs did cause COPD, as measured by histologic changes of the alveoli, increased pulmonary resistance and enlargement of the right heart ventricle that pumps blood to the lung.
The activated PMN exosomes were covered with enzymatically active surface-bound NE, while quiescent PMN exosomes had none. This surface NE was resistant to alpha1-antitrypsin inhibition; the exosomes from activated PMNs degraded collagen, they caused emphysema when put into mouse lungs, and they carried the PMN cell-surface markers CD63 and CD66b that identify them as coming from PMNs. Human COPD lung-derived exosomes carrying those PMN cell-surface markers conferred COPD to mice.
A very large dose of purified NE — enough to overwhelm the alpha1-antitrypsin barrier — can cause alveolar enlargement in mice. Because the exosome-bound NE was protected against apha1-antitrypsin inhibition, researchers found that the dose of activated PMN exosomes needed to cause the same damage as purified NE was 10,000 times less.
The activated PMN exosomes had another cause for their aggressive proteolysis — they carried integrin Mac-1 on their surface. Integrin Mac-1 allowed the exosomes to bind directly to collagen fibrils, a second mechanism besides protected NE for why the proteolytic exosomes exert an outsized degradative capacity in relation to their size and protease load.
“This investigation reveals an entirely unappreciated aspect of the interplay between inflammation, proteolysis and matrix remodeling with far-reaching implications for future research,” Blalock said. “Our report significantly expands the biological repertoire of the exosome, demonstrating potent biological effects of these particles ex cellula.”
The study also suggests therapeutic strategies to interrupt pathogenic aspects of PMN exosome function: 1) disrupting the ionic binding of the NE to the exosome, to dislodge the NE and make it susceptible to alpha1-antitrypsin; 2) inhibiting the exosomal integrin Mac-1 to block collagen binding; and 3) directly inhibiting the exosomal NE with small-molecule compounds.
Blalock is also interested in another big question — exosome activity in healthy smokers.
“Only one in seven or one in eight smokers gets COPD,” he said. “It would be an amazing outcome if we found activated PMN exosomes in a subpopulation of people who smoke.” Those people could then be warned of the risk they faced.
This Cell study took six years of work.
Significant research was done by co-first authors Kristopher Genschmer, Ph.D., and Derek W. Russell, M.D., who were NIH T32 grant trainees with Blalock. Both are assistant professors in the UAB Division of Pulmonary, Allergy and Critical Care Medicine. Amit Gaggar, M.D., Ph.D., a professor of pulmonary, allergy and critical care medicine, is co-senior author with Blalock, and he is a former trainee who did his Ph.D. with Blalock. Co-author Charitharth Vivek Lal, M.D., assistant professor in the UAB Pediatrics Division of Neonatology, is the physician who collected the lung fluid from neonates and performed all of the bronchopulmonary dysplasia work.
Co-authors with Genschmer, Russell, Gaggar, Lal and Blalock of the paper “Activated PMN exosomes: Pathogenic entities causing matrix destruction and disease in the lung” are Tomasz Szul, Mojtaba Abdul Roda, Xin Xu, Liliana Viera, Tarek H. Abdalla, Robert W. King, J. Michael Wells and Mark T. Dransfield, UAB Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine; Preston E. Bratcher, National Jewish Medical Center, Denver, Colorado; Brett D. Noerager, University of Montevallo, Montevallo, Alabama; Gabriel Rezonzew, UAB Department of Pediatrics; Brian S. Dobosh, Camilla Margaroli and Rabindra Tirouvanziam, Department of Pediatrics, Emory University, Atlanta, Georgia; and Carmel M. McNicholas, UAB Department of Cell, Developmental and Integrative Biology.
This study was supported by National Institutes of Health grants HL135710, HL077783, HL114439, HL110950, HL126596, HL102371, HL126603, HL123940, HL105346-07 and HL105346-05; American Heart Association grant 17SDG32720009; and Veterans Affairs grant BX001756.
Blalock is a distinguished professor in the UAB School of Medicine, and he holds the Nancy E. Dunlap, M.D., Endowed Chair in Pulmonary Disease.
Genschmer KR, Russell DW, et al. Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung. Cell (2019) 176: 113-126. doi: 10.1016/j.cell.2018.12.002. PMID: 30633902.
This blog post comes from the Myotonic Dystrophy Foundation.
Pharmacodynamic Biomarkers and DM
There is now strong support for the concept that a panel of splicing events may serve as a pharmacodynamic biomarker for go/no go decisions in drug development for myotonic dystrophy type 1 (DM1) and Duchenne muscular dystrophy (DMD). Data establishing splicing event sensitivity to free MBNL levels has converged with the natural history of alternative splicing patterns in DM patients to yield a subset of splicing events with the sensitivity and reproducibility to evaluate candidate therapeutics in early stage clinical trials. Quantitative pharmacodynamic biomarkers are invaluable in de-risking industry drug discovery and development, as they facilitate early stage assessment of molecular target engagement and modulation and may inform dose ranging studies. The only caveat is the dependence of these measures upon repeated muscle biopsies (a risk reduced, but not eliminated, by more tolerable needle biopsies). The identification and validation of a non-invasive assay of patient splicing status would be a valuable step forward for clinical trials in DM.
Early Support for a Non-Invasive Biomarker for DM1
Dr. Thurman Wheeler and colleagues at Massachusetts General, Harvard Medical, and Boston Children’s have explored the concept that a subset of extracellular RNAs (exRNAs) released into blood or urine may: (a) reflect alternative splicing status in DM-affected tissues and (b) thereby serve as an easily accessible pharmacodynamic biomarker platform for DM1 (Antoury et al., 2018). These studies were supported in part by a grant to facilitate “Development of Biomarkers for Myotonic Studies” from Myotonic Dystrophy Foundation/Wyck Foundation.
The research team initially found that > 30 transcripts that are alternatively spliced in DM1 muscle biopsies were detectable in human blood and urine samples; follow-up studies confirmed the presence of RNAs in extracellular fluids/exosomal particles. Normalized DMPK expression levels in urine from DM1 patients, by droplet digital PCR, were ~50% of unaffected controls. Assessments of DM1-established alternative splicing events showed that a subset (10/33) also occurred in urine exRNA, including being conserved in longitudinal (6-26 month) studies of the same patients. Assessments of alternative splicing events in blood exRNA did not yield the same value.
Using principal component analysis of 10 alternative splicing events observed in urine exRNA, the research team then generated a putative composite biomarker panel for DM1. The ensuing predictive model of alternative splicing in DM1 proved to be 100% accurate in comparisons of training and independent validation data sets to distinguish DM1 from unaffected controls and in distinguishing disease status of subsequently enrolled subjects. The research team also linked alternative splicing patterns in urine exRNA to variation in DM1 clinical phenotypes, suggesting that modeling of urine exRNA alternative splicing may allow both the tracking of disease progression and the impact of candidate therapeutics.
Finally, to address questions as to the source of urine exRNA, the team assessed alternative splicing in urinary tract cells of DM1 mouse models (the ubiquitous Mbnl1 ko and the tissue-specific HSALR). While kidney and bladder cells of the Mbnl1 ko reflected patterns in skeletal muscle, assessments of the same tissues in the HSALR showed no differences from control mice. These data strongly suggested that the exRNAs assessed in urine reflect exosomes released from urinary tract cells. Some of the alternatively spliced transcripts in urine exRNA also were shown to be altered by antisense oligonucleotide drugs previously shown to correct splicing patterns in DM1 mouse models. The research team’s parallel studies of Duchenne muscular dystrophy also supported the concept that urine exRNA has utility as a pharmacodynamic biomarker in drug intervention studies.
Towards a Non-Invasive Biomarker for DM1
Taken together, these data provide compelling proof of concept that a panel of alternative splicing events assessed in urine may serve as a robust composite biomarker of DM1 progression and as a tool for assessment of candidate therapeutics. A non-invasive biomarker such as this would greatly extend the ability to perform repeated measurements in longitudinal natural history studies (as a disease progression biomarker) and in interventional clinical trials (as patient stratification and pharmacodynamic biomarkers), including making assessment of pediatric DM1 patient cohorts feasible. Although it is not essential to formally qualify a biomarker, existing regulatory agency guidance documents (see References below) provide a valuable evidentiary framework for moving non-invasive biomarker work towards an accepted clinical tool for DM1.
Antoury L, Hu N, Balaj L, Das S, Georghiou S, Darras B, Clark T, Breakefield XO, Wheeler TM. Analysis of extracellular mRNA in human urine reveals splice variant biomarkers of muscular dystrophies. Nat Commun. (2018) 9: 3906. doi: 10.1038/s41467-018-06206-0. PMID: 30254196
Guidance for Industry and FDA Staff: Qualification Process for Drug Development Tools. (pdf)
Updated guidelines on Minimal Information for Studies of Extracellular Vesicles have now been published in the Journal of Extracellular Vesicles (JEV, Taylor & Francis) as MISEV2018.
The original MISEV2014 guidelines were released in 2014 by the Board of Directors of the International Society for Extracellular Vesicles (ISEV) to provide guidance in standardization of protocols and reporting in the EV field. Accumulating more than 800 citations since its release, the MISEV2014 guidelines have achieved the aim of becoming a guiding standard for researchers. A 2016 survey of ISEV members reaffirmed the need for guidelines and recommended that they be updated regularly…but with broad community input to accommodate and shape the quickly developing field.
MISEV2018 updates the topics of nomenclature, separation, characterization, and functional analysis, integrating the contributions of over 380 ISEV members, a strong tribute to the commitment of ISEV members. A two-page checklist summarizing the main points is also included.
So what’s new? MISEV2018 recommends the use of ‘extracellular vesicle’ as the preferred generic terminology for use in publications, in part due to challenges in confirming the biogenesis mechanisms of exosomes, microvesicles, and other particles, and in part due to the vague and varied uses of other terms. Separation and concentration options are now many and diverse; researchers should pick the methods most fit for downstream purpose and, more importantly, report these clearly and accurately. The EV-TRACK database (van Deun et al., Nature Methods, 2017) is supported as a means to record these details in order to improve clarity and reproducibility. To establish presence of EVs, examples of EV-enriched markers are provided, but the need for “negative” (better: “depleted”) markers is also highlighted. MISEV2018 adds topology as a recommended form of EV characterization, for example identifying where in or on a vesicle your favorite protein or RNA resides. It also recommends functional analysis of the ‘non-EV’ fractions to confirm EV-specific function (or not!). An appreciation of EV heterogeneity is included with a reminder that ‘larger EVs matter’ and a request to explore a range of EV subtypes in functional studies. Finally, although some of the specific details contained in MISEV2018 are focused on mammalian components, it is appreciated that the guidelines are applicable to non-mammalian and non-eukaryote research.
Lotvall J, Hill AF, Hochberg F, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the international society for extracellular vesicles. J. Extracell. Vesicles. (2014) 3: 26913. doi:10.1080/20013078.2018.1535750. PMID:25536934.
Witwer KW, Soekmadji C, Hill AF, et al. Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility. J. Extracell. Vesicles. (2017) 6: 1396823. doi:10.1080/20013078.2017.1396823. PMID:29184626.
Théry C, Kenneth W Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles. (2018) 7: 1535750. doi:10.1080/20013078.2018.1535750.