An unfortunate truth about the use of mechanical ventilation to save the lives of patients in respiratory distress is that the pressure used to inflate the lungs is likely to cause further lung damage.
In a new study, scientists identified a molecule that is produced by immune cells during mechanical ventilation to try to decrease inflammation, but isn’t able to completely prevent ventilator-induced injury to the lungs.
The team is working on exploiting that natural process in pursuit of a therapy that could lower the chances for lung damage in patients on ventilators. Delivering high levels of the helpful molecule with a nanoparticle was effective at fending off ventilator-related lung damage in mice on mechanical ventilation.
“Our data suggest that the lungs know they’re not supposed to be overinflated in this way, and the immune system does its best to try to fix it, but unfortunately it’s not enough,” said Dr. Joshua A. Englert, assistant professor of pulmonary, critical care and sleep medicine at The Ohio State University Wexner Medical Center and co-lead author of the study.
“How can we exploit this response and take what nature has done and augment that? That led to the therapeutic aims in this study.”
The work builds upon findings from the lab of co-lead author Samir Ghadiali, professor and chair of biomedical engineering at Ohio State, who for years has studied how the physical force generated during mechanical ventilation activates inflammatory signaling and causes lung injury.
Efforts in other labs to engineer ventilation systems that could reduce harm to the lungs haven’t panned out, Ghadiali said.
“We haven’t found ways to ventilate patients in a clinical setting that completely eliminates the injurious mechanical forces,” he said. “The alternative is to use a drug that reduces the injury and inflammation caused by mechanical stresses.”
The research is published today (Jan. 12, 2021) in Nature Communications.
Though a therapy for humans is years away, the progress comes at a time when more patients than ever before are requiring mechanical ventilation: Cases of acute respiratory distress syndrome (ARDS) have skyrocketed because of the ongoing COVID-19 pandemic.
ARDS is one of the most frequent causes of respiratory failure that leads to putting patients on a ventilator.
“Before COVID, there were several hundred thousand cases of ARDS in the United States each year, most of which required mechanical ventilation. But in the past year there have been 21 million COVID-19 patients at risk,” said Englert, a physician who treats ICU patients.
The immune response to ventilation and the inflammation that comes with it can add to fluid build-up and low oxygen levels in the lungs of patients already so sick that they require life support.
The molecule that lessens inflammation in response to mechanical ventilation is called microRNA-146a (miR-146a).
MicroRNAs are small segments of RNA that inhibit genes’ protein-building functions – in this case, turning off the production of proteins that promote inflammation.
The researchers found that immune cells in the lungs called alveolar macrophages – whose job is to protect the lungs from infection – activate miR-146a when they’re exposed to pressure that mimics mechanical ventilation.
This action makes miR-146a part of the innate, or immediate, immune response launched by the body to begin its fight against what it is perceiving as an infection – the mechanical ventilation.
“This means an innate regulator of the immune system is activated by mechanical stress. That makes me think it’s there for a reason,” Ghadiali said. That reason, he said, is to help calm the inflammatory nature of the very immune response that is producing the microRNA.
The research team confirmed the moderate increase of miR-146a levels in alveolar macrophages in a series of tests on cells from donor lungs that were exposed to mechanical pressure and in mice on miniature ventilators.
The lungs of genetically modified mice that lacked the microRNA were more heavily damaged by ventilation than lungs in normal mice – pointing to miR-146a’s protective role in lungs during mechanical breathing assistance.
Finally, the researchers examined cells from lung fluid of ICU patients on ventilators and found miR-146a levels in their immune cells were increased as well.
The problem: The expression of miR-146a under normal circumstances isn’t high enough to stop lung damage from prolonged ventilation.
The intended therapy would be introducing much higher levels of miR-146a directly to the lungs to ward off inflammation that can lead to injury. When overexpression of miR-146a was prompted in cells that were then exposed to mechanical stress, inflammation was reduced.
To test the treatment in mice on ventilators, the team delivered nanoparticles containing miR-146a directly to mouse lungs – which resulted in a 10,000-fold increase in the molecule that reduced inflammation and kept oxygen levels normal.
In the lungs of ventilated mice that received “placebo” nanoparticles, the increase in miR-146a was modest and offered little protection.
From here, the team is testing the effects of manipulating miR-146a levels in other cell types – these functions can differ dramatically, depending on each cell type’s job.
Inflammation is a key component of myocardial ischemic injury (1). Studies have shown that nucleic acids such as cellular RNAs, including microRNAs (miRNAs), are released from initial tissue injury within the myocardium (2–4) and may contribute to the increased myocardial inflammation following ischemia-reperfusion (I/R) injury (4, 5).
Systemic administration of nucleic acid binding nanoprobe (5) or RNase (4, 6) leads to reduced myocardial inflammation and infarction, likely by reducing extracellular (ex) RNA levels. Moreover, several of these ex-miRNAs (e.g., ss–miR-146a-5p and miR-133a) induce potent innate immune responses, including cytokine and complement factor production in cultured cells and acute peritonitis after local injection (7, 8).
However, it remains unknown how ex-miRNAs such as miR-146a-5p interact with various cardiac cells and whether they induce myocardial inflammation and cardiomyocyte (CM) dysfunction.
miRNAs are small and ss noncoding RNAs that bind to the 3′ untranslated region of a target gene and induce degradation of mRNA or inhibition of translation. miR-146a is among one of the well-studied miRNAs that reportedly regulate innate immune function.
When acting in its conventional role within the cell, miR-146a is known as an innate immune regulator, downregulating TRAF-6 and IRAK-1 expression, two key molecules involved in TLR 4 signaling, and attenuating host proinflammatory response to endotoxin (9, 10). In contrast, mature ss–miR-146a-5p, when added to cell cultures or administered in vivo, exhibits a remarkable proinflammatory effect such as cytokine and complement production and immune cell activation (7, 8).
TLRs are a group of innate immune receptors that recognize conserved pathogen-associated molecular patterns presented in different types of pathogens such as bacteria, fungi, and viruses (11). They are expressed either on the surface of cell plasma membrane or intracellular endosomes.
TLRs located in endosomes recognize microbial and endogenous nucleic acids such as DNA and RNA (12, 13). TLR 7 in particular is originally known for sensing ssRNA of viral origins such as influenza A and HIV (14, 15), but it can also sense certain miRNAs (7) and other small RNA (15, 16).
In this study, we tested the hypothesis that exogenous ss–miR-146a-5p is an innate immune mediator capable of inducing myocardial inflammation and CM contractile dysfunction. We showed that mouse CMs produced proinflammatory cytokines in response to ex vesicles (EVs) loaded with miR-146a-5p.
We demonstrated that intracardiac injection of miR-146a-5p induced a robust cardiac innate immune response via a TLR7-dependent mechanism. Moreover, we identified that miR-146a-5p activated heterocellular cross-talk signaling among various cardiac cell types in vitro and led to impairment of coronary artery endothelial cell barrier function and CM contractile function. These findings provide new insight into the effect of ex-miRNAs on innate immune response and function of cardiac cells.
We and others have demonstrated that cellular nucleic acids such as RNA and miRNA are released from the heart following myocardial ischemic injury (4, 5, 7, 34–36).
The cellular RNA, once released from the cells, can be highly proinflammatory by inducing cytokine production and innate immune cell activation (35). Systemic administration of RNase (4, 6) or a nucleic acid binding nanoprobe (5) reduces ex-RNA and attenuates myocardial cytokine production, innate immune cell infiltration, and cardiac injury following myocardial ischemia.
These observations support the notion that ex-RNA may play a pivotal role in cardiac innate immune responses. Further analysis using miRNA array by our group identified a panel of plasma miRNAs that were upregulated following cardiac ischemia, some of which, such as miR-146a-5p, were highly proinflammatory in innate immune cells (7).
But whether ex–miR-146a-5p is capable of inducing myocardial innate immune response and affecting cardiac function has been unclear. In this study, we demonstrated that miR-146a-5p loaded in engineered EVs induced a dose-dependent proinflammatory cytokine response in isolated CMs.
A single dose of intracardiac injection of miR-146a-5p induced myocardial cytokine expression and robust monocyte/neutrophil recruitment into the heart.
These innate immune responses appear to be mediated by TLR7 signaling. Moreover, miR-146a-5p induced HCAEC impairment by interrupting its barrier function.
However, this effect appeared to be indirect and mediated through activated CMs and cardiac fibroblasts treated with miR-146a-5p. Finally, we demonstrated that miR-146a-5p was able to suppress CM sarcomere shortening through TLR7-mediated activation of myeloid-derived macrophages.
All together, these data suggest that ex–miR-146a-5p may be an innate immune mediator capable of activating multiple cellular functions in the heart and modulate myocardial inflammation and CM function via TLR7 sensing.
Both adult mouse and neonatal rat CMs produced cytokines in response to miR-146a-5p treatment. The loss-of-function experiments in TLR7 KO CMs suggest that the cytokine response in adult CMs was clearly mediated through TLR7. Our initial pilot experiments using lipofectamine as the delivery vehicle of miRNAs failed to show any cytokine response in adult CMs, likely due to poor transfection (23).
However, miR-146a-5p loaded in EVs exhibited a strong proinflammatory effect in adult CMs. This is relevant because in vivo, circulating plasma miRNAs are carried in part by EVs. In fact, this has been implied in our previous work that demonstrated plasma EVs isolated from sepsis mice promoted significant CXCL2 and IL-6 via miRNA- and TLR7-dependent mechanisms (37).
In addition to the physiological relevance of the EVs, engineered EVs could potentially be a means to deliver therapeutics to the heart following cardiac injury. Several studies have already demonstrated that delivery of EVs to the heart can lead to improved cardiac recovery after myocardial infarction and I/R injury (38, 39).
Although these EV studies did not demonstrate that targeting miRNAs or other nucleic acids are potential treatments for cardiac injury, the study by Chen and colleagues used dextran-thiazole orange, a multivalent nucleic acid–scavenging nanoprobe, to illustrate ex localization of nucleic acids in the heart and, at the same time, to attenuate proinflammatory cytokine production and myocardial injury in a mouse model of I/R (5).
Therefore, it is probable EVs containing molecules that target miRNAs and nucleic acids could be developed into therapies for cardiac injury.
We tested the impact of miR-146a-5p activation of CMs and cardiac fibroblasts on coronary artery endothelial barrier function. This is important because cardiovascular disease aggravates endothelial barrier dysfunction resulting in a significant increase in invading immune cells and myocardial inflammation (28, 29, 40).
To examine miR-146a–mediated inflammatory signaling on endothelial function, we treated both CMs and cardiac fibroblasts with miR-146a and harvested the conditioned media to test endothelial permeability using an ECIS assay. We found that HCAEC monolayers responded with increased intercellular permeability to the conditioned media from both CMs and fibroblasts treated with miR-146a-5p.
This indirect mechanism may be important because HCAECs do not express TLR7 and did not respond to miR-146a-5p directly (31). This data suggest that miR146a-5p–induced activation of CMs and fibroblasts may represent a potential mechanism that regulates cardiac endothelial barrier function.
Although we speculate that both CMs and fibroblasts may secrete soluble mediators that cause endothelial dysfunction in response to ex-miRNAs, we do not know the nature of the mediators. Several cytokines have been reported to induce endothelial dysfunction, including TNF-α, IL-6, and IL-1β42.
These cytokines exert their effects on endothelial cells through a variety of mechanisms such as increased expression of endothelial adhesion molecules to facilitate leukocyte transmigration or inhibition of NO synthase, both of which increase endothelial barrier permeability (41–43).
In addition to regulating endothelial barrier function, myocardial inflammation can contribute to cardiac dysfunction through complex inflammatory pathways. Proinflammatory cytokines initiate a localized inflammatory response (44), which triggers the expression of chemokines (45), cell adhesion molecules, and recruitment of leukocytes (46).
Of the leukocytes, macrophages are of notable interest as inflammatory myeloid-derived macrophages produce large amounts of cytokines such as IL-6 and IL-18 through TLR signaling pathways (47, 48). Both IL-6 and IL-18 induce CM hypertrophy (47), and in both the noninfarcted and infarcted heart, IL-18 is also known to exacerbate fibrosis and cardiac dysfunction (48).
In our study, we simulated interaction between macrophages and CMs and investigated whether activation of macrophages by miR-146a → TLR7 signaling could affect adult CM contractility. We discovered that the conditioned media from WT, but not TLR7 KO, macrophages treated with miR-146a induced CM depression.
These data suggest that TLR7 is necessary for miR-146a–induced cellular responses in macrophages and subsequent depression of CM contractility. We speculate that cytokines manufactured in macrophages following the miR-146a-5p treatment are likely the cause of CM depression as cytokines such as both IL-6 and TNF-α are known cardiac depressants (49, 50).
Our study suggests that endogenous ex–miR-146a could play a role in mediating cardiac cell cross-talk following cardiac injury. Although direct in vivo evidence is needed, we speculate that ex–miR-146a released from damaged tissue may be taken into endosomes in multiple cardiac cell types, including cardiac resident macrophages, fibroblasts, and CMs, activating the TLR7 signaling pathway and stimulating production of proinflammatory cytokines and chemokines such as IL-6 and CXCL-2.
Cross-talk between these cells may then release additional cytokines and chemokines, resulting in disrupted endothelial barrier permeability or reduced contractility of the heart. This could, in turn, exacerbate the damage done by the initial tissue injury and lead to infiltration of innate immune cells that may contribute further to inflammatory responses following initial cardiac injury.
A number of limitations should be acknowledged. For one, this study was primarily in vitro. We did not establish the role of ex–miR-146a-5p in myocardial inflammation in vivo after transient ischemia. Another limitation was the use of intracardiac injections to deliver miR-146a to the heart, which was less physiologically relevant, but useful to test the myocardial response to exogenous miRNAs.
Our goal was to test the concept that activation of miR-146a-5p → TLR7 signaling is sufficient to induce myocardial inflammation and CM contractile dysfunction. Loss-of-function and overexpression studies will help to define the exact role of miR-146a in myocardial inflammation and dysfunction after cardiac ischemic injury in vivo.
In summary, we identified that exogenous delivery of ss–miR-146a-5p was able to drive an innate immune response in the heart and on various cardiac cells, including CMs, coronary artery endothelial cells, and cardiac fibroblasts, thereby leading to myocardial inflammatory and cardiac cellular dysfunction. Heterocellular cross-talk between various cardiac cells leads to impairment of endothelial barrier function and CM depression following miR-146a → TLR7 activation. Overall, this study establishes a pivotal role of miR-146a → TLR7 signaling in myocardial innate immune response and CM dysfunction.
reference link: https://www.immunohorizons.org/content/4/9/561
What obstacles need to be negotiated before microRNA-based therapeutics can be used in COPD? http://ow.ly/fM4n30aD4BX
Chronic obstructive pulmonary disease (COPD) represents a major cause of chronic morbidity and mortality, affecting more than 200 million people worldwide and leading to approximately 3 million deaths each year.
COPD is mainly caused by cigarette smoking and is characterised by a chronic inflammation leading to obstruction of the small airways and destruction of lung parenchyma (emphysema). Therapies that slow down the accelerated decline in lung function in patients with COPD are still lacking. Therefore, it is essential to unravel the mechanistic processes that underlie the inflammatory reaction and subsequent structural changes in COPD .
Aberrant cross-talk between epithelial and mesenchymal cells has been associated with inflammatory and remodelling processes in COPD. Osei et al.  previously demonstrated that airway epithelial cells (AECs) from COPD patients release more interleukin (IL)-1α upon in vitro exposure to cigarette smoke extract.
Moreover, using an elegant co-culture model, they demonstrated that this epithelial-derived IL-1α induced a pro-inflammatory lung fibroblast phenotype, releasing high amounts of the neutrophil attracting chemokine IL-8 . Interestingly, we and others have reported increased levels of IL-1α in the lungs of patients with COPD and have shown in in vivo cigarette smoke models that neutrophilic inflammation is strongly dependent on IL-1α [3, 4].
In the current issue of the European Respiratory Journal, Osei et al.  demonstrate that the dysfunctional cross-talk between AECs and fibroblasts in COPD is due to the impaired ability of COPD fibroblasts to upregulate microRNA-146a-5p. MicroRNAs (miRNAs) are endogenous, small noncoding RNAs with a regulatory function on gene expression.
They bind in a sequence-specific manner to sites with imperfect complementarity in target messenger RNAs (mRNAs), leading to direct inhibition of protein translation or degradation of the transcript. In this way, miRNAs can interact with hundreds of genes simultaneously and regulate several developmental and physiological processes, including cellular proliferation, differentiation, apoptosis and innate and adaptive immune responses .
miR-146a-5p has frequently emerged as a regulator of inflammation . Upon activation of several inflammatory pathways, such as Toll-like receptor (TLR) or IL-1R signalling, miR-146a-5p is induced in a nuclear factor (NF)-κB-dependent manner . By targeting key molecules downstream of TLR and IL-1R pathways like tumour necrosis factor receptor-associated factor-6 and IL-1 receptor-associated kinase (IRAK)-1, it functions as a negative feedback regulator, limiting the intensity and duration of the inflammatory response . In their co-cultures of airway epithelial cells and primary lung fibroblasts, Osei et al.  demonstrated that epithelial-derived IL-1α induces miR-146a-5p expression in fibroblasts.
Importantly, this increase of miR-146a-5p was impaired in fibroblasts from patients with COPD, which releases the brake on the IL-1R/NF-κB pathway and is likely to contribute to the abnormal inflammation in COPD. Interestingly, treating fibroblasts with an miR-146a-5p mimic downregulated the expression of IRAK-1, resulting in a reduced release of IL-8 and confirming the anti-inflammatory role of miR-146a-5p .
It is worth noting that the inability of COPD fibroblasts to upregulate miR-146a-5p not only leads to an impaired negative feedback regulation of NF-κB signalling, but also results in a reduced mRNA degradation and thus prolonged half-life of cyclooxygenase (COX)-2, another target of miR-146a-5p . COX-2 is a key enzyme in biosynthesis of prostaglandin E2, a promoter of neutrophil recruitment and an inhibitor of the repair function of fibroblasts.
In an attempt to find the mechanism explaining the inability of COPD lung fibroblasts to upregulate miR-146a-5p, Osei et al.  assessed the presence of the single nucleotide polymorphism (SNP) rs2910164 in their primary lung fibroblasts. This common G>C SNP in pre-miR-146a-5p has been associated with decreased expression of the mature miR-146a-5p .
Unexpectedly, in the present study, a lower miR-146a-5p expression was observed in fibroblasts from donors homozygous for the G allele of SNP rs2910164, which were all but one COPD patient . A validation study in a large independent cohort will be needed to verify these results. Moreover, despite the strong association of this SNP with the presence of COPD, no such association was found in any of the large genome-wide association studies of lung-function parameters of airflow obstruction (i.e. forced expiratory volume in 1 s or its ratio to forced vital capacity) .
Since miRNAs are key regulators of the human transcriptome and are differentially expressed in nearly every disease, there is an enormous therapeutic potential in both miRNA inhibitors and mimics. In recent years, several miRNA therapeutics have gone into preclinical development and have been used in clinical trials .
To date, the most clinically advanced miRNA therapy is miravirsen, a locked nucleic acid modified miR-122 inhibitor used for the treatment of hepatitis C virus (HVC) infections . miR-122 is a liver-specific miRNA and is required for the propagation of HCV. Phase II clinical trials with miravirsen reported a significant, dose-dependent decrease in HCV, which sustained long after the administration , demonstrating the effectiveness of miRNA therapy in humans.
Whether or not miRNA-based therapeutics will ever be used for the treatment of such a complex and heterogeneous disease as COPD depends on the hurdles that still lie ahead. First, we need a clear picture of the candidate miRNAs. In the last decade, several miRNA profiling studies have been published, yielding lists of miRNAs that are differentially expressed between control subjects and smokers with or without COPD [15–17].
Unfortunately, large discrepancies exist between the different studies. These can be due to differences in sample material, ranging from whole lung tissue to epithelial brushings and induced sputum. We were the first to show a significant differential expression of miRNAs in the induced sputum of patients with COPD and current smokers compared with never-smokers, including downregulation of miR-146a-5p .
Next to sample material, the characteristics of the study population (including chronic bronchitis versus emphysematous phenotype, COPD severity, smoking history, age, sex) may also substantially influence the outcome. And finally, the different platforms used for miRNA profiling, ranging from microarrays to RT-qPCR and, the current gold standard, small RNA sequencing, do not always deliver the same results .
Once differentially expressed miRNAs have been identified, the next step is to validate the biological significance of these findings. In vitro and in vivo functional studies have already implicated several miRNAs in the pathogenesis of COPD (figure 1) [24, 25].
Recently, using in vivo perturbation experiments and gene set-enrichment analysis, we were able to demonstrate a protective role of miR-218-5p in cigarette smoke-induced inflammatory responses and COPD . Importantly, miR-218-5p is significantly down-regulated in bronchial epithelium of patients with COPD .
Translation of these in vitro and animal studies to humans will require substantial validation and mechanistic studies, with an important focus on the dosing of miRNA therapy. Many of the functional studies, including the current study by Osei et al. , use doses of miRNA inhibitors or mimics that are beyond physiological relevance. Additionally, it seems likely that miRNA-based therapy for a complex disease like COPD, encompassing several immunological pathways and cell types, will have to be directed at multiple miRNAs. Moreover, the interplay between different miRNAs, which may share several targets, should be taken into account.
Another hurdle is the delivery of the miRNA inhibitors or mimics. Targeting of miRNA-based therapeutics to a particular tissue or specific cell type is a challenging area of research . It seems obvious that, for example, targeting the airway epithelium is more feasible than the underlying fibroblasts.
Therefore, the route of administration (local versus systemic) as well as the delivery method (from viral or lipid vectors to nanoparticle and polymer systems) should be considered carefully. Importantly, nonspecific targeting of miRNA therapeutics may lead to toxicity. Indeed, while the ability of miRNAs to target multiple mRNAs and pathways is key to their enormous therapeutic potential, it may also be a limitation, giving rise to unwanted effects in other cell types.
Taken together, profiling studies have yielded several miRNAs that are dysregulated in COPD. The need for functional studies like the current work by Osei et al.  is great, for demonstration of the biological relevance of these candidate miRNAs. Ideally, to establish miRNAs as promising therapeutic targets in COPD, in vivo studies should be performed, taking into account physiologically relevant dosing and targeted delivery to the desired lung compartment or cell type.
reference link : https://erj.ersjournals.com/content/49/5/1700431
More information: Nature Communications (2021). DOI: 10.1038/s41467-020-20449-w