Macrophage-Derived Extracellular DNA Initiates Heterotopic Ossification.

Heterotopic ossification (HO) severely affects people's lives; however, its pathological mechanism remains poorly understood. Although extracellular DNA (ecDNA) has been shown to play important roles in pathological calcification, its effects in HO development and progression remain unknown. The in vivo rat Achilles tendon injury model and in vitro collagen I calcification model were used to evaluate the effects of ecDNA in the ectopic calcifications and the main cell types involved in those pathological process. Histology, immunofluorescent staining, reverse transcriptase-polymerase chain reaction analysis and micro-computed tomography were used to identify the distribution of macrophage-derived ecDNA and elucidate their roles in HO. The results showed that the amount of ecDNA and ectopic calcification increased significantly and exhibited a strong correlation in the injured tendons of HO model compared with those of the controls, which was accompanied by a significantly increased number of M2 macrophages in the injured tendon. During in vitro co-culture experiments, M2 macrophages calcified the reconstituted type I collagen and ectopic bone collected from the injured tendons of HO rats, while those effects were inhibited by deoxyribonuclease. More importantly, deoxyribonuclease reversed the pathological calcification in the injured rat tendon HO model. The present study showed that ecDNA from M2 macrophages initiates pathological calcification in HO, and the elimination of ecDNA might be developed into a clinical strategy to prevent ectopic mineralization diseases. The use of deoxyribonuclease for the targeted degradation of ecDNA at affected tissue sites provides a potential solution to treat diseases associated with ectopic mineralization.

An in vitro method for the analysis of hemocyte-derived extracellular traps in shrimp.

The formation of extracellular traps (ETs) is a cell death mechanism relying on the release of nucleic acids in response to different stimuli. More recently, ETs have been recognized as an important cellular immune response since they are able to entrap and kill various microorganisms. The main goal was to describe a methodology to induce and visualize the in vitro formation of ETs by shrimp hemocytes. ETs formation was induced by the incubation of hemocyte monolayers from naïve shrimp (Penaeus vannamei) with a standard dose of Vibrio parahaemolyticus M0905. Following fixation, slides were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged by fluorescence microscopy. The methodology proposed in this study successfully induced the formation and release of hemocyte-derived ETs in penaeid shrimp. The procedure described here can be used as a novel immune marker to assess shrimp health status.

Functions and cellular signaling by ribosomal extracellular RNA (rexRNA): Facts and hypotheses on a non-typical DAMP.

Upon microbial infections with the subsequent host response of innate immunity, a variety of fragmented RNA- and DNA-based "Pathogen-associated molecular patterns" (PAMPs) are recognized mainly by endosomal or cytoplasmic host cell "Pattern recognition receptors" (PRRs), particularly "Toll-like receptors" (TLRs). Concomitantly, various self-extracellular RNA species (exRNAs) are present in extracellular body fluids where they contribute to diverse physiological and homeostatic processes. In principle, such exRNAs, including the most abundant one, ribosomal exRNA (rexRNA), are designated as "Danger-associated molecular patterns" (DAMPs) and are prevented by e.g. natural modifications from uncontrolled signaling via TLRs to avoid hyper-inflammatory responses or autoimmunity. Upon cellular stress or tissue damage/necrosis, the levels and composition of released self-exRNA species, either in free form, in complex with proteins or in association with extracellular vesicles (EVs), can change considerably. Among the self-exRNAs, rexRNA is considered as a non-typical DAMP, since it may induce inflammatory responses by cell membrane receptors, both in the absence or presence of PAMPs. Yet, its mode of receptor activation to mount inflammatory responses remains obscure. RexRNA also serves as a universal damaging factor in cardiovascular and other diseases independent of PRRs. In general, RNase1 provides a profound antagonist in these pathologies and in rexRNA-mediated inflammatory cell responses. Based on the extrapolation of the here described aspects of rexRNA-biology, further activities of this molecular entity are hypothesized that may stimulate additional research in this area.

Extracellular DNA: A Missing Link in the Pathogenesis of Ectopic Mineralization.

Although deoxyribonucleic acid (DNA) is the genetic coding for the very essence of life, these macromolecules or components thereof are not necessarily lost after a cell dies. There appears to be a link between extracellular DNA and biomineralization. Here the authors demonstrate that extracellular DNA functions as an initiator of collagen intrafibrillar mineralization. This is confirmed with in vitro and in vivo biological mineralization models. Because of their polyanionic property, extracellular DNA molecules are capable of stabilizing supersaturated calcium phosphate solution and mineralizing 2D and 3D collagen matrices completely as early as 24 h. The effectiveness of extracellular DNA in biomineralization of collagen is attributed to the relatively stable formation of amorphous liquid droplets triggered by attraction of DNA to the collagen fibrils via hydrogen bonding. These findings suggest that extracellular DNA is biomimetically significant for fabricating inorganic-organic hybrid materials for tissue engineering. DNA-induced collagen intrafibrillar mineralization provides a clue to the pathogenesis of ectopic mineralization in different body tissues. The use of DNase for targeting extracellular DNA at destined tissue sites provides a potential solution for treatment of diseases associated with ectopic mineralization.

Extracellular Nucleic Acids Present in the Candida albicans Biofilm Trigger the Release of Neutrophil Extracellular Traps.

Neutrophils, the first line of the host's defense, use a variety of antimicrobial mechanisms to fight invading pathogens. One of the most crucial is the production of neutrophil extracellular traps (NETs) in the process called NETosis. The unique structure of NETs effectively inhibits the spread of pathogens and ensures their exposure to a high concentration of NET-embedded antimicrobial compounds. NETosis strategy is often used by the host to defend against fungal infection caused by Candida albicans. In immunocompromised patients, this microorganism is responsible for developing systemic fungal infections (candidiasis). This is correlated with the use of a vast array of virulence factors, leading to the acquisition of specific resistance to host defense factors and available drug therapies. One of the most important features favoring the development of drug resistance is a C. albicans ability to form biofilms that protect fungal cells mainly through the production of an extracellular matrix (ECM). Among the main ECM-building macromolecules extracellular nucleic acids have been identified and their role is probably associated with the stbilization of the biofilm structure. The complex interactions of immune cells with the thick ECM layer, comprising the first line of contact between these cells and the biofilm structure, are still poorly understood. Therefore, the current studies aimed to assess the release of extracellular nucleic acids by C. albicans strains at different stages of biofilm formation, and to determine the role of these molecules in triggering the NETosis. We showed for the first time that fungal nucleic acids, purified directly from mature C. albicans biofilm structure or obtained from the whole fungal cells, have the potential to induce NET release in vitro. In this study, we considered the involvement of TLR8 and TLR9 in NETosis activation. We showed that DNA and RNA molecules initiated the production of reactive oxygen species (ROS) by activation of the NADPH oxidase complex, essential for ROS-dependent NETosis. Furthermore, analysis of the cell migration showed that the nucleic acids located in the extracellular space surrounding the biofilm may be also effective chemotactic factors, driving the dynamic migration of human neutrophils to the site of ongoing fungal infection.

The role of extracellular nucleic acids in the immune system modulation of Rhodnius prolixus (Hemiptera: Reduviidae).

Extracellular traps (ETs) are extracellular nucleic acids associated with cytoplasmic proteins that may aid in the capture and killing of pathogens. To date, only a few insects were shown to display this kind of immune response. Jaburetox, a peptide derived from jack bean urease, showed toxic effects in Rhodnius prolixus, affecting its immune response. The present study aims to evaluate the role of extracellular nucleic acids in R. prolixus' immune response, using Jaburetox as a model entomotoxin. The insects were treated with extracellular nucleic acids and/or Jaburetox, and the cellular and humoral responses were assessed. We also evaluated the release of extracellular nucleic acids induced by toxins, and performed immunocompetence assays using pathogenic bacteria. Our results demonstrated that extracellular nucleic acids can modulate the insect immune responses, either alone or associated with the toxin. Although RNA and DNA induced a cellular immune response, only DNA was able to neutralize the Jaburetox-induced aggregation of hemocytes. Likewise, the activation of the humoral response was different for RNA and DNA. Nevertheless, it was observed that both, extracellular DNA and RNA, immunocompensated the Jaburetox effects on insect defenses upon the challenge of a pathogenic bacterium. The toxin was not able to alter cellular viability, in spite of inducing an increase in the reactive species of oxygen formation. In conclusion, we have demonstrated a protective role for extracellular nucleic acids in R. prolixus´ immune response to toxins and pathogenic bacteria.

Extracellular nucleic acid scavenging rescues rats from sulfur mustard analog-induced lung injury and mortality.

Sulfur mustard (SM) is a highly toxic war chemical that causes significant morbidity and mortality and lacks any effective therapy. Rats exposed to aerosolized CEES (2-chloroethyl ethyl sulfide; 10% in ethanol), an analog of SM, developed acute respiratory distress syndrome (ARDS), which is characterized by increased inflammation, hypoxemia and impaired gas exchange. We observed elevated levels of extracellular nucleic acids (eNA) in the bronchoalveolar lavage fluid (BALF) of CEES-exposed animals. eNA can induce inflammation, coagulation and barrier dysfunction. Treatment with hexadimethrine bromide (HDMBr; 10 mg/kg), an eNA neutralizing agent, 2 h post-exposure, reduced lung injury, inhibited disruption of alveolar-capillary barrier, improved blood oxygenation (PaO2/FiO2 ratio), thus reversing ARDS symptoms. HDMBr treatment also reduced lung inflammation in the CEES-exposed animals by decreasing IL-6, IL-1A, CXCL-1 and CCL-2 mRNA levels in lung tissues and HMGB1 protein in BALF. Furthermore, HDMBr treatment also reduced levels of lung tissue factor and plasminogen activator inhibitor-1 indicating reduction in clot formation and increased fibrinolysis. Fibrin was reduced in BALF of the HDMBr-treated animals. This was further confirmed by histology that revealed diminished airway fibrin, epithelial sloughing and hyaline membrane in the lungs of HDMBr-treated animals. HDMBr completely rescued the CEES-associated mortality 12 h post-exposure when the survival rate in CEES-only group was just 50%. Experimental eNA treatment of cells caused increased inflammation that was reversed by HDMBr. These results demonstrate a role of eNA in the pathogenesis of CEES/SM-induced injury and that its neutralization can serve as a potential therapeutic approach in treating SM toxicity.

Extracellular RNA as a Versatile DAMP and Alarm Signal That Influences Leukocyte Recruitment in Inflammation and Infection.

Upon vascular injury, tissue damage, ischemia, or microbial infection, intracellular material such as nucleic acids and histones is liberated and comes into contact with the vessel wall and circulating blood cells. Such "Danger-associated molecular patterns" (DAMPs) may thus have an enduring influence on the inflammatory defense process that involves leukocyte recruitment and wound healing reactions. While different species of extracellular RNA (exRNA), including microRNAs and long non-coding RNAs, have been implicated to influence inflammatory processes at different levels, recent in vitro and in vivo work has demonstrated a major impact of ribosomal exRNA as a prominent DAMP on various steps of leukocyte recruitment within the innate immune response. This includes the induction of vascular hyper-permeability and vasogenic edema by exRNA via the activation of the "vascular endothelial growth factor" (VEGF) receptor-2 system, as well as the recruitment of leukocytes to the inflamed endothelium, the M1-type polarization of inflammatory macrophages, or the role of exRNA as a pro-thrombotic cofactor to promote thrombosis. Beyond sterile inflammation, exRNA also augments the docking of bacteria to host cells and the subsequent microbial invasion. Moreover, upon vessel occlusion and ischemia, the shear stress-induced release of exRNA initiates arteriogenesis (i.e., formation of natural vessel bypasses) in a multistep process that resembles leukocyte recruitment. Although exRNA can be counteracted for by natural circulating RNase1, under the conditions mentioned, only the administration of exogenous, thermostable, non-toxic RNase1 provides an effective and safe therapeutic regimen for treating the damaging activities of exRNA. It remains to be investigated whether exRNA may also influence viral infections (including COVID-19), e.g., by supporting the interaction of host cells with viral particles and their subsequent invasion. In fact, as a consequence of the viral infection cycle, massive amounts of exRNA are liberated, which can provoke further tissue damage and enhance virus dissemination. Whether the application of RNase1 in this scenario may help to limit the extent of viral infections like COVID-19 and impact on leukocyte recruitment and emigration steps in immune defense in order to limit the extent of associated cardiovascular diseases remains to be studied.

Marine Purple Photosynthetic Bacteria as Sustainable Microbial Production Hosts.

Photosynthetic microorganisms can serve as the ideal hosts for the sustainable production of high-value compounds. Purple photosynthetic bacteria are typical anoxygenic photosynthetic microorganisms and are expected to be one of the suitable microorganisms for industrial production. Purple photosynthetic bacteria are reported to produce polyhydroxyalkanoate (PHA), extracellular nucleic acids and hydrogen gas. We characterized PHA production as a model compound in purple photosynthetic bacteria, especially focused on marine strains. PHA is a family of biopolyesters synthesized by a variety of microorganisms as carbon and energy storage materials. PHA have recently attracted attention as an alternative to conventional petroleum-based plastics. Production of extracellular nucleic acids have been studied in Rhodovulum sulfidophilum, a marine purple non-sulfur bacterium. Several types of artificial RNAs have been successfully produced in R. sulfidophilum. Purple photosynthetic bacteria produce hydrogen via nitrogenase, and genetic engineering strategies have been investigated to enhance the hydrogen production. This mini review describes the microbial production of these high-value compounds using purple photosynthetic bacteria as the host microorganism.

Cell-Free Circulating Nucleic Acids as Early Biomarkers for NAFLD and NAFLD-Associated Disorders.

Non-alcoholic fatty liver disease (NAFLD) is the worldwide most common cause of chronic liver pathology, which prevalence strongly correlates with the increasing incidence of diabetes, obesity and metabolic syndrome in the general population. Simple steatosis, the earliest NAFLD stage, usually remains asymptomatic, and appropriate changes in the lifestyle, as well as the diet, can reverse the affected liver into the healthy state. The potential of simple steatosis to progress into severe fibrotic stages and to facilitate carcinogenesis necessitates timely NAFLD detection and risk stratification in community-based healthcare settings. Since their initial discovery a decade ago, extracellular circulating miRNAs have been found in all human biological fluids including blood and shown to hold great promises as non-invasive biomarkers. Normally, intracellular miRNAs participate in the regulation of gene expression, but once released by dying/dead cells they remain highly stable in the extracellular environment for prolonged periods. Therefore, circulating miRNA profiles can reflect the ongoing pathogenic processes in body's tissues and organs, and enable highly sensitive non-invasive diagnosis of multiple disorders. A non-urgent character of the NAFLD-related decision-making justifies the use of chronic liver diseases as an excellent test case for examining the practical utility of circulating miRNAs as biomarkers for longitudinal monitoring of human health. In this review, we summarize the state-of-the-art in the field of early diagnosis of NAFLD using circulating blood miRNAs, and stress the necessity of additional experimental validation of their diagnostic potential. We further emphasize on the potential diagnostics promises of other cell-free RNA species found in human biological fluids.

Human plasma and serum extracellular small RNA reference profiles and their clinical utility.

Circulating extracellular RNAs (exRNAs) have the potential to serve as biomarkers for a wide range of medical conditions. However, limitations in existing exRNA isolation methods and a lack of knowledge on parameters affecting exRNA variability in human samples may hinder their successful discovery and clinical implementation. Using combinations of denaturants, reducing agents, proteolysis, and revised organic extraction, we developed an automated, high-throughput approach for recovery of exRNAs and exDNA from the same biofluid sample. We applied this method to characterize exRNAs from 312 plasma and serum samples collected from 13 healthy volunteers at 12 time points over a 2-month period. Small RNA cDNA library sequencing identified nearly twofold increased epithelial-, muscle-, and neuroendocrine-cell-specific miRNAs in females, while fasting and hormonal cycle showed little effect. External standardization helped to detect quantitative differences in erythrocyte and platelet-specific miRNA contributions and in miRNA concentrations between biofluids. It also helped to identify a study participant with a unique exRNA phenotype featuring a miRNA signature of up to 20-fold elevated endocrine-cell-specific miRNAs and twofold elevated total miRNA concentrations stable for over 1 year. Collectively, these results demonstrate an efficient and quantitative method to discern exRNA phenotypes and suggest that plasma and serum RNA profiles are stable over months and can be routinely monitored in long-term clinical studies.

Extracellular nucleic acids of the marine bacterium Rhodovulum sulfidophilum and recombinant RNA production technology using bacteria.

Extracellular nucleic acids of high molecular weight are detected ubiquitously in seawater. Recent studies have indicated that these nucleic acids are, at least in part, derived from active production by some bacteria. The marine bacterium Rhodovulum sulfidophilum is one of those bacteria. Rhodovulumsulfidophilum is a non-sulfur phototrophic marine bacterium that is known to form structured communities of cells called flocs, and to produce extracellular nucleic acids in culture media. Recently, it has been revealed that this bacterium produces gene transfer agent-like particles and that this particle production may be related to the extracellular nucleic acid production mechanism. This review provides a summary of recent physiological and genetic studies of these phenomena and also introduces a new method for extracellular production of artificial and biologically functional RNAs using this bacterium. In addition, artificial RNA production using Escherichia coli, which is related to this topic, will also be described.

Pattern Recognition Receptors and Control of Innate Immunity: Role of Nucleic Acids.

The innate immune system protects against infectious microbes by the recognition of pathogen- associated molecular patterns, which serve to detect pathogens on the host cell surface or in endosomes by pattern recognition receptors such as Toll-like receptors, nucleotide-binding oligomerization domain-containing protein-1-like receptors, mannose-receptor, or retinoic acid-inducible gene-1- like receptors that initiate proper host defense mechanisms. In addition to pathogen-associated molecular patterns, a series of endogenous danger-associated molecular patterns, such as nucleic acids, are recognized by pattern recognition receptors, which serve as body´s own alarm signals under sterile conditions, such as ischemic injuries, trauma, tumors, tissue transplants, or autoimmune diseases. Thus, exogenous as well as endogenous nucleic acids can function as "alarmins" to alert the body about danger or disease by triggering inflammation, dendritic cell maturation, and stimulate the immune response resulting in the release of cytokines, which in turn can augment the local inflammatory environment. Moreover, danger-associated molecular patterns such as nucleic acids can act as cofactor in the activation of pattern recognition receptors in situations of cellular stress or upon infection leading to a massive amplification of the inflammatory response. As a consequence, acute and also chronic inflammatory diseases such as rheumatoid arthritis, cancer, or atherosclerosis may depend on such perpetuated proinflammatory responses involving activities of nucleic acids. As antagonists, RNase1 or DNase administration or nucleic acid complexing agents may result in a significant blockade of the outcome of particular pathological situations and in considerable tissue protection.

The gene transfer agent-like particle of the marine phototrophic bacterium Rhodovulum sulfidophilum.

Gene transfer agents (GTAs) are shaped like bacteriophage particles but have many properties that distinguish them from bacteriophages. GTAs play a role in horizontal gene transfer in nature and thus affect the evolution of prokaryotic genomes. In the course of studies on the extracellular production of designed RNAs using the marine bacterium Rhodovulum sulfidophilum, we found that this bacterium produces a GTA-like particle. The particle contains DNA fragments of 4.5 kb, which consist of randomly fragmented genomic DNA from the bacterium. This 4.5-kb DNA production was prevented while quorum sensing was inhibited. Direct observation of the particle by transmission electron microscopy revealed that the particle resembles a tailed phage and has a head diameter of about 40 nm and a tail length of about 60 nm. We also identified the structural genes for the GTA in the genome. Translated amino acid sequences and gene positions are closely related to those of the genes that encode the Rhodobacter capsulatus GTA. This is the first report of a GTA-like particle from the genus Rhodovulum. However, gene transfer activity of this particle has not yet been confirmed. The differences between this particle and other GTAs are discussed.