DANGER Signals Activate G -Protein Receptor Kinases Suppressing Neutrophil Function and Predisposing to Infection After Tissue Trauma.

OBJECTIVE: Injured tissue predisposes the subject to local and systemic infection. We studied injury-induced immune dysfunction seeking novel means to reverse such predisposition. BACKGROUND: Injury mobilizes primitive "DANGER signals" [danger-associated molecular patterns (DAMPs)] activating innate immunocyte (neutrophils, PMN) signaling and function. Mitochondrial formyl peptides activate G -protein coupled receptors (GPCR) like formyl peptide receptor-1. Mitochondrial DNA and heme activate toll-like receptors (TLR9 and TLR2/4). GPCR kinases (GRKs) can regulate GPCR activation. METHODS: We studied human and mouse PMN signaling elicited by mitochondrial DAMPs (GPCR surface expression; protein phosphorylation, or acetylation; Ca 2+ flux) and antimicrobial functions [cytoskeletal reorganization, chemotaxis (CTX), phagocytosis, bacterial killing] in cellular systems and clinical injury samples. Predicted rescue therapies were assessed in cell systems and mouse injury-dependent pneumonia models. RESULTS: Mitochondrial formyl peptides activate GRK2, internalizing GPCRs and suppressing CTX. Mitochondrial DNA suppresses CTX, phagocytosis, and killing through TLR9 through a novel noncanonical mechanism that lacks GPCR endocytosis. Heme also activates GRK2. GRK2 inhibitors like paroxetine restore functions. GRK2 activation through TLR9 prevented actin reorganization, implicating histone deacetylases (HDACs). Actin polymerization, CTX, bacterial phagocytosis, and killing were also rescued, therefore, by the HDAC inhibitor valproate. Trauma repository PMN showed GRK2 activation and cortactin deacetylation, which varied with severity and was most marked in patients developing infections. Either GRK2 or HDAC inhibition prevented loss of mouse lung bacterial clearance, but only the combination rescued clearance when given postinjury. CONCLUSIONS: Tissue injury-derived DAMPs suppress antimicrobial immunity through canonical GRK2 activation and a novel TLR-activated GRK2-pathway impairing cytoskeletal organization. Simultaneous GRK2/HDAC inhibition rescues susceptibility to infection after tissue injury.

Investigation of the Presence of DNA in Human Blood Plasma Small Extracellular Vesicles.

Extracellular DNA (ecDNA) is DNA outside of cells, which is a result of various mechanisms. EcDNA is believed to be a cause of various pathogeneses as well as their potential biomarker. EcDNA is believed to also be part of small extracellular vesicles (sEVs) from cell cultures. If ecDNA is present in sEVs in plasma, their membrane may protect it from degradation by deoxyribonucleases. Moreover, sEVs play a role in the intercellular communication, and they can therefore transfer ecDNA between cells. The aim of this study was to investigate the presence of ecDNA in sEVs isolated from fresh human plasma by the ultracentrifugation and density gradient, which serves to exclude the co-isolation of non-sEVs compartments. The novelty of the current study is the investigation of the localization and subcellular origin of the ecDNA associated with sEVs in plasma, as well as the estimation of the approximate concentration. The cup-shaped sEVs were confirmed by transmission electron microscopy. The highest concentration of particles was in the size of 123 nm. The presence of the sEVs markers CD9 and TSG101 was confirmed by western blot. It was found that 60-75% of DNA is on the surface of sEVs, but a part of the DNA is localized inside the sEVs. Moreover, both nuclear and mitochondrial DNA were present in plasma EVs. Further studies should focus on the potential harmful autoimmune effect of DNA carried by plasma EVs or specifically sEVs.

The Effect of Melatonin on Periodontitis.

BACKGROUND: Periodontitis is a chronic disease with a complex etiology that includes bacterial colonization, excessive inflammation, and oxidative stress. The hormone melatonin has antioxidant properties and might contribute to alleviating chronic conditions by reducing oxidative stress. The aim of this study was to analyze the effect of exogenous melatonin on periodontitis in an animal model of the disease as well as in patients with periodontitis. METHODS: In rats with ligature-induced periodontitis, melatonin was administered in drinking water for two weeks. In the human study, patients with treatment-resistant periodontitis were asked to rinse their mouths with a solution containing melatonin or placebo every evening for two weeks. Periodontal status as well as salivary markers of oxidative stress were assessed at the end of the study. RESULTS: Neither radiography nor µCT revealed any significant effects of melatonin on alveolar bone loss. Gum recession was the only improved macroscopic measure in rats (p < 0.05). Analysis of salivary markers of oxidative stress revealed no effects of treatment in rats or humans despite clearly elevated melatonin concentrations in melatonin treated groups. CONCLUSION: Our results do not support the use of melatonin for the treatment of periodontitis. However, the negative outcome is limited by the short duration of the study and the chosen route of application as well as the dose of melatonin.

Urea and creatinine levels in saliva of patients with and without periodontitis.

Despite the fact that saliva contains measurable concentrations of urea and creatinine, it is not widely used in clinical nephrology. One of the reasons is the high inter- and intra-individual variability in the salivary markers of kidney function. We hypothesized that gingival bleeding in patients with periodontitis could contribute to this variability by increasing the concentration of salivary urea or creatinine. Samples were collected from 25 patients with periodontitis and 29 healthy controls. In addition, saliva samples from five healthy volunteers were artificially contaminated with blood. The concentration of urea, but not that of creatinine, was more than twice as high in patients with periodontitis than in controls. Artificial contamination of saliva with blood did not affect the salivary concentration of creatinine. Salivary urea increased only with very high levels of contamination (≥2.5% blood in saliva), but that did not occur in patients. In conclusion, periodontitis increases the concentration of salivary urea, but this is not likely to be a result of contamination with blood. Future studies should investigate the composition of the oral microbiome, specifically regarding how it affects the concentration of salivary urea. Salivary creatinine seems to be a more robust non-invasive marker of renal functions than salivary urea.

Exosomes-Associated DNA-New Marker in Pregnancy Complications?

Despite a large number of studies, the etiology of pregnancy complications remains unknown. The involvement of cell-free DNA or fetal cell-free DNA in the pathogenesis of pregnancy complications is currently being hypothesized. Cell-free DNA occurs in different forms-free; part of neutrophil extracellular traps; or as recently discovered, carried by extracellular vesicles. Cell-free DNA is believed to activate an inflammatory pathway, which could possibly cause pregnancy complications. It could be hypothesized that DNA in its free form could be easily degraded by nucleases to prevent the inflammatory activation. However, recently, there has been a growing interest in the role of exosomes, potential protectors of cell-free DNA, in pregnancy complications. Most of the interest from recent years is directed towards the micro RNA carried by exosomes. However, exosome-associated DNA in relation to pregnancy complications has not been truly studied yet. DNA, as an important cargo of exosomes, has been so far studied mostly in cancer research. This review collects all the known information on the topic of not only exosome-associated DNA but also some information on vesicles-associated DNA and the studies regarding the role of exosomes in pregnancy complications from recent years. It also suggests possible analysis of exosome-associated DNA in pregnancy from plasma and emphasizes the importance of such analysis for future investigations of pregnancy complications. A major obstacle to the advancement in this field is the proper uniformed technique for exosomes isolation. Similarly, the sensitivity of methods analyzing a small fraction of DNA, potentially fetal DNA, carried by exosomes is variable.

Sex, Age, and Bodyweight as Determinants of Extracellular DNA in the Plasma of Mice: A Cross-Sectional Study.

Extracellular DNA (ecDNA) is studied as a possible biomarker, but also as a trigger of the immune responses important for the pathogenesis of several diseases. Extracellular deoxyribonuclease (DNase) activity cleaves ecDNA. The aim of our study was to describe the interindividual variability of ecDNA and DNase activity in the plasma of healthy mice, and to analyze the potential determinants of the variability, including sex, age, and bodyweight. In this experiment, 58 adult CD1 mice (41 females and 31 males) of a variable age (3 to 16 months old) and bodyweight (females 25.7 to 52.1 g, males 24.6 to 49.6 g) were used. The plasma ecDNA was measured using a fluorometric method. The nuclear ecDNA and mitochondrial ecDNA were quantified using real-time PCR. The deoxyribonuclease activity was assessed using the single radial enzyme diffusion method. The coefficient of variance for plasma ecDNA was 139%, and for DNase 48%. Sex differences were not found in the plasma ecDNA (52.7 ± 73.0 ηg/mL), but in the DNase activity (74.5 ± 33.5 K.u./mL for males, and 47.0 ± 15.4 K.u./mL for females). There were no associations between plasma ecDNA and bodyweight or the age of mice. Our study shows that the variability of plasma ecDNA and DNase in adult healthy mice is very high. Sex, age, and bodyweight seem not to be major determinants of ecDNA variability in healthy mice. As ecDNA gains importance in the research of several diseases, it is of importance to understand its production and cleavage. Further studies should, thus, test other potential determinants, taking into account cleavage mechanisms other than DNase.

Anti-cytokine therapy and plasma DNA in patients with rheumatoid arthritis.

BACKGROUND: Extracellular DNA (ecDNA) is increased in inflammation and it also induces inflammation. In patients with rheumatoid arthritis (RA), plasma ecDNA is higher than in healthy controls. Due to low specificity, it cannot be used for screening, but it might be useful for monitoring and prognosis of therapy success. The effect of treatment with biological disease-modifying antirheumatic drugs (bDMARDs) on plasma ecDNA in RA patients with regards to its subcellular origin has not been analyzed yet. The aim of this study was to describe the effects of bDMARDs on plasma ecDNA and its nuclear (nDNA) and mitochondrial (mtDNA) fractions in patients with RA. METHODS: Plasma samples of 32 patients with RA were collected before, as well as 3 and 6 months after starting the treatment with bDMARDs. Total plasma ecDNA was quantified fluorometrically. The subcellular origin of ecDNA was assessed using real time PCR. Treatment success was monitored using DAS28 and C-reactive protein (CRP). RESULTS: The clinical status of patients improved. Both DAS28 and CRP decreased by 52 and 73% after 3 months of treatment. Plasma ecDNA decreased significantly only after 6 months (by 26%). Real-time PCR showed that both, nDNA and mtDNA decreased by 63 and by 45% after 6 months. CONCLUSION: Treatment with bDMARDs decreases plasma ecDNA of both nuclear and mitochondrial origin. Dynamics of ecDNA is slower than dynamics of standard clinical markers. Therefore, it is likely to be not useful for monitoring of the disease progress, at least for RA.

Mitochondria-induced formation of neutrophil extracellular traps is enhanced in the elderly via Toll-like receptor 9.

Neutrophil extracellular traps are potent antimicrobial weapons; however, their formation during sterile inflammation is detrimental, and the mechanism of induction is still unclear. Since advanced age is the primary clinical risk factor for poor outcomes in inflammatory diseases, we hypothesized that sterile stimuli, represented by mitochondria, would induce neutrophil extracellular trap formation in an age-dependent manner. Therefore, we analyzed induction of neutrophil extracellular traps in patients grouped according to age or immune status and observed that neutrophils from elderly patients responded to the presence of mitochondria with enhanced neutrophil extracellular trap formation. These neutrophil extracellular traps were also found to be more oxidized and exhibited higher resistance to DNase I degradation. Additionally, a higher concentration of residual neutrophil extracellular traps was detected in the plasma of the elderly. This plasma was capable of priming neutrophils through TLR9-mediated signaling, leading to further neutrophil extracellular trap formation, which was successfully inhibited with chloroquine. Finally, in a mouse model of mitochondria-induced acute lung injury, we observed that neutrophils from aged mice displayed impaired chemotactic activity but exhibited a trend of higher neutrophil extracellular trap formation. Thus, we propose that residual neutrophil extracellular traps circulating in the elderly preactivate neutrophils, making them more prone to enhanced neutrophil extracellular trap formation when exposed to mitochondria during sterile inflammation. Further investigation is needed to determine whether this vicious circle could be a suitable therapeutic target.

Severe gestational diabetes mellitus in lean dams is associated with low IL-1α levels and affects the growth of the juvenile mouse offspring.

We investigated how maternal gestational diabetes (GDM) impacts the metabolic status of offspring. GDM was induced in CD1 mice consuming a fast-food diet (FFD) by repeated low-dose streptozotocin injections before mating. Offspring of normoglycemic standard chow or the FFD consuming dams served as controls. In 4-week-old offspring weaned to standard chow, plasma concentrations of extracellular DNA, inflammatory markers, and parameters of the cardiometabolic status (glycemia, liver lipid content; body, organ, and fat weight) were determined. Two-factor analysis of variance indicated that the male offspring of GDM dams manifest postnatal growth retardation and lower relative kidney weight. Regardless of sex, GDM offspring manifest the lowest IL-1α levels, and other inflammatory markers showed mild and inconsistent alterations. Offspring of dams consuming the FFD displayed higher liver triacylglycerols content. The three groups of offspring showed no significant differences in glycemia and extracellular DNA. Partial least squares-discriminant analysis indicated that male GDM offspring present lower kidney, body, and brown adipose tissue weights; lower IL-1α levels, and higher concentrations of GM-CSF and IL-10 compared with their FFD counterparts. The model failed to select discriminative variables in females. In conclusion, in mice, maternal GDM in the absence of obesity adversely affects the early growth of juvenile male offspring.

Effective stabilization of electrochemically prepared ecological oxidizing agent-ferrate(VI)-by encapsulation in zeolite and its application to water containing SARS-CoV-2 virus.

Improper and insufficient treatment of infectious hospital wastewater could seriously endanger public health and the environment. Ferrate(VI), a strong oxidizing, disinfecting, and coagulating agent, has the potential as a green solution for decontamination of water and wastewater. In this paper, electrochemically prepared potassium ferrate (K2 FeO4 ) with high purity was successfully encapsulated and applied to the water contaminated by SARS-CoV-2. Natural zeolite was chosen as an appropriate ecological material for ferrate encapsulation. The stability of encapsulated ferrate (in tablet form) was monitored for an extended time period (290 days) and has significantly increased in contrast with free potassium ferrate by almost 30%. Subsequently, the K2 FeO4 encapsulated with zeolite in tablet form was applied to the water and municipal water samples containing the SARS-CoV-2 virus. The removal efficiency reached up to 98.5% and 86.7%, respectively, under natural conditions. Combination of environmentally friendly oxidizing agent and natural excellent adsorbent leads to the creation of very effective water treatment matter. These findings are essentially immediate and especially important for immediate water treatment in urgent situations such as natural disasters or military conflict. PRACTITIONER POINTS: Electrochemical preparation of oxidizing agent, K2 FeO4 , in high purity by own constructed electrolyzer. Encapsulation of ferrate(VI) to natural zeolite threefold improving the stability during 9 months. SARS-CoV-2 virus was successfully removed from various contaminated types of water. High degradation efficiency of virus fragments by Fe(VI) was achieved without additional water adjustment, in natural pH range.

Neutrophil extracellular traps formation and clearance is enhanced in fever and attenuated in hypothermia.

Fever and hypothermia represent two opposite strategies for fighting systemic inflammation. Fever results in immune activation; hypothermia is associated with energy conservation. Systemic Inflammatory Response Syndrome (SIRS) remains a significant cause of mortality worldwide. SIRS can lead to a broad spectrum of clinical symptoms but importantly, patients can develop fever or hypothermia. During infection, polymorphonuclear cells (PMNs) such as neutrophils prevent pathogen dissemination through the formation of neutrophil extracellular traps (NETs) that ensnare and kill bacteria. However, when dysregulated, NETs also promote host tissue damage. Herein, we tested the hypothesis that temperature modulates NETs homeostasis in response to infection and inflammation. NETs formation was studied in response to infectious (Escherichia coli, Staphylococcus aureus) and sterile (mitochondria) agents. When compared to body temperature (37°C), NETs formation increased at 40°C; interestingly, the response was stunted at 35°C and 42°C. While CD16+ CD49d+ PMNs represent a small proportion of the neutrophil population, they formed ~45-85% of NETs irrespective of temperature. Temperature increased formyl peptide receptor 1 (FPR1) expression to a differential extent in CD16+ CD49d- vs. CD49d+ PMNSs, suggesting further complexity to neutrophil function in hypo/hyperthermic conditions. The capacity of NETs to induce Toll-like receptor 9 (TLR9)-mediated NF-κB activation was found to be temperature independent. Interestingly, NET degradation was enhanced at higher temperatures, which corresponded with greater plasma DNase activity in response to temperature increase. Collectively, our observations indicate that NETs formation and clearance are enhanced at 40°C whilst temperatures of 35°C and 42°C attenuate this response. Targeting PMN-driven immunity may represent new venues for intervention in pathological inflammation.

Wastewater Sequencing-An Innovative Method for Variant Monitoring of SARS-CoV-2 in Populations.

The SARS-CoV-2 outbreak has already affected more than 555 million people, and 6.3 million people have died. Due to its high infectivity, it is crucial to track SARS-CoV-2 outbreaks early to prevent the spread of infection. Wastewater monitoring appears to be a powerful and effective tool for managing epidemiological situations. Due to emerging mutations of SARS-CoV-2, there is a need to monitor mutations in order to control the pandemic. Since the sequencing of randomly chosen individuals is time-consuming and expensive, sequencing of wastewater plays an important role in revealing the dynamics of infection in a population. The sampling method used is a crucial factor and significantly impacts the results. Wastewater can be collected as a grab sample or as a 24 h composite sample. Another essential factor is the sample volume, as is the method of transport used. This review discusses different pretreatment procedures and RNA extraction, which may be performed using various methods, such as column-based extraction, TRIzol, or magnetic extraction. Each of the methods has its advantages and disadvantages, which are described accordingly. RT-qPCR is a procedure that confirms the presence of SARS-CoV-2 genes before sequencing. This review provides an overview of currently used methods for preparing wastewater samples, from sampling to sequencing.

The dynamics of extracellular DNA associates with treatment response in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) as a chronic autoimmune inflammatory disease increases extracellular DNA (ecDNA). Our previous study has shown that anti-inflammatory treatment reduces ecDNA, but it is unclear whether there is an association with treatment response. The aim of this study was to analyze the changes of ecDNA induced by biological disease-modifying antirheumatic drugs (bDMARDs) in RA patients with an emphasis on the subcellular origin of ecDNA. Plasma samples from 40 RA patients were collected in three different time-points: before treatment with bDMARDs as well as 3 and 12 months following treatment initiation. Total, nuclear and mitochondrial ecDNA was quantified using fluorometry and real-time PCR. Disease activity score (DAS28) and C-reactive protein (CRP) were used to monitor the clinical status and the response to treatment. Treatment with bDMARDs elicited an overall improvement of the clinical status: DAS28 and CRP showed a significant decrease by 54% and 43%, respectively, after 3 months of treatment. A significant decrease of total ecDNA by 60% and nuclear ecDNA by 58% was detected only in good responders after 3 months of bDMARDs treatment. No significant changes of plasma ecDNA concentration were observed in moderate and non-responders. Deoxyribonuclease activity was not affected by the treatment. None of the analyzed biomarkers differed between the groups at baseline. Plasma ecDNA especially of nuclear origin could potentially be useful to monitor the treatment response in RA. Further studies should shed light on disease-treatment interplay implicated in ecDNA origin potentially linked to neutrophil extracellular traps.

Plasma and wound fluids from trauma patients suppress neutrophil extracellular respiratory burst.

BACKGROUND: Trauma increases susceptibility to secondary bacterial infections. The events suppressing antimicrobial immunity are unclear. Polymorphonuclear neutrophils (PMNs) migrate toward bacteria using chemotaxis, trap them in extracellular neutrophil extracellular traps, and kill them using respiratory burst (RB). We hypothesized that plasma and wound fluids from trauma patients alter PMN function. METHODS: Volunteer PMNs were incubated in plasma or wound fluids from trauma patients (days 0 and 1, days 2 and 3), and their functions were compared with PMNs incubated in volunteer plasma. Chemotaxis was assessed in transwells. Luminometry assessed total and intracellular RB responses to receptor-dependent and independent stimulants. Neutrophil extracellular trap formation was assessed using elastase assays. The role of tissue necrosis in creating functionally suppressive systemic PMN environments was assessed using a novel pig model where PMNs were incubated in uninjured pig plasma or plasma from pigs undergoing intraperitoneal instillation of liver slurry. RESULTS: Both plasma and wound fluids from trauma patients markedly suppress total PMN RB. Intracellular RB is unchanged, implicating suppression of extracellular RB. Wound fluids are more suppressive than plasma. Biofluids suppressed RB maximally early after injury and their effects decayed with time. Chemotaxis and neutrophil extracellular trap formation were suppressed by biofluids similarly. Lastly, plasma from pigs undergoing abdominal liver slurry instillation suppressed PMN RB, paralleling suppression by human trauma biofluids. CONCLUSION: Trauma plasma and wound fluids suppress RB and other key PMNs antimicrobial functions. Circulating suppressive signals can be derived from injured or necrotic tissue at wound sites, suggesting a key mechanism by which tissue injuries can put the host at risk for infection.

Ferrate (VI), Fenton Reaction and Its Modification: An Effective Method of Removing SARS-CoV-2 RNA from Hospital Wastewater.

The outbreak of the coronavirus disease 2019 (COVID-19) raises questions about the effective inactivation of its causative agent, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in medical wastewater by disinfectants. For this reason, our study of wastewater from a selected hospital evaluated several different advanced oxidation methods (Fenton reaction and Fenton-like reaction and ferrate (VI)) capable of effectively removing SARS-CoV-2 RNA. The obtained results of all investigated oxidation processes, such as ferrates, Fenton reaction and its modifications achieved above 90% efficiency in degradation of SARS-CoV-2 RNA in model water. The efficiency of degradation of real SARS-CoV-2 from hospital wastewater declines in following order ferrate (VI) > Fenton reaction > Fenton-like reaction. Similarly, the decrease of chemical oxygen demand compared to effluent was observed. Therefore, all of these methods can be used as a replacement of chlorination at the wastewater effluent, which appeared to be insufficient in SARS-CoV-2 removal (60%), whereas using of ferrates showed efficiency of up to 99%.

Role of Mitochondria-Derived Danger Signals Released After Injury in Systemic Inflammation and Sepsis.

Significance: Sepsis is a major public health concern, with high mortality and morbidity, especially among patients undergoing trauma. It is characterized by a systemic inflammatory response syndrome (SIRS) occurring in response to infection. Although classically associated with pathogens, many patients with SIRS do not have infection. The variability of the disease course cannot be fully explained by our current understanding of its pathogenesis. Thus, other factors are likely to play key roles in the development and progression of SIRS/sepsis. Recent Advances: Circulating levels of damage-associated molecular patterns (DAMPs) seem to correlate with SIRS/sepsis morbidity and mortality. Of the known DAMPs, those of mitochondrial (mt) origin have been of particular interest, since their DNA (mtDNA) and formyl peptides (mtFPs) resemble bacterial DNA and peptides, and hence, when released, may be recognized as "danger signals." Critical Issues: mtDAMPs released after tissue injury trigger immune responses similar to those induced by pathogens. Thus, they can result in systemic inflammation and organ damage, similar to that observed in SIRS/sepsis. We will discuss recent findings on the roles of mtDAMPs, particularly regarding the less recognized mtFPs, in the activation of inflammatory responses and development of SIRS/sepsis. Future Directions: There are no established methods to predict the course of SIRS/sepsis, but clinical studies reveal that plasma levels of mtDAMPs may correlate with the outcome of the disease. We propose that non-pathogen-initiated, mtDAMPs-induced SIRS/sepsis events need further studies aimed at early clinical recognition and better treatment of this disease.

Sorption of SARS-CoV-2 Virus Particles to the Surface of Microplastics Released during Washing Processes.

The research aims at washing processes as possible sources of microplastics, specifical microfibers in wastewater, and the behavior of the virus particles SARS-CoV-2 in wastewater after the washing process as well as their ability to sorb to the surface of microfibers, released from washing processes. The conclusions of the research point to the ability of the virus to attach to possible solid impurities such as textile fibers (microfibers) occurring in the sewer and to the ability of wash water to influence their possible occurrence in the sewer. The highest efficiency (more than 99%) of removal virus particles was after washing process, using liquid washing powder, and washing soda. These findings may gradually contribute to a better understanding of the behavior of the virus particles in the sewer.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Monocyte exocytosis of mitochondrial danger-associated molecular patterns in sepsis suppresses neutrophil chemotaxis.

BACKGROUND: Trauma and sepsis both increase the risk for secondary infections. Injury mobilizes mitochondrial (MT) danger-associated molecular patterns (mtDAMPs) directly from cellular necrosis. It is unknown, however, whether sepsis can cause active MT release and whether mtDAMPs released by sepsis might affect innate immunity. METHODS: Mitochondrial release from human monocytes (Mo) was studied after LPS stimulation using electron microscopy and using fluorescent video-microscopy of adherent Mo using Mito-Tracker Green (MTG) dye. Release of MTG+ microparticles was studied using flow cytometry after bacterial stimulation by size exclusion chromatography of supernatants with polymerase chain reaction (PCR) for mitochondrial DNA (mtDNA). Human neutrophil (PMN), chemotaxis, and respiratory burst were studied after PMN incubation with mtDNA. RESULTS: LPS caused Mo to release mtDAMPs. Electron microscopy showed microparticles containing MT. mtDNA was present both in microvesicles and exosomes as shown by PCR of the relevant size exclusion chromatography bands. In functional studies, PMN incubation with mtDNA suppressed chemotaxis in a dose-dependent manner, which was reversed by chloroquine, suggesting an endosomal, toll-like receptor-9-dependent mechanism. In contrast, PMN respiratory burst was unaffected by mtDNA. CONCLUSION: In addition to passive release of mtDAMPs by traumatic cellular disruption, inflammatory and infectious stimuli cause active mtDAMP release via microparticles. mtDNA thus released can have effects on PMN that may suppress antimicrobial function. mtDAMP-mediated "feed-forward" mechanisms may modulate immune responses and potentially be generalizable to other forms of inflammation. Where they cause immune dysfunction the effects can be mitigated if the pathways by which the mtDAMPs act are defined. In this case, the endosomal inhibitor chloroquine is benign and well tolerated. Thus, it may warrant study as a prophylactic antiinfective after injury or prior sepsis.