Polymorphonuclear Neutrophil Infiltration Research Articles

IRG1/ACOD1 Promotes Neutrophil Reverse Migration and Alleviates Local Inflammation.

Polymorphonuclear neutrophil (PMN) infiltration at inflammatory site plays a critical role in inflammation. PMN reverse migration (rM) describes the phenomenon that PMNs migrate away from inflammatory site back into the vasculature, and its role within inflammatory scenarios remains to be fully determined. This study aimed to investigate the mechanism underlying PMN rM and its role in inflammation. First, we demonstrated PMN rM in a mouse model of LPS-induced acute lung inflammation. By single-cell RNA sequencing (scRNA-seq), we demonstrated that reverse migrated (rM-ed) PMNs in blood expressed high level of immuneresponsive gene 1 (Irg1), the encoding gene of cis-aconitate decarboxylase (ACOD1). Using a mouse air pouch model, which enables us to directly track rM-ed PMNs in vivo, we detected higher expression of ACOD1 in the rM-ed PMNs in circulation. Furthermore, mice with Irg1 knockout exhibited decreased PMN rM and higher levels of inflammatory cytokine in inflammatory site. Mechanistically, we found that itaconate, the product of ACOD1 catalyzation, decreased PMN ICAM-1 expression at the inflammation site. Furthermore, inflammatory site showed a high level of shed CD11a, the ligand of ICAM-1. Neutralization of either ICAM-1 or CD11a leading to increased PMN rM. These findings suggest that the binding of ICAM-1 and shed CD11a serves as a retaining force to hold PMNs in the site of inflammation, and ACOD1-decreased PMN surface expression of ICAM-1 weakens the retaining force, so promoting PMNs to leave the inflammatory site. These results indicate a regulatory role of IRG1 in PMN rM and subsequent contributions to inflammation resolution.

Myeloperoxidase induces monocyte migration and activation after acute myocardial infarction.

Myocardial infarction (MI) is a significant contributor to morbidity and mortality worldwide. Many individuals who survive the acute event continue to experience heart failure (HF), with inflammatory and healing processes post-MI playing a pivotal role. Polymorphonuclear neutrophils (PMN) and monocytes infiltrate the infarcted area, where PMN release high amounts of the heme enzyme myeloperoxidase (MPO). MPO has numerous inflammatory properties and MPO plasma levels are correlated with prognosis and severity of MI. While studies have focused on MPO inhibition and controlling PMN infiltration into the infarcted tissue, less is known on MPO's role in monocyte function. Here, we combined human data with mouse and cell studies to examine the role of MPO on monocyte activation and migration. We revealed a correlation between plasma MPO levels and monocyte activation in a patient study. Using a mouse model of MI, we demonstrated that MPO deficiency led to an increase in splenic monocytes and a decrease in cardiac monocytes compared to wildtype mice (WT). In vitro studies further showed that MPO induces monocyte migration, with upregulation of the chemokine receptor CCR2 and upregulation of inflammatory pathways identified as underlying mechanisms. Taken together, we identify MPO as a pro-inflammatory mediator of splenic monocyte recruitment and activation post-MI and provide mechanistic insight for novel therapeutic strategies after ischemic injury.

Investigation of the rhythmic recruitment of tear neutrophils to the ocular surface and their phenotypes

Hundreds of thousands of polymorphonuclear neutrophils (PMNs) are collected from the ocular surface upon waking, while few are harvested during daytime. This study aimed to investigate potential factors contributing to the circadian infiltration of tear PMNs, including changes in IL-8 and C5a in tears, and their phenotypes across different time points in a 24-h cycle. Tear PMNs were collected using a gentle eyewash after 2-h and 7-h of sleep (eye closure, EC) at night, after 2-h EC during the day, and towards the end of the afternoon. Significantly fewer cells were collected after 2-h EC during the day compared to 2-h EC at night. A positive correlation between IL-8 and PMN numbers existed, but not with C5a. Tear PMNs collected after 2-h EC at night were less degranulated and possessed a larger activation potential compared to 7-h EC. Tear PMNs from 7-h EC at night exhibited hyper-segmented nuclei and more NETosis compared to 2 h EC night, indicating an aged and activated phenotype. The diurnal-nocturnal recruitment pattern of tear PMNs may be driven by increased IL-8 in nighttime tears. Higher degranulation and NETs point to the significant activation of tear PMNs on the ocular surface during prolonged eye closure at night.

Mesenchymal Stromal/Stem Cells Know Best: The Remarkable Complexities of Its Interactions With Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs), the predominant immune cell type in humans, have long been known as first-line effector cells against bacterial infections mainly through phagocytosis and production of reactive oxygen species (ROS). However, recent research has unveiled novel and pivotal roles of these abundant but short-lived granulocytes in health and disease. Human mesenchymal stromal/stem cells (MSCs), renowned for their regenerative properties and modulation of T lymphocytes from effector to regulatory phenotypes, exhibit complex and context-dependent interactions with PMNs. Regardless of species or source, MSCs strongly abrogate PMN apoptosis, a critical determinant of PMN function, except if PMNs are highly stimulated. MSCs also have the capacity to fine-tune PMN activation, particularly in terms of CD11b expression and phagocytosis. Moreover, MSCs can modulate numerous other PMN functions, spanning migration, ROS production, and neutrophil extracellular trap (NET) formation/NETosis, but directionality is remarkably dependent on the underlying context: in normal nondiseased conditions, MSCs enhance PMN migration and ROS production, whereas in inflammatory conditions, MSCs reduce both these functions and NETosis. Furthermore, the state of the MSCs themselves, whether isolated from diseased or healthy donors, and the specific secreted products and molecules, can impact interactions with PMNs; while healthy MSCs prevent PMN infiltration and NETosis, MSCs isolated from patients with cancer promote these functions. This comprehensive analysis highlights the intricate interplay between PMNs and MSCs and its profound relevance in healthy and pathological conditions, shedding light on how to best strategize the use of MSCs in the expanding list of diseases with PMN involvement.

Clearance of Neutrophils From ICH-Affected Brain by Macrophages Is Beneficial and Is Assisted by Lactoferrin and CD91.

Within hours after intracerebral hemorrhage (ICH) onset, masses of polymorphonuclear neutrophils (PMNs) infiltrate the ICH-affected brain. After degranulation involving controlled release of many toxic antimicrobial molecules, the PMNs undergo rapid apoptosis and then are removed by phagocytic microglia/macrophages (MΦ) through a process called efferocytosis. Effective removal of PMNs may limit secondary brain damage and inflammation; however, the molecular mechanisms governing these cleanup activities are not well understood. We propose that scavenger receptor CD91 on myeloid phagocytes especially in presence of CD91 ligand, LTF (lactoferrin, protein abundant in PMNs), plays an important role in clearance of dead apoptotic PMNs (ANs). Mice/rats were subjected to an autologous blood injection model of ICH. Primary cultured microglia were used to assess phagocytosis of ANs. Immunohistochemistry was employed to assess CD91 expression and PMN infiltration. CD91 knockout mice selectively in myeloid phagocytes (Mac-CD91-KO) were used to establish the CD91/LTF function in phagocytosis and in reducing ICH-induced injury, as assessed using behavioral tests, hematoma resolution, and oxidative stress. Masses of PMNs are found in ICH-affected brain, and they contain LTF. MΦ at the outer border of hematoma are densely packed, expressing CD91 and phagocytosing ANs. Microglia deficient in CD91 demonstrate defective phagocytosis of ANs, and mice deficient in CD91 (Mac-CD91-KO) subjected to ICH injury have increased neurological dysfunction that is associated with impaired hematoma resolution (hemoglobin and iron clearance) and elevated oxidative stress. LTF that normally ameliorates ICH injury in CD91-proficient control mice shows reduced therapeutic effects in Mac-CD91-KO mice. Our study suggests that CD91 plays a beneficial role in improving ANs phagocytosis and ultimately post-ICH outcome and that the beneficial effect of LTF in ICH is in part dependent on presence of CD91 on MΦ.

Reinforcement of colon anastomosis healing with leukocyte platelet-rich fibrin in rabbit model

AimThis study investigated the regenerative efficacy of leukocyte platelet-rich fibrin (L-PRF) on colon anastomotic healing in rabbits. Main methodsThirty-six healthy male white New Zealand rabbits were subjected to complete transactions of the ascending colon. The rabbits were equally divided into two groups: the control group, where the transected colon ends were anastomosed by a simple interrupted suture pattern, and the L-PRF-treated group, in which L-PRF was wrapped entirely around the anastomotic line. The postoperative acute pain scale was assessed using the Bristol Rabbit Pain Scale before surgery and at each four-hour interval post-operatively. After euthanizing the rabbits, the adhesion degree score, anastomotic bursting pressure, and stenosis degree of the anastomotic colon were assessed, and histopathological examination at the 7th, 14th, and 28th days postoperatively. Key findingsRabbits in both groups showed a significant increase in pain scores compared to baseline. Postoperatively, the L-PRF group exhibited significantly lower pain scores, adhesion scores, and stenosis degrees than the control group. However, the anastomotic bursting pressure was significantly higher in the L-PRF group. Re-epithelialization, polymorphonuclear neutrophil infiltration, granulation tissue formation, and collagen deposition scores were improved considerably in the L-PRF group compared to the control group. Immunostaining of growth factor expression was significantly lower in the control than in the L-PRF group. SignificanceThe L-PRF can augment collagen deposition, re-epithelialize the mucosa, promote angiogenesis, reduce adhesions, and diminish the stenosis degree scores. Therefore, it can be considered a promising aid in healing bowel anastomoses.

0.01% Hypochlorous Acid Treats Aspergillus fumigatus Keratitis in Rats by Reducing Fungal Load and Inhibiting the Inflammatory Response.

To investigate the antifungal and anti-inflammatory effects of 0.01% hypochlorous acid (HCLO) on rats with Aspergillus fumigatus keratitis. The time-kill assay and broth microdilution procedures were used in vitro to demonstrate that 0.01% HCLO was fungicidal and fungistatic. The severity of the disease was evaluated in vivo using a clinical score and slit-lamp photographs. Fungal load, polymorphonuclear neutrophil infiltration, and the production of related proteins were determined using colony plate counting, in vivo confocal microscopy, periodic acid-Schiff staining, fungal fluorescence staining, immunofluorescence staining, myeloperoxidase assay, and Western blotting. In vitro, 0.01% HCLO can destroy A. fumigatus spores in 1minute. The optical density of the 0.01% HCLO group was significantly lower than that of the phosphate-buffered saline control group (P < 0.01), and no visible mycelium was observed using a fluorescence microscope. 0.01% HCLO reduced the severity of A. fumigatus keratitis in rats by decreasing the clinical score, fungal loading (periodic acid-Schiff, plate count, and fungal fluorescence staining), and inhibiting neutrophil infiltration and activity (immunofluorescence staining and myeloperoxidase). Furthermore, the Western blot analysis revealed that 0.01% HCO decreased protein expression levels of tumor necrosis factor-α and IL-1β. According to our findings, 0.01% HCLO can kill A. fumigatus spores in vitro. It has antifungal and anti-inflammatory effects on A. fumigatus keratitis in rats. It also inhibited A. fumigatus growth; decreased neutrophil infiltration, tumor necrosis factor-α, and IL-1β expression; and provided a potential treatment for fungal keratitis. This study provides a potential treatment for fungal keratitis in the clinic.

Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis

BackgroundExcess polymorphonuclear neutrophil (PMN) recruitment or excessive neutrophil extracellular trap (NET) formation can lead to the development of multiple organ dysfunction during sepsis. M2 macrophage-derived exosomes (M2-Exos) have exhibited anti-inflammatory activities in some inflammatory diseases to mediate organ functional protection, but their role in treating sepsis-related acute lung injury (ALI) remains unclear. In this study, we sought to investigate whether M2-Exos could prevent potentially deleterious inflammatory effects during sepsis-related ALI by modulating abnormal PMN behaviours.MethodsC57BL/6 wild-type mice were subjected to a caecal ligation and puncture (CLP) mouse model to mimic sepsis in vivo, and M2-Exos were administered intraperitoneally 1 h after CLP. H&E staining, immunofluorescence and immunohistochemistry were conducted to investigate lung tissue injury, PMN infiltration and NET formation in the lung. We further demonstrated the role of M2-Exos on PMN function and explored the potential mechanisms through an in vitro coculture experiment using PMNs isolated from both healthy volunteers and septic patients.ResultsHere, we report that M2-Exos inhibited PMN migration and NET formation, alleviated lung injury and reduced mortality in a sepsis mouse model. In vitro, M2-Exos significantly decreased PMN migration and NET formation capacity, leading to lipid mediator class switching from proinflammatory leukotriene B4 (LTB4) to anti-inflammatory lipoxin A4 (LXA4) by upregulating 15-lipoxygenase (15-LO) expression in PMNs. Treatment with LXA4 receptor antagonist attenuated the effect of M2-Exos on PMNs and lung injury. Mechanistically, prostaglandin E2 (PGE2) enriched in M2-Exos was necessary to increase 15-LO expression in PMNs by functioning on the EP4 receptor, upregulate LXA4 production to downregulate chemokine (C-X-C motif) receptor 2 (CXCR2) and reactive oxygen species (ROS) expressions, and finally inhibit PMN function.ConclusionsOur findings reveal a previously unknown role of M2-Exos in regulating PMN migration and NET formation through lipid mediator class switching, thus highlighting the potential application of M2-Exos in controlling PMN-mediated tissue injury in patients with sepsis.

Melanoma-derived soluble mediators modulate neutrophil biological properties and the release of neutrophil extracellular traps

Polymorphonuclear neutrophils (PMNs) are the main effector cells in the inflammatory response. The significance of PMN infiltration in the tumor microenvironment remains unclear. Metastatic melanoma is the most lethal skin cancer with an increasing incidence over the last few decades. This study aimed to investigate the role of PMNs and their related mediators in human melanoma. Highly purified human PMNs from healthy donors were stimulated in vitro with conditioned media (CM) derived from the melanoma cell lines SKMEL28 and A375 (melanoma CM), and primary melanocytes as controls. PMN biological properties (chemotaxis, survival, activation, cell tracking, morphology and NET release) were evaluated. We found that the A375 cell line produced soluble factors that promoted PMN chemotaxis, survival, activation and modification of morphological changes and kinetic properties. Furthermore, in both melanoma cell lines CM induced chemotaxis, activation and release of neutrophil extracellular traps (NETs) from PMNs. In contrast, the primary melanocyte CM did not modify the biological behavior of PMNs. In addition, serum levels of myeloperoxidase, matrix metalloprotease-9, CXCL8/IL-8, granulocyte and monocyte colony-stimulating factor and NETs were significantly increased in patients with advanced melanoma compared to healthy controls. Melanoma cell lines produce soluble factors able to “educate” PMNs toward an activated functional state. Patients with metastatic melanoma display increased circulating levels of neutrophil-related mediators and NETs. Further investigations are needed to better understand the role of these “tumor-educated neutrophils” in modifying melanoma cell behavior.

FOXO3 Deficiency in Neutrophils Drives Colonic Inflammation and Tumorigenesis.

Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs' coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3389 presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

Dexamethasone's effect on endometrial inflammatory response in jennies after artificial insemination with frozen donkey semen

Artificial insemination (AI) with frozen donkey semen leads to low fertility rates in jennies. This might be due to an exacerbation of the acute inflammatory endometrial response to frozen semen, as seen in mares with persistent post breeding endometritis. Higher pregnancy rates were obtained in these mares when treated with dexamethasone at breeding time. The aim of this study was to evaluate the effect of dexamethasone on endometrial inflammatory responses in jennies after AI with frozen donkey semen. This study was performed from August to December 2022 in Argentina. Six healthy jennies of proven fertility were included in a crossover experimental design. All animals passed through an estrous cycle with no treatment before crossover between treatments. Jennies were subjected to 1) no AI and no treatment in estrus (control, C); 2) AI with no treatment (D-); and 3) AI with administration of dexamethasone, 50 mg IV at AI time (D+). Post ovulation. deep AI was performed with frozen donkey semen (700 × 106 progressively motile sperm (PMS). Endometrial samples (cytology and biopsies) were taken at estrus and 24 hours post AI. Cytology smears were stained with Diff-Quick™; 200 cells were counted under an optical microscope (Nikon Eclipse™ NI-U) and the percentage (%) of polymorphonuclear neutrophils (PMN) and eosinophils was calculated.Endometrial biopsies were taken from the bifurcation of the uterine body, fixed in 10% buffered formalin prior to hematoxylin-eosin staining. Five fields were selected per sample at 400x and average cell counts were calculated. The effect of treatments on % PMN and % eosinophils in cytology and biopsies were evaluated by the Kruskal Wallis test followed by the Dunn test for multiple comparisons. Values are expressed as median and interquartile ranges. The cytology smears of all animals showed an inflammatory response to semen deposition. There were differences (P = 0.003) between % PMN in C [0.625 (0.50 - 0.94)] and the other two groups [D-: 35.00 (22.50 - 46.00); D+: 39.50 (28.63 - 40.25)]. No eosinophils were observed. The inflammatory pattern in biopsies showed a diffuse infiltration of PMN and eosinophils in the luminal epithelium, stratum compactum and stratum spongiosum. There were differences (P = 0.003) between % PMN in C [0.30 (0.05 - 0.40) and the other two groups [D-: 41.90 (28.60 - 43.50); D+: 28.70 (10.35 - 49.00)]. Eosinophils were different (P = 0.005) for the three groups [C: 0.40 (0.20 - 1.20); D-: 21.50 (11.35 - 60.60); D+: 4.60 (1.30 - 7.75)]. In conclusion, a single dose of dexamethasone at AI in jennies did not decrease endometrial PMN but decreased eosinophils 24 hours after insemination.

Amniotic Mesenchymal-Derived Extracellular Vesicles and Their Role in the Prevention of Persistent Post-Breeding Induced Endometritis.

Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose-response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE.

Oxidative Stress Induces Bovine Endometrial Epithelial Cell Damage through Mitochondria-Dependent Pathways

Simple SummaryPolymorphonuclear neutrophil (PMN) count is the main diagnostic method of bovine endometritis. High neutrophil PMN counts in the endometrium of cows affected by endometritis suggest the involvement of oxidative stress among the causes of impaired fertility. The damage mechanism of oxidative stress on bovine endometrial epithelial cells (BEECs) is still unelucidated. The objective of this experiment was to investigate the relationship between oxidative stress and graded endometritis in dairy uteri and the molecular mechanism of oxidative stress injury to BEECs. Our research showed that there was an imbalance of antioxidant stress in dairy cow uterine with endometritis, oxidative stress damaged dairy cow endometrial epithelial cells through mitochondria-dependent pathways. These findings may provide new insight into the therapeutic target of bovine endometrial cell injury.Bovine endometritis is a mucosal inflammation that is characterized by sustained polymorphonuclear neutrophil (PMN) infiltration. Elevated PMN counts in the uterine discharge of dairy cows affected by endometritis suggest that oxidative stress may be among the causes of impaired fertility due to the condition. Nevertheless, the effects of oxidative stress-mediated endometritis in dairy cows largely remain uninvestigated. Therefore, fresh uterine tissue and uterine discharge samples were collected to diagnose the severity of endometritis according to the numbers of inflammatory cells in the samples. Twenty-six fresh uteri were classified into healthy, mild, moderate, and severe endometritis groups based on hematoxylin and eosin stain characteristics and the percentage of PMNs in discharge. BEECs were treated with graded concentrations of H2O2 from 50 μM to 200 μM in vitro as a model to explore the mechanism of oxidative stress during bovine graded endometritis. The expressions of antioxidant stress kinases were detected by quantitative fluorescence PCR to verify the oxidative stress level in uteri with endometritis. Reactive oxygen species were detected by fluorescence microscope, and inflammation-related mRNA expression increased significantly after H2O2 stimulation. Moreover, mRNA expression levels of antioxidant oxidative stress-related enzymes (glutathione peroxidase, superoxide dismutase, and catalase) and mitochondrial membrane potential both decreased. Further investigation revealed that expression of the apoptosis regulator Bcl-2/Bax decreased, whereas expression of the mitochondrial apoptosis-related proteins cytochrome c and caspase-3 increased in response to oxidative stress. Our results indicate that an imbalance exists between oxidation and antioxidation during bovine endometritis. Moreover, apoptosis induced in vitro by oxidative stress was characterized by mitochondrial damage in BEECs.

Helicobacter pylori-mediated gastric pathogenesis is attenuated by treatment of 2-deoxyglucose and metformin.

Helicobacter pylori infection causes chronic inflammation in the stomach, which is linked to the development of gastric cancer. The anti-inflammatory and anticancer effects of a glycolysis inhibitor 2-deoxyglucose (2DG) and an antidiabetic medication metformin (Met) have gotten attention. Using a Mongolian gerbil animal model, we investigated H. pylori-mediated gastric pathogenesis and how this pathogenesis is influenced by 2DG and Met. Five-week-old male gerbils were infected with H. pylori strain 7.13. After 2 weeks of infection, gerbils were fed 2DG-containing food (0.03% w/w), Met-containing water (0.5% w/v), or both (Combi) for 2 (short-term) or 10 weeks (long-term). Gastric pathogenesis and host response to H. pylori infection were examined by macroscopic and histopathologic analysis of gerbils' stomach. As a result, indicators of gastric pathogenesis by H. pylori infection including infiltration of polymorphonuclear neutrophils and lymphocytes, intestinal metaplasia, atrophy, and proliferation of gastric epithelial cells were attenuated by short-term administration of 2DG, Met, or Combi. When the infection was sustained for long-term, gastric pathogenesis in drug-treated gerbils was equivalent to that in untreated gerbils, with the exception that the infiltration of neutrophil was reduced by 2DG. Colonization of H. pylori in stomach was unaffected by both short- and long-term treatments. Our findings demonstrate that the progression of gastric pathogenesis induced by H. pylori infection can be attenuated by the short-term individual or combinational treatment of 2DG and Met, implying that 2DG or Met could be considered as a treatment option for gastric diseases in the early stages of infection.

Morphological changes without histological myocarditis in hearts of COVID-19 deceased patients

Objective. Patients with underlying heart diseases have a higher risk of dying from Covid-19. It has also been suggested that Covid-19 affects the heart through myocarditis. Despite the rapidly growing research on the management of Covid-19 associated complications, most of the ongoing research is focused on the respiratory complications of Covid-19, and little is known about the prevalence of myocarditis. Design. This study aimed to characterize myocardial involvement by using a panel of antibodies to detect hypoxic and inflammatory changes and the presence of SARS-CoV-2 proteins in heart tissues obtained during the autopsy procedure of Covid-19 deceased patients. Thirty-seven fatal COVID-19 cases and 21 controls were included in this study. Results. Overall, the Covid-19 hearts had several histopathological changes like the waviness of myocytes, fibrosis, contract band necrosis, infiltration of polymorphonuclear neutrophils, vacuolization, and necrosis of myocytes. In addition, endothelial damage and activation were detected in heart tissue. However, viral replication was not detected using RNA in situ hybridization. Also, lymphocyte infiltration, as a hallmark of myocarditis, was not seen in this study. Conclusion. No histological sign of myocarditis was detected in any of our cases; our findings are thus most congruent with the hypothesis of the presence of a circulating endothelium activating factor such as VEGF, originating outside of the heart, probably from the hypoxic part of the Covid-19 lungs.

Ginkgolide C Alleviates Acute Lung Injury Caused by Paraquat Poisoning via Regulating the Nrf2 and NF-κB Signaling Pathways.

Paraquat (PQ), a highly toxic herbicide and primary attack for lung, results in severe acute lung injury (ALI) appeared as evident oxidative stress, inflammation, and apoptosis. Increasing evidence elucidates that nuclear factor erythroid-2-related factor 2 (Nrf2) and its associated nuclear factor-κB (NF-κB) exhibit many merits for protection of ALI by coordinating a fine-turned response to oxidative stress, inflammation, and apoptosis. Ginkgolide C (GC) has been reported to be a safe and potent therapeutic agent against ALI. However, whether GC could protect ALI induced by PQ poisoning and the possible underlining mechanisms have remained not to be fully elucidated. A rat model of ALI and a model of acute type II alveolar epithelial cell (RLE-6TN) injury constructed by exposure to PQ were applied to discuss the protective effect of GC. Furthermore, Nrf2 gene silencing RLE-6TN cells were used to discuss the exact mechanism. We confirmed that GC significantly ameliorated the histopathological damages, ultrastructural changes, lung injury score, W/D ratio, and Hyp activity of lung tissue and inhibited polymorphonuclear neutrophil (PMN) infiltration after PQ poisoning. Further results revealed that GC remarkably activated Nrf2-based cytoprotective system and inhibited NF-κB-induced inflammatory injury as well as apoptosis. Taken together, we concluded that GC preserved protection of PQ-induced ALI via the Nrf2-NF-κB dependent signal pathway, which may provide us novel insights into the treatment strategies for PQ poisoning.

Myeloid-Specific Pyruvate-Kinase-Type-M2-Deficient Mice Are Resistant to Acute Lung Injury.

Infiltration of polymorphonuclear neutrophils (PMNs) plays a central role in acute lung injury (ALI). The mechanisms governing PMN inflammatory responses, however, remain incompletely understood. Based on our recent study showing a non-metabolic role of pyruvate kinase type M2 (PKM2) in controlling PMN degranulation of secondary and tertiary granules and consequent chemotaxis, here we tested a hypothesis that Pkm2-deficient mice may resist ALI due to impaired PMN inflammatory responses. We found that PMN aerobic glycolysis controlled the degranulation of secondary and tertiary granules induced by fMLP and PMA. Compared to WT PMNs, Pkm2-deficient (Pkm2-/-) PMNs displayed significantly less capacity for fMLP- or PMA-induced degranulation of secondary and tertiary granules, ROS production, and transfilter migration. In line with this, myeloid-specific Pkm2-/- mice exhibited impaired zymosan-induced PMN infiltration in the peritoneal cavity. Employing an LPS-induced ALI mouse model, LPS-treated Pkm2-/- mice displayed significantly less infiltration of inflammatory PMNs in the alveolar space and a strong resistance to LPS-induced ALI. Our results thus reveal that PKM2 is required for PMN inflammatory responses and deletion of PKM2 in PMN leads to an impaired PMN function but protection against LPS-induced ALI.

Некротичний ентероколіт у передчасно народжених дітей, які померли: причини, фактори ризику летальності, гістологічні зміни в стінці кишки

Despite advances in the diagnosis and treatment of necrotizing enterocolitis (NEC), the associated morbidity and mortality rates remain high. Purpose - to establish risk factors for mortality of necrotizing enterocolitis in preterm born infants, as well as to analyze histological changes of the intestinal lining. Materials and methods. The course of NEC in 21 preterm neonates who died of this disease (group 1, n=21) over a period of 3 years was analyzed. To establish risk factors for mortality rate health indicators of children in group 1 were compared with the course of NEC in children who survived with similar stages of the disease (group 2, n=43). The following research methods were used: general clinical, laboratory, instrumental, histological and statistical. Results. Our data show that the main causes of severe stages of NEC in preterm infants is infection, often in combination with severe asphyxia. The identified risk factors for mortality allowed to establish that the risk of death for children with NEC was associated with: male sex (OR=4.675; χ2=7.679; p=0.006) - increases the risk for mortality by 4 time; inflammatory changes in the placenta (OR=6.139; χ2=10.501; p=0.002) - increases the risk by 6 times; red blood cell transfusion in children (OR=8.262; χ2=8.557; p=0.004) - increases the risk by 8 times; thrombocytopenia (OR=4.320; χ2=4.866; p=0.028) - increases the risk by 4 time; the developmen of multiple organ system failure (OR=12.364; χ2=17.578; p&lt;0.001) and DIC syndrome (OR=10.725; χ2=14.592; p&lt;0.001) - increases the risk by 12 and 11 times, respectively; the positive symptoms - oedema of the anterior abdominal wall (OR=14.025; χ2=19.258; p&lt;0.001) and vasodilation of the anterior abdominal l wall (OR=5.333; χ2=5.444; p=0.02) - increases the risk by 14 and 5 times, respectively; the intestinal pneumatosis on abdominal when x-ray detected (OR=6.840; χ2=6.867; p=0.009) and the peritoneal effusion detected by abdominal ultrasound (OR=8.750; χ2=14.448; p&lt;0.001) - increases the risk of mortality by 7 and 9 times, respectively. During histological examination of the intestinal wall with NEC lymphohistiocytic infiltration of submucosa indicates perinatal hypoxia and its crucial role in the thanatogenesis of the disease, while polymorphonuclear segmental neutrophil infiltration is associated with perinatal infection. In 15 children (71.4%) changes of both types were noted, which indicates mixed etiology of intestinal lesions. Conclusions. Study results confirmed that necrotizing enterocolitis is a serious disease of newborns with a high mortality rate. The severe forms of NEC occur against the background of infection in combination with hypoxia. The obtained risk factors for the mortality rate of NEC allow to improve the prognosis of the course of this disease, will provide an opportunity to identify children who need increased attention of doctors to the treatment and further management of these patients with the use of preventive technologies that can prevent catastrophic consequences. The presence of congenital intestinal defects in combination with premature birth contribute to the development and aggravate the course of NEC, up to the development of stage III and a negative prognosis of the disease. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local ethics committee of all participating institutions. The informed consent of the parents of patient was obtained for conducting the studies. No conflict of interests was declared by the authors. Key words: necrotizing enterocolitis, preterm neonates, risk factors for mortality, histological changes.

Sevoflurane Dampens Acute Pulmonary Inflammation via the Adenosine Receptor A2B and Heme Oxygenase-1.

Acute respiratory distress syndrome is a life-threatening disease associated with high mortality. The adenosine receptor A2B (Adora2b) provides anti-inflammatory effects, which are also associated with the intracellular enzyme heme oxygenase-1 (HO-1). Our study determined the mechanism of sevoflurane’s protective properties and investigated the link between sevoflurane and the impact of a functional Adora2b via HO-1 modulation during lipopolysaccharide (LPS)-induced lung injury. We examined the LPS-induced infiltration of polymorphonuclear neutrophils (PMNs) into the lung tissue and protein extravasation in wild-type and Adora2b−/− animals. We generated chimeric animals, to identify the impact of sevoflurane on Adora2b of hematopoietic and non-hematopoietic cells. Sevoflurane decreased the LPS-induced PMN-infiltration and diminished the edema formation in wild-type mice. Reduced PMN counts after sevoflurane treatment were detected only in chimeric mice, which expressed Adora2b exclusively on leukocytes. The Adora2b on hematopoietic and non-hematopoietic cells was required to improve the permeability after sevoflurane inhalation. Further, sevoflurane increased the protective effects of HO-1 modulation on PMN migration and microvascular permeability. These protective effects were abrogated by specific HO-1 inhibition. In conclusion, our data revealed new insights into the protective mechanisms of sevoflurane application during acute pulmonary inflammation and the link between sevoflurane and Adora2b, and HO-1 signaling, respectively.

TNF-α Induces Neutrophil Apoptosis Delay and Promotes Intestinal Ischemia-Reperfusion-Induced Lung Injury through Activating JNK/FoxO3a Pathway.

Background Intestinal ischemia is a common clinical critical illness. Intestinal ischemia-reperfusion (IIR) leads to acute lung injury (ALI), but the causative factors of ALI are unknown. The aim of this study was to reveal the causative factors and mechanisms of IIR-induced lung injury. Methods A mouse model of IIR was developed using C57BL/6 mice, followed by detection of lung injury status and plasma levels of inflammatory factors in sham-operated mice and model mice. Some model mice were treated with a tumor necrosis factor-α (TNF-α) inhibitor lenalidomide (10 mg/kg), followed by observation of lung injury status through hematoxylin and eosin staining and detection of neutrophil infiltration levels through naphthol esterase and Ly6G immunohistochemical staining. Additionally, peripheral blood polymorphonuclear neutrophils (PMNs) were cultured in vitro and then stimulated by TNF-α to mimic in vivo inflammatory stimuli; this TNF-α stimulation was also performed on PMNs after knockdown of FoxO3a or treatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125. PMN apoptosis after stimulation was detected using flow cytometry. Finally, the role of PMN apoptosis in IIR-induced lung injury was evaluated in vivo by detecting the ALI status in the model mice administered with ABT-199, a Bcl-2 inhibitor. Results IIR led to pulmonary histopathological injury and increased lung water content, which were accompanied by increased plasma levels of inflammatory factors, with the TNF-α plasma level showing the most pronounced increase. Inhibition of TNF-α led to effective reduction of lung tissue injury, especially that of the damaging infiltration of PMNs in the lung. In vitro knockdown of FoxO3a or inhibition of JNK activity could inhibit TNF-α-induced PMN apoptosis. Further in vivo experiments revealed that ABT-199 effectively alleviated lung injury and decreased inflammation levels by promoting PMN apoptosis during IIR-induced lung injury. Conclusion TNF-α activates the JNK/FoxO3a pathway to induce a delay in PMN apoptosis, which promotes IIR-induced lung injury.