Investigation of Allogeneic Neutrophil Transfusion in Improving Survival Rates of Severe Infection Mice.

The management of granulocytopenia-associated infections is challenging, and a high mortality rate is associated with traditional supportive therapies. Neutrophils-the primary defenders of the human immune system-have potent bactericidal capabilities. Here, we investigated the dynamic in vivo distribution of neutrophil transfusion and their impact on the treatment outcome of severe granulocytopenic infections. We transfused 89Zr-labeled neutrophils in the C57BL/6 mice and observed the dynamic neutrophil distribution in mice for 24 h using the micro-positron emission tomography (Micro-PET) technique. The labeled neutrophils were predominantly retained in the lungs and spleen up to 4 h after injection and then redistributed to other organs, such as the spleen, liver, and bone marrow. Neutrophil transfusion did not elicit marked inflammatory responses or organ damage in healthy host mice. Notably, allogeneic neutrophils showed rapid chemotaxis to the infected area of the host within 1 h. Tail vein infusion of approximately 107 neutrophils substantially bolstered host immunity, ameliorated the inflammatory state, and increased survival rates in neutrophil-depleted and infected mice. Overall, massive allogeneic neutrophil transfusion had a therapeutic effect in severe infections and can have extensive applications in the future.

Stomatin promotes neutrophil degranulation and vascular leakage in the early stage after severe burn via enhancement of the intracellular binding of neutrophil primary granules to F-actin.

BACKGROUND: The pathophysiology of severe burn injuries in the early stages involves complex emergency responses, inflammatory reactions, immune system activation, and a significant increase in vascular permeability. Neutrophils, crucial innate immune cells, undergo rapid mobilization and intricate pathophysiological changes during this period. However, the dynamic alterations and detailed mechanisms governing their biological behavior remain unclear. Stomatin protein, an essential component of the cell membrane, stabilizes and regulates the membrane and participates in cell signal transduction. Additionally, it exhibits elevated expression in various inflammatory diseases. While Stomatin expression has been observed in the cell and granule membranes of neutrophils, its potential involvement in post-activation functional regulation requires further investigation. METHODS: Neutrophils were isolated from human peripheral blood, mouse peripheral blood, and mouse bone marrow using the magnetic bead separation method. Flow cytometry was used to assess neutrophil membrane surface markers, ROS levels, and phagocytic activity. The expression of the Stomatin gene and protein was examined using quantitative real-time polymerase chain reaction and western blotting methods, respectively. Furthermore, the enzyme-linked immunosorbent assay was used to evaluate the expression of neutrophil-derived inflammatory mediators (myeloperoxidase (MPO), neutrophil elastase (NE), and matrix metalloproteinase 9 (MMP9)) in the plasma. Images and videos of vascular leakage in mice were captured using in vivo laser confocal imaging technology, whereas in vitro confocal microscopy was used to study the localization and levels of the cytoskeleton, CD63, and Stomatin protein in neutrophils. RESULTS: This study made the following key findings: (1) Early after severe burn, neutrophil dysfunction is present in the peripheral blood characterized by significant bone marrow mobilization, excessive degranulation, and impaired release and chemotaxis of inflammatory mediators (MPO, NE, and MMP9). (2) After burn injury, expression of both the stomatin gene and protein in neutrophils was upregulated. (3) Knockout (KO) of the stomatin gene in mice partially inhibited neutrophil excessive degranulation, potentially achieved via reduced production of primary granules and weakened binding of primary granules to the cell skeleton protein F-actin. (4) In severely burned mice, injury led to notable early-stage vascular leakage and lung damage, whereas Stomatin gene KO significantly ameliorated lung injury and vascular leakage. CONCLUSIONS: Stomatin promotes neutrophil degranulation in the early stage of severe burn injury via increasing the production of primary granules and enhancing their binding to the cell skeleton protein F-actin in neutrophils. Consequently, this excessive degranulation results in aggravated vascular leakage and lung injury.

DNase aggravates intestinal microvascular injury in IBD patients by releasing NET-related proteins.

Neutrophils accumulate in the inflammatory mucosa of patients with inflammatory bowel disease (IBD), and excessive release of NETs (neutrophil extracellular traps may be one of the important factors that cause IBD progression. However, the specific mechanism underlying vascular injury caused by NETs remains unclear. Immunofluorescence, ELISA, and flow cytometry were used in this study to detect the expression of NETs and DNase in the tissue and peripheral blood samples of patients with IBD. DSS mouse model was used to detect colon injury and vascular permeability. We found that NETs and DNase levels increased in the colon of patients with IBD. We found an increase in the activity of NET-related MPO released by DNase. DNase released NET-related proteins and damaged vascular endothelial cells in vitro. In DSS mouse model, the synchronous increase of DNase and NETs in the colon leads to an increase in vascular injury markers (CD44, sTM). DNase aggravated colon injury and increased vascular permeability in vivo, which was inhibited by gentamicin sulfate (GS). GS does not reduce the expression of DNase, but rather reduces the release of NET-related proteins to protect vascular endothelium by inhibiting DNase activity. MPO and histones synergistically damaged the vascular endothelium, and vascular injury can be improved by their active inhibitors. We further found that H2 O2 is an important substrate for MPO induced vascular damage. In conclusion, in IBD, DNase, and NET levels increased synchronously in the lesion area and released NET-related proteins to damage the vascular endothelium. Therefore, targeting DNase may be beneficial for the treatment of IBD.

Prospective study and validation of early warning marker discovery based on integrating multi-omics analysis in severe burn patients with sepsis.

Background: Early detection, timely diagnosis and rapid response are essential for case management and precautions of burn-associated sepsis. However, studies on indicators for early warning and intervention have rarely been conducted. This study was performed to better understand the pathophysiological changes and targets for prevention of severe burn injuries. Methods: We conducted a multi-center, prospective multi-omics study, including genomics, microRNAomics, proteomics and single-cell transcriptomics, in 60 patients with severe burn injuries. A mouse model of severe burn injuries was also constructed to verify the early warning ability and therapeutic effects of potential markers. Results: Through genomic analysis, we identified seven important susceptibility genes (DNAH11, LAMA2, ABCA2, ZFAND4, CEP290, MUC20 and ENTPD1) in patients with severe burn injuries complicated with sepsis. Through plasma miRNAomics studies, we identified four miRNAs (hsa-miR-16-5p, hsa-miR-185-5p, hsa-miR-451a and hsa-miR-423-5p) that may serve as early warning markers of burn-associated sepsis. A proteomic study indicated the changes in abundance of major proteins at different time points after severe burn injury and revealed the candidate early warning markers S100A8 and SERPINA10. In addition, the proteomic analysis indicated that neutrophils play an important role in the pathogenesis of severe burn injuries, as also supported by findings from single-cell transcriptome sequencing of neutrophils. Through further studies on severely burned mice, we determined that S100A8 is also a potential early therapeutic target for severe burn injuries, beyond being an early warning indicator. Conclusions: Our multi-omics study identified seven susceptibility genes, four miRNAs and two proteins as early warning markers for severe burn-associated sepsis. In severe burn-associated sepsis, the protein S100A8 has both warning and therapeutic effects.

Neutrophil extracellular traps contribute to myofibroblast differentiation and scar hyperplasia through the Toll-like receptor 9/nuclear factor Kappa-B/interleukin-6 pathway.

Background: Inflammation is an important factor in pathological scarring. The role of neutrophils, one of the most important inflammatory cells, in scar hyperplasia remains unclear. The purpose of this article is to study the correlation between neutrophil extracellular traps (NETs) and scar hyperplasia and identify a new target for inhibiting scar hyperplasia. Methods: Neutrophils were isolated from human peripheral blood by magnetic-bead sorting. NETs in plasma and scars were detected by enzyme-linked immunosorbent assays (ELISAs), immunofluorescence and flow cytometry. Immunohistochemistry was used to assess neutrophil (CD66B) infiltration in hypertrophic scars. To observe the entry of NETs into fibroblasts we used immunofluorescence and flow cytometry. Results: We found that peripheral blood neutrophils in patients with hypertrophic scars were more likely to form NETs (p < 0.05). Hypertrophic scars showed greater infiltration with neutrophils and NETs (p < 0.05). NETs activate fibroblasts in vitro to promote their differentiation and migration. Inhibition of NETs with cytochalasin in wounds reduced the hyperplasia of scars in mice. We induced neutrophils to generate NETs with different stimuli in vitro and detected the proteins carried by NETs. We did not find an increase in the expression of common scarring factors [interleukin (IL)-17 and transforming growth factor-ß (TGF-ß), p > 0.05]. However, inhibiting the production of NETs or degrading DNA reduced the differentiation of fibroblasts into myofibroblasts. In vitro, NETs were found to be mediated by Toll-like receptor 9 (TLR-9) in fibroblasts and further phosphorylated nuclear factor Kappa-B (NF-κB). We found that IL-6, which is downstream of NF-κB, was increased in fibroblasts. Additionally, IL-6 uses autocrine and paracrine signaling to promote differentiation and secretion. Conclusions: Our experiments found that NETs activate fibroblasts through the TLR-9/NF-κB/IL-6 pathway, thereby providing a new target for regulating hypertrophic scars.

Primitive genotypic characteristics in umbilical cord neutrophils identified by single-cell transcriptome profiling and functional prediction.

The function and heterogeneity of neutrophils in neonatal umbilical cord blood (UCB) have not been characterized. In this study, we analyzed the neutrophils in UCB and healthy adults using single-cell RNA sequencing analysis for the first time. We found that neutrophils divided into six subpopulations (G2, G3, G4, G5a, G5b, and G5c) with different marker genes and different functions under homeostasis. Compared with healthy adults, neutrophils of UCB were more naïve and have more obvious degranulation and activation functions. Moreover, we found significant differences in the amount and function of G5b cells between healthy adults and UCB. The amount of G5b group in UCB was lower, but it has more degranulation, secretion and activation functions. In addition, we noted a new subset of G5c labeled by CD52, which almost did not exist in UCB. Besides, its differential genes were enriched in terms such as protein synthesis and mRNA transcription. Furthermore, uncharacteristic transcription factors ZNF-276, ZNF-319 and ZNF-354A were identified in our study. In summary, we first examined the heterogeneity and functional diversity of neutrophils in UCB, and these data provided new insights into the mechanism of neutrophil-mediated diseases of neonates and the wider use of neutrophils in UCB.

Remedial repair of extensive distal muscle secondary necrosis due to high level crush injury of the upper thighs: a case report.

Extensive necrosis of lower extremity muscles through the clinical practice of limb salvage after a case of high level crushing injury. A case of car accident injury complicated with necrotizing fasciitis, myositis, and septic shock was admitted to our hospital. The pathogenic factors, clinical characteristics, and surgical repair of this case were analyzed. Septic shock, pulmonary infection and atelectasis, and skin and soft tissue injury of lower limbs were all effectively treated, and limbs were saved successfully. After wound healing, the patient was discharged from hospital and entered the follow-up rehabilitation treatment. Although there is no direct trauma to the distal extremity below the cross-section of both lower extremities, there are still hypoxic ischemic changes, which can easily be ignored in the early stage. If not treated in time, myofascial and osteofascial compartment syndrome, necrotizing fasciitis, myositis, and sepsis are often secondary in the later stage, which should be warned against. During surgical debridement, attention should be paid to the protection of the source artery, and debridement and surgical exploration should be carried out according to the trend of blood vessels. The interecological muscle tissue between the intersections should be kept as far as possible, and the main nerves, blood vessels, and musculocutaneous perforators should be kept to ensure the blood supply of the skin flap.

A novel computer vision-based assessment of neutrophil chemotaxis in patients with severe infection.

OBJECTIVES: To evaluate the value of chemotactic function of neutrophils in patients with severe infections. METHODS: A computer vision-based cellular chemotaxis analysis platform was established for the dynamic assessment of neutrophil chemotaxis. Fifty-three patients in the intensive care unit were eligible for the study. In parallel, 142 healthy volunteers were recruited to detect and establish the normal values for chemotactic function. Four chemotactic function indicators were determined-chemotaxis distance (CD), chemotaxis cell ratio (CCR), chemotaxis index (CI) and maximum speed of chemotaxis (Vmax). The chemotaxis function scores (CFS) were calculated for further correlation analysis with clinical data. RESULTS: The normal ranges of indicators were established as CD ≥ 1755.85 µm, CCR ≥ 3.34%, CI ≥ 39.63, Vmax ≥ 14.63 µm min-1 and CFS ≥ 15. We found that the chemotactic function of neutrophils in patients suffering from infections was significantly impaired. The mean values of CD, CCR, CI, Vmax and CFS were 1452.8 µm (P < 0.0001), 3.1% (P < 0.0001), 34.5 (P < 0.0001), 12.2 µm min-1 (P < 0.0001) and 9 (P < 0.0001), respectively. CD and CFS were significantly negatively correlated with the APACHE II score (rCD = -0.55, rCFS = -0.39), SOFA score (rCD = -0.68, rCFS = -0.56), procalcitonin concentration (rCD = -0.60, rCFS = -0.5) and the expression of P2RX1 (rCD = -0.76, rCFS = -0.56), respectively. CONCLUSIONS: CD, CCR, CI and Vmax can well reflect the neutrophil chemotactic function in patients with severe infections. CFS systematically indicated neutrophil function and has promising clinical application prospects.

A narrative review of research progress on the relationship between hypoxia-inducible factor-2α and wound angiogenesis.

OBJECTIVE: This article aims to pay attention to the latest research on the expression, activation and function of hypoxia-inducible factor-2α (HIF-2α) under hypoxia and non-hypoxia conditions, and summarizes the current knowledge about the interaction between hypoxia-inducible factor-2 and angiogenesis, hoping to understand its actions in physiology and disease, with the goal of providing a new strategy for the diagnosis and treatment of wounds. BACKGROUND: Wound healing is a complex and continuous process, involving coagulation, inflammation, angiogenesis, new tissue formation and extracellular matrix remodeling. Of these, angiogenesis is an essential step. One of the main reasons for non-healing or delayed healing of wounds in peripheral vascular diseases and diabetes is the reduced ability to regenerate microvessels through the process of angiogenesis, which has become the focus of new methods for treating chronic wounds. HIF-2α regulates many aspects of angiogenesis, including vascular maturation, cell migration, proliferation and metastasis. METHODS: Throughout extensive search of PubMed, summarize the medical research on HIF-2α to 2020. CONCLUSIONS: HIF-2α is necessary for normal embryonic development by stimulating the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF). It is essential for the formation of new blood vessels in physiological and pathophysiological environments. Targeting HIF-2α in wound healing has much clinical significance for tissue repair.

Investigation and assessment of neutrophil dysfunction early after severe burn injury.

BACKGROUND: Extensive burn injury results in a complex immune response that is associated with mortality and prognosis. Studies on acquired immune and the development of sepsis in burn patients have been reported. However, one of the main cells in innate immune, neutrophil dysfunction in the burn shock stage has not been thoroughly characterized. METHODS: Neutrophil chemotaxis, expression of neutrophil surface markers (P2X1 receptor, [P2RX1]), degranulation (myeloperoxidase [MPO], heparin-binding protein [HBP], matrix metalloproteinase-9 [MMP-9] and neutrophil elastase [NE]), oxidative burst capacity, neutrophil extracellular trap (NET) generation, phagocytosis and apoptosis were measured in 18 patients with major burns (≥30% total body surface area [TBSA]) within 48 h after burn injury. In addition, circulating neutrophils and vascular permeability in mice model with 30% TBSA third-degree burns were also observed and investigated. RESULTS: Neutrophil functions were reduced considerably in burn shock stage, which was characterized by decreased chemotaxis, phagocytosis and abnormal bactericidal function. Increased release of heparin-binding protein (HBP) and the expression of P2RX1 on the neutrophil surface are related to fluid leakage and decreased chemotaxis during burn shock stage, respectively. The combination of HBP concentration in plasma and P2RX1 expression on neutrophils gives a better prediction of neutrophil dysfunction in burn-injured patients. CONCLUSION: Neutrophil dysfunction plays a key role in the development of burn injury. Targeting the restoration of neutrophil function may be a feasible therapeutic intervention to help reduce fluid loss during shock and the severity of subsequent infection.

Expression changes in protein-coding genes and long non-coding RNAs in denatured dermis following thermal injury.

OBJECTIVE: Thermal injury repair is a complex process during which maintaining the proliferation of human dermis fibroblasts (HDFs) and synthesis of extracellular matrix (ECM) plays a critical role. In the present study, we analyzed potential molecular markers and the probable association between differentially-expressed lncRNAs and protein-coding genes within denatured dermis following thermal injury, attempting to provide further insights to thermal injury repair pathogenesis. METHODS AND MAIN RESULTS: We found that the expression of 3940 lncRNAs was increased, while that of 1438 lncRNAs was reduced in the denatured dermis following thermal injury when compared to normal tissue. Of them, 338 were upregulated and 154 were downregulated by more than 128 times. Via cross-check with another microarray profile analysis on differentially-expressed lncRNAs after thermal injury, LINC00302 was found to be downregulated after thermal injury; more importantly, this skin-specially expressed lncRNA is located near a series of genes related to multiple skin inflammation and skin barrier-associated genomes. LINC00302 overexpression promoted the cell viability and the protein levels of α-SMA and Collagen I in HDFs. CONCLUSIONS: In conclusion, mRNAs and lncRNAs could be differentially expressed in the denatured dermis following thermal injury. mRNA and lncRNA regulatory signaling pathways could participate in thermal injury repair pathogenesis. More importantly, LINC00302 may play a critical role in thermal injury repair.