Release Of Neutrophil Research Articles

Baricitinib inhibits the activation of innate immune cells and exerts therapeutic effects on acute peritonitis and systemic inflammatory response syndrome

Timely treatment of acute inflammatory diseases induced by bacteria or fungi is essential to prevent infectious damage. Baricitinib is an inhibitor of Janus kinases (JAKs) which was approved to treat rheumatoid arthritis, atoptic dermatitis, and alopecia areata. It is also known that JAKs play important roles in innate immunity and inflammatory response. In this study, we investigated the regulatory effects of baricitinib on the activation of macrophages and neutrophils and its therapeutic effects on acute peritonitis and systemic inflammatory response syndrome (SIRS). In addition, we also studied its anti-inflammatory mechanisms by transcriptome and immunoblotting analyses. The results showed that baricitinib inhibited the expression and secretion of multiple inflammatory factors in macrophages induced by multiple Toll-like receptor (TLR) agonists. Baricitinib also moderately suppressed superoxide release of neutrophils stimulated by TLR agonists. In zymosan-induced acute peritonitis, baricitinib significantly reduced the infiltration of neutrophils into peritoneal cavity and the production of inflammatory factors in peritoneal cavity. In addition, baricitinib also slightly decreased the production of inflammatory factors in SIRS. Transcriptome analysis revealed that baricitinib markedly inhibited the mRNA transcription of many interferon-inducible genes, antiviral immune-related genes, transcription factors in JAKs-STATs signaling pathways, inflammatory factors, chemokines, colony-stimulating factor, and immunoglobulin receptors in macrophages induced by lipopolysaccharide (LPS). Furthermore, immunoblotting analysis showed that baricitinib selectively inhibited the phosphorylation of STAT1 and STAT3 in macrophages stimulated by LPS, and almost completely blocked the phosphorylation of STAT1 and STAT3 induced by IFN-γ and IL-6. Collectively, baricitinib can moderately inhibit the activation of macrophages and superoxide production of neutrophils, and exert anti-inflammatory effects by blocking JAKs-STATs signaling pathways, and has potential to be developed into therapeutic drug for acute inflammatory diseases, especially local ones.

BTK and MMP9 regulate NLRP3 inflammasome-dependent cytokine and NET responses in primary neutrophils.

Inflammation is a double-edged state of immune activation required to resolve threats harmful to the host but can also cause severe collateral damage. Polymorphonuclear neutrophils (PMN) the primary leukocyte population in humans, mediate inflammation through the release of cytokines and neutrophil extracellular traps (NETs). Whilst the pathophysiological importance of NETs is unequivocal, the multiple molecular pathways driving NET release are not fully defined. Recently, NET release was linked to the NLRP3 inflammasome which is regulated by Bruton's tyrosine kinase in macrophages. As NLRP3 inflammasome regulation by BTK has not been studied in neutrophils, we here explored a potential regulatory role of BTK in primary murine and human neutrophils and matched monocytes or macrophages from Btk-deficient vs WT mice or healthy donors (HD) vs BTK-deficient X-linked agammaglobulinemia (XLA) patients, respectively. Cytokine, MPO and MMP-9 release were quantified by ELISA, NET release and inflammasome formation by immunofluorescence microscopy. Surprisingly, in both mouse and human primary neutrophils, we observed a significant increase in NLRP3 inflammasome-dependent IL-1β and NETs when BTK was absent or inhibited, whereas IL-1β release was decreased in corresponding primary mouse macrophages or human PBMC, respectively. This suggests a novel negative regulatory role of BTK in terms of neutrophil NLRP3 activation. Both IL-1β and NET release in mouse and human primary neutrophils were strictly dependent of NLRP3, caspase-1 and, surprisingly, MMP-9. This highlights BTK and MMP-9 as novel and versatile inflammasome regulators and may have implications for the clinical use of BTK inhibitors.

Highly sensitive magnetic particle imaging of abdominal aortic aneurysm NETosis with anti-Ly6G iron oxide nanoparticles

Abdominal aortic aneurysms (AAA) are a significant health concern in developed countries due to their considerable mortality rate. The crucial factor of the progression of AAA is the release of neutrophils and neutrophil extracellular traps (NETs). Magnetic particle imaging (MPI) is a new imaging technique that offers the capability to detect superparamagnetic iron oxide nanoparticles (SPION) with exceptional sensitivity. We aimed to investigate the functional imaging of MPI for the detection and monitoring of neutrophil infiltration within AAA. A novel multimodal imaging agent targeting neutrophils, PEG-Fe3O4-Ly6G–Cy7 nanoparticles (Ly6G NPs), were designed by coupling Fe3O4 nanoparticles with Ly6G antibodies and Cy7. The targeting and sensitivity of Ly6G NPs were assessed using MPI and fluorescence imaging (FLI) in the AAA mouse model. After the inhibition of NETosis, the degree of neutrophil infiltration and AAA severity were assessed using MPI with Ly6G NPs. Ly6G NPs accurately localized and quantitatively analyzed AAA lesion sites in mice using MPI/FLI/CT. Compared to the control group, elevated MPI and FLI signal intensities were detected at the abdominal aortic lesion site, and neutrophil infiltration and NETs accumulation were detected by histological analysis in the AAA models. After the inhibition of NETs accumulation in vivo, pathological damage in the abdominal aorta was significantly reduced, along with a decrease in the accumulation of Ly6G NPs and MPI signals. This multimodal MPI strategy revealed that nanoparticles targeting Ly6G can be used to detect neutrophil infiltration within AAA and monitor AAA severity.

Unexplained fever with consumptive syndrome in the elderly: two cases of VEXAS syndrome with inflammasome dysregulation.

The aim of this study is to investigate the inflammasome dysregulation in peripheral blood leukocytes of VEXAS patients. The constitutive and in vitro triggered activation of inflammasome in PBMC and neutrophils was analyzed in two Brazilian patients with typical UBA1 mutations, and compared with healthy donors. Our findings highlight the constitutive activation of caspase-1 in VEXAS leukocytes, accompanied by increased plasma levels of IL-18. Furthermore, upon stimulation of isolated peripheral blood mononuclear cells (PBMC) and neutrophils, we observed not only the exhaustion of NLRP3 and NLRP1/CARD8 pathways in VEXAS PBMC but also a significant increase in NLRP3-mediated NETs release in VEXAS neutrophils. These findings support previous studies on the contribution of the inflammasome to VEXAS pathogenesis, identifying at least two profoundly affected pathways (NLRP3 and NLRP1/CARD8) in VEXAS peripheral blood.

Deficiency of hasB accelerated the clearance of Streptococcus equi subsp. Zooepidemicus through gasdermin d-dependent neutrophil extracellular traps

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus, SEZ) is an essential zoonotic bacterial pathogen that can cause various inflammation, such as meningitis, endocarditis, and pneumonia. UDP-glucose dehydrogenase (hasB) is indispensable in synthesizing SEZ virulence factor hyaluronan capsules. Our study investigated the infection of hasB on mice response to SEZ by employing a constructed capsule-deficient mutant strain designated as the ΔhasB strain. This deficiency was associated with a reduced SEZ bacterial load in the mice’s blood and peritoneal lavage fluid (PLF) post-infection. Besides, the ΔhasB SEZ strain exhibited a higher propensity for neutrophil infiltration and release of cell-free DNA (cfDNA) in vivo compared to the wild-type (WT) SEZ strain. In vitro experiments further revealed that ΔhasB SEZ more effectively induced the formation of neutrophil extracellular traps (NETs) containing histone 3 (H3), neutrophil elastase (NE), and DNA, than its WT counterpart. Moreover, the release of NETs was determined to be gasdermin D (GSDMD)-dependent during the infection process. Taken together, these findings underscore that the deficiency of the hasB gene in SEZ leads to enhanced GSDMD-dependent NET release from neutrophils, thereby reducing SEZ’s capacity to resist NETs-mediated eradication during infection. Our finding paves the way for the development of innovative therapeutic strategies against SEZ.

Effect of Oridonin on Experimental Animal Model of Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is a serious disease that occurs in premature and low-birth-weight infants. In recent years, the incidence of BPD has not decreased, and there is no effective treatment for it. Oridonin (Ori)is a traditional Chinese medicine with a wide range of biological activities, especially pharmacological and anti-inflammatory. It is well known that inflammation plays a key role in BPD. However, the therapeutic effect of Ori on BPD has not been studied. Therefore, in the present study, we will observe the anti-inflammatory activity of Ori in an experimental animal model of BPD. Here, we showed that Ori could significantly decrease hyperoxia-induced alveolar injury, inhibit neutrophil recruitment, myeloperoxidase concentrations, and release inflammatory factors in BPD neonatal rats. Taken together, the experimental results suggested that Ori can significantly improve BPD in neonatal rats by inhibiting inflammatory response.

Neutrophils cultured ex vivo from CD34+ stem cells are immature and genetically tractable

BackgroundNeutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation, emergency granulopoiesis leads to release of immature neutrophils from the bone marrow, serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood, partly due to a lack of genetically amenable models of neutrophil development.MethodsWe optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry, we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis, and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors.ResultsWe obtain high yields of ex vivo cultured neutrophils, which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation, cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins, as demonstrated by proteomic analysis.ConclusionEx vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.

Astilbin inhibited neutrophil extracellular traps in gouty arthritis through suppression of purinergic P2Y6 receptor

BackgroundGouty arthritis (GA), a common inflammatory condition triggered by monosodium urate crystal accumulation, often necessitates safer treatment alternatives due to the limitations of current therapies. Astilbin, a flavonoid from Smilax glabra Roxb, has demonstrated potential in traditional Chinese medicine for its anti-inflammatory properties. However, the anti-GA effect and its underlying mechanism have not been fully elucidated. PurposeThis study aimed to investigate the therapeutic potential of astilbin in GA, focusing on its effects on neutrophil extracellular traps (NETs), as well as the potential molecular target of GA both in vitro and in vivo. Study designFirstly, astilbin inhibited the citrullinated histone H3 (Cit h3) protein levels and reduced the NETs formation in neutrophils stimulated by monosodium urate (MSU). Secondly, we wondered the effect of astilbin on migration of neutrophils and dimethyl-sulfoxide (DMSO)-differentiated HL-60 (dHL-60) cells under the stimulation of MSU. Then, the effect of astilbin on suppressing NETs through purinergic P2Y6 receptor (P2Y6R) and Interlukin-8 (IL-8)/ CXC chemokine receptor 2 (CXCR2) pathway was investigated. Also, the relationship between P2Y6R and IL-8/CXCR2 was explored in dHL-60 cells under stimulation of MSU. Finally, we testified the effect of astilbin on reducing NETs in GA through suppressing P2Y6R and then down-regulating IL-8/CXCR2 pathway. MethodsMSU was used to induce NETs in neutrophils and dHL-60 cells. Real-time formation of NETs and migration of neutrophils were monitored by cell living imaging with or without MSU. Then, the effect of astilbin on NETs formation, P2Y6R and IL-8/CXCR2 pathway were detected by immunofluorescence (IF) and western blotting. P2Y6R knockdown dHL-60 cells were established by small interfering RNA to investigate the association between P2Y6R and IL-8/CXCR2 pathway. Also, plasmid of P2Y6R was used to overexpress P2Y6R in dHL-60 cells, which was employed to explore the role of P2Y6R in astilbin inhibiting NETs. Within the conditions of knockdown and overexpression of P2Y6R, migration and NETs formation were assessed by transmigration assay and IF staining, respectively. In vivo, MSU-induced GA mice model was established to assess the effect of astilbin on inflammation by haematoxylin-eosin and ELISA. Additionally, the effects of astilbin on neutrophils infiltration, NETs, P2Y6R and IL-8/CXCR2 pathway were analyzed by IF, ELISA, immunohistochemistry (IHC) and western blotting. ResultsUnder MSU stimulation, astilbin significantly suppressed the level of Cit h3 and NETs formation including the fluorescent expressions of Cit h3, neutrophils elastase, myeloperoxidase, and intra/extracellular DNA. Also, results showed that MSU caused NETs release in neutrophils as well as a trend towards recruitment of dHL-60 cells to MSU. Astilbin could markedly decrease expressions of P2Y6R and IL-8/CXCR2 pathway which were upregulated by MSU. By silencing P2Y6R, the expression of IL-8/CXCR2 pathway and migration of dHL-60 cells were inhibited, leading to the suppression of NETs. These findings indicated the upstream role of P2Y6R in the IL-8/CXCR2 pathway. Moreover, overexpression of P2Y6R was evidently inhibited by astilbin, causing a downregulation in IL-8/CXCR2 pathway, migration of dHL-60 cells and NETs formation. These results emphasized that astilbin inhibited the IL-8/CXCR2 pathway primarily through P2Y6R. In vivo, astilbin administration led to marked reductions in ankle swelling, inflammatory infiltration as well as neutrophils infiltration. Expressions of P2Y6R and IL-8/CXCR2 pathway were evidently decreased by astilbin and P2Y6R inhibitor MRS2578 either alone or in combination. Also, astilbin and MRS2578 showed notable effect on reducing MSU-induced NETs formation and IL-8/CXCR2 pathway whether used alone or in combination, parallelly demonstrating that astilbin decreased NETs formation mainly through P2Y6R. ConclusionThis study revealed that astilbin suppressed NETs formation via downregulating P2Y6R and subsequently the IL-8/CXCR2 pathway, which evidently mitigated GA induced by MSU. It also highlighted the potential of astilbin as a promising natural therapeutic for GA.

Circulating Neutrophil Profiles Undergo a Dynamic Shift during Metabolic Dysfunction-Associated Steatohepatitis (MASH) Progression.

Neutrophils play a crucial role in host defense against infection. Aberrant neutrophil activation may induce tissue damage via sterile inflammation. Neutrophil accumulation has been identified as a feature of the inflammatory response observed in metabolic dysfunction-associated steatohepatitis (MASH) and has been associated with liver fibrosis and cirrhosis. Here, we performed the transcriptomic analysis of circulating neutrophils from mild and advanced MASH patients to identify the potential mechanism behind neutrophil contribution to MASH progression. Our findings demonstrated that circulating neutrophils from mild and advanced MASH display an increased activated transcriptional program, with the expression of pro-inflammatory factors and an amplified lifespan compared to cells from non-diseased controls. Our results also suggest that MASH progression is associated with a dynamic shift in the profile of circulating neutrophils. In the early stages of MASH, mature neutrophils predominate in the bloodstream. As hepatic inflammation and fibrosis progress, the premature release of immature neutrophils into the circulation occurs. These immature neutrophils exhibit a pro-inflammatory profile that may exacerbate inflammation and promote fibrosis in MASH.

Perturbation of azurophilic granule integrity drives NLRP3-independent IL-1b processing and release in neutrophils

Abstract IL-1β is an inflammatory cytokine secreted by myeloid cells in response to infection or sterile tissue damage. Non-canonical secretion of IL-1β from monocytes downstream of activated NLRP3 inflammasomes is the best-characterized model; this is mediated by caspase-1 cleavage of GSDMD and N-GSDMD plasma membrane (PM) pore formation to permit release of IL-1β and induction of pyroptosis. NLRP3 is a cytosolic sensor of cellular homeostasis including [K+]. In monocytes, K+ efflux mediated NLRP3 activation is triggered by agents that disrupt lysosomal integrity. Neutrophils assemble competent NLRP3 inflammasomes and release IL-1β via GSDMD-dependent mechanisms, but they resist N-GSDMD PM pore formation and pyroptosis. We tested whether lysosome-disrupting stimuli in neutrophils would phenocopy the monocyte mechanism of NLRP3-dependent IL-1β release. Surprisingly, our data indicates that lysosome disruption induces release of mature IL-1β and lytic cell death in an NLRP3-independent manner. Kinetic analysis demonstrates an alternate mechanism of release such that early phase IL-1β release is NLRP3-dependent, but prolonged stimulation leads to NLRP3-independent release. Collectively, our data supports a signaling mechanism by which azurophilic granule disruption and cytosolic accumulation of neutrophil serine proteases disrupt canonical NLRP3 inflammasome assembly and directly cleave proIL-1β as part of a novel neutrophil-specific signaling mechanism of IL-1β processing and export.

The genetic architecture of complete blood counts in lactating Holstein dairy cows.

Complete blood counts (CBCs) measure the abundance of individual immune cells, red blood cells, and related measures such as platelets in circulating blood. These measures can indicate the health status of an animal; thus, baseline circulating levels in a healthy animal may be related to the productive life, resilience, and production efficiency of cattle. The objective of this study is to determine the heritability of CBC traits and identify genomic regions that are associated with CBC measurements in lactating Holstein dairy cattle. The heritability of CBCs was estimated using a Bayes C0 model. The study population consisted of 388 cows with genotypes at roughly 75,000 markers and 16 different CBC phenotypes taken at one to three time points (n = 33, 131, and 224 for 1, 2, and 3 time points, respectively). Heritabilities ranged from 0.00 ± 0.00 (red cell distribution width) to 0.68 ± 0.06 (lymphocytes). A total of 96 different 1-Mb windows were identified that explained more than 1% of the genetic variance for at least one CBC trait, with 10 windows explaining more than 1% of the genetic variance for two or more traits. Multiple genes in the identified regions have functions related to immune response, cell differentiation, anemia, and disease. Positional candidate genes include RAD52 motif-containing protein 1 (RDM1), which is correlated with the degree of immune infiltration of immune cells, and C-X-C motif chemokine ligand 12 (CXCL12), which is critically involved in neutrophil bone marrow storage and release regulation and enhances neutrophil migration. Since animal health directly impacts feed intake, understanding the genetics of CBCs may be useful in identifying more disease-resilient and feed-efficient dairy cattle. Identification of genes responsible for variation in CBCs will also help identify the variability in how dairy cattle defend against illness and injury.

Leukotriene B4-induced neutrophil extracellular traps impede the clearance of Pneumocystis.

Pneumocystis pneumonia (PCP) is a fungal pulmonary disease with high mortality in immunocompromised patients. Neutrophils are essential in defending against fungal infections; however, their role in PCP is controversial. Here we aim to investigate the effects of neutrophil extracellular traps (NETs) on Pneumocystis clearance and lung injury using a mouse model of PCP. Intriguingly, although neutrophils play a fundamental role in defending against fungal infections, NETs failed to eliminate Pneumocystis, but instead impaired the killing of Pneumocystis. Mechanically, Pneumocystis triggered Leukotriene B4 (LTB4)-dependent neutrophil swarming, leading to agglutinative NET formation. Blocking Leukotriene B4 with its receptor antagonist Etalocib significantly reduced the accumulation and NET release of neutrophils in vitro and in vivo, enhanced the killing ability of neutrophils against Pneumocystis, and alleviated lung injury in PCP mice. This study identifies the deleterious role of agglutinative NETs in Pneumocystis infection and reveals a new way to prevent NET formation, which provides new insights into the pathogenesis of PCP.

A Narrative Review: The Role of NETs in Acute Respiratory Distress Syndrome/Acute Lung Injury.

Nowadays, acute respiratory distress syndrome (ARDS) still has a high mortality rate, and the alleviation and treatment of ARDS remains a major research focus. There are various causes of ARDS, among which pneumonia and non-pulmonary sepsis are the most common. Trauma and blood transfusion can also cause ARDS. In ARDS, the aggregation and infiltration of neutrophils in the lungs have a great influence on the development of the disease. Neutrophils regulate inflammatory responses through various pathways, and the release of neutrophils through neutrophil extracellular traps (NETs) is considered to be one of the most important mechanisms. NETs are mainly composed of DNA, histones, and granuloproteins, all of which can mediate downstream signaling pathways that can activate inflammatory responses, generate immune clots, and cause damage to surrounding tissues. At the same time, the components of NETs can also promote the formation and release of NETs, thus forming a vicious cycle that continuously aggravates the progression of the disease. NETs are also associated with cytokine storms and immune balance. Since DNA is the main component of NETs, DNase I is considered a viable drug for removing NETs. Other therapeutic methods to inhibit the formation of NETs are also worthy of further exploration. This review discusses the formation and mechanism of NETs in ARDS. Understanding the association between NETs and ARDS may help to develop new perspectives on the treatment of ARDS.

The granulocyte colony-stimulating factor produced during Streptococcus suis infection controls neutrophil recruitment in the blood without affecting bacterial clearance.

Streptococcus suis causes diseases in pigs and has emerged as a zoonotic agent. When infected, the host develops an exacerbated inflammation that can lead to septic shock and meningitis. Although neutrophils greatly infiltrate the lesions, their dynamics during S. suis infection remain poorly described. Moreover, very few studies reported on the production and role of a key factor in the regulation of neutrophils: the colony-stimulating granulocyte factor (G-CSF). In this study, we characterized the G-CSF-neutrophil axis in the pathogenesis of S. suis induced disease. Using a mouse model of S. suis infection, we first evaluated the recruitment of neutrophils and their activation profile by flow cytometry. We found that infection provokes a massive neutrophil recruitment from the bone marrow to the blood and spleen. In both compartments, neutrophils displayed multiple activation markers. In parallel, we observed high systemic levels of G-CSF, with a peak of production coinciding with that of neutrophil recruitment. We then neutralized the effects of G-CSF and highlighted its role in the release of neutrophils from the bone marrow to the blood. However, it did not affect bacteremia nor the cytokine storm induced by S. suis. In conclusion, systemic G-CSF induces the release of neutrophils from the bone marrow to the blood, but its role in inflammation or bacterial clearance seems to be compensated by unknown factors. A better understanding of the role of neutrophils and inflammatory mediators could lead to better strategies for controlling the infection caused by S. suis.

Analysis of Changes in Variation of Neutrophil and Monocyte Parameters, Including Volume, Conductivity and Scatter in Sepsis Patients and Healthy Controls: A Cross-sectional Study

Introduction: Sepsis continues to be a leading cause of mortality and prolonged hospitalisation. The conventional method of blood culture, while considered the gold standard, has limitations such as contamination and delayed reporting. The examination of peripheral smears has uncovered signs suggestive of septicaemia; however, these findings suffer from inter-observer variability and reliance on staining quality. Aim: To investigate the variation of neutrophil and monocyte parameters, including Volume, Conductivity, and Scatter (VCS), in sepsis compared to healthy controls. Materials and Methods: A cross-sectional analytical study was conducted at the Himalayan Institute of Medical Sciences, Swami Rama Himalayan University in Dehradun, Uttarakhand, over the course of one year, from January 2021 to December 2021, involving patients over 18 years of age categorised into sepsis group based on clinical suspicion, sepsis screen, blood culture, and Sequential Organ Failure Assessment (SOFA) score (n=117). A group of healthy controls was also included (n=140). Haematological investigations were performed using the DXH 800 Haematology Analyser (Beckman Coulter, CA, USA) with VCS Technology. Categorical variables were analysed using the Chi-square test, while non parametric data was compared using the Mann-Whitney U test. Results: The average age in the sepsis group was 50.17±13.17 years, while in the control group, it was 38.14±8.78 years. The results revealed higher White Blood Cell (WBC) counts (16.76±7.39)×103 / cumm in the sepsis group compared to healthy controls (6.68±1.42)×103 , absolute neutrophil counts (13.74±7.280)×103 in sepsis patients, and eosinopenia in the sepsis group (0.0114±0.0104)×103 compared to controls (0.23±0.116)×103 . Moreover, mean neutrophilic volumes (158.00±14.840) and monocytic volumes (182.58±18.64) were higher in the sepsis group, while they were lower in healthy controls, which were (149.52±5.23 and 171.17±6.28), respectively. Axial light loss for neutrophil and monocyte was 142.40±11.78 and 121.50±17.93, respectively, while it was lower in healthy controls showing a value of 135.51±7.63 and 119.45±8.25, respectively. Furthermore, mean neutrophilic and monocytic conductivity and scatter were decreased in sepsis. The observed higher WBC counts and absolute neutrophil counts in sepsis patients suggest a premature release of neutrophils from the bone marrow. The alterations in cell volume reflect an immune response. Additionally, the overall scatter of neutrophils and monocytes was reduced, accompanied by increased cellular transparency. Conclusion: The present study contributes valuable insights into the pathophysiological mechanisms underlying sepsis, emphasising the dynamic interplay between immune cells and their functional characteristics. Understanding these variations in cellular parameters could potentially aid in the development of more targeted diagnostic and therapeutic approaches for sepsis, ultimately improving patient outcomes. Further research is warranted to delve deeper into the specific mechanisms driving these observed changes and to explore their clinical implications in the context of sepsis management.

Changes in the cellular composition of blood in horses in acute laminitis

Relevance. Equine laminitis is a multi-organ disease that can affect important systems of the horse’s body in the most neglected cases, but most often the hooves are affected, which in turn affects the economic component of the owner of the sick animal.Methods. The work was performed on two groups of horses. The first group included clinically healthy horses, the second group had clinical signs of laminitis. Blood for tests was taken in compliance with a starvation diet and the rules of asepsis and antiseptics from the jugular. The calculation of the leukocyte formula was carried out manually. The results were systematically evaluated and compared with each other to calculate the percentage ratio.Results. It was found that with laminitis there is a significant change in the cellular composition of the blood, in horses of the experimental group there is an increase in leukocytes (by about 302.5%), which may be associated with an inflammatory process in the body that triggers the active production of lymphocytes into the bloodstream. There is also an increase in segmented neutrophils when calculating the leukocyte formula (by 41.52%). This change is most likely caused by traumatic damage to the sole of the hoof. In this case, the increase in segmented neutrophils will be influenced by granulocyte colony stimulating factor (G-CSF), a hormone that stimulates the production and release of neutrophils from the bone marrow.

The RNA m6A demethylase ALKBH5 drives emergency granulopoiesis and neutrophil mobilization by upregulating G-CSFR expression.

Emergency granulopoiesis and neutrophil mobilization that can be triggered by granulocyte colony-stimulating factor (G-CSF) through its receptor G-CSFR are essential for antibacterial innate defense. However, the epigenetic modifiers crucial for intrinsically regulating G-CSFR expression and the antibacterial response of neutrophils remain largely unclear. N6-methyladenosine (m6A) RNA modification and the related demethylase alkB homolog 5 (ALKBH5) are key epigenetic regulators of immunity and inflammation, but their roles in neutrophil production and mobilization are still unknown. We used cecal ligation and puncture (CLP)-induced polymicrobial sepsis to model systemic bacterial infection, and we report that ALKBH5 is required for emergency granulopoiesis and neutrophil mobilization. ALKBH5 depletion significantly impaired the production of immature neutrophils in the bone marrow of septic mice. In addition, Alkbh5-deficient septic mice exhibited higher retention of mature neutrophils in the bone marrow and defective neutrophil release into the circulation, which led to fewer neutrophils at the infection site than in their wild-type littermates. During bacterial infection, ALKBH5 imprinted production- and mobilization-promoting transcriptome signatures in both mouse and human neutrophils. Mechanistically, ALKBH5 erased m6A methylation on the CSF3R mRNA to increase the mRNA stability and protein expression of G-CSFR, consequently upregulating cell surface G-CSFR expression and downstream STAT3 signaling in neutrophils. The RIP-qPCR results confirmed the direct binding of ALKBH5 to the CSF3R mRNA, and the binding strength declined upon bacterial infection, accounting for the decrease in G-CSFR expression on bacteria-infected neutrophils. Considering these results collectively, we define a new role of ALKBH5 in intrinsically driving neutrophil production and mobilization through m6A demethylation-dependent posttranscriptional regulation, indicating that m6A RNA modification in neutrophils is a potential target for treating bacterial infections and neutropenia.

L-Borneol promotes skin flap survival by regulating HIF-1α/NF-κB pathway

Ethnopharmacological relevanceThe clinical application of skin flaps in surgical reconstruction is frequently impeded by the occurrence of distant necrosis. L-Borneol exhibits myogenic properties in traditional Chinese medicine and is used in clinical settings to promote wound healing and conditions such as stroke. Nevertheless, the precise mechanism by which borneol exerts its protective effects on skin flap survival remains unclear. Aim of the studyTo explore the potential of L-borneol to promote skin flap survival and elucidate the underlying mechanisms. Materials and methodsThirty-six male Sprague–Dawley rats were randomly divided into three groups: a high-dose (200 mg/kg L-borneol per day), a low-dose (50 mg/kg/day), and control group (same volume of solvent). In each rat, a modified rectangular McFarlane flap model measuring 3 × 9 cm was constructed. Daily intragastric administration of L-borneol or solvent was performed. The flap was divided into three square sections of equal size, namely Zone I (the proximal zone), Zone II (the intermediate zone), and Zone III (the distal zone). The survival rate was quantified, and the histological state of each flap was evaluated on the seventh day following the surgical procedure. The assessment of angiogenesis was conducted using lead oxide/gelatin angiography, whereas the evaluation of blood flow in the free flap was performed using laser Doppler flow imaging. Superoxide dismutase activity was detected using the water-soluble tetrazolium salt-8 method. The quantities of vascular endothelial growth factor, interleukin (IL)-1β, IL-6, and tumour necrosis factor-α were determined using immunohistochemistry. The levels of nuclear transcription factor-κB, hypoxia-inducible factor-1, B-cell lymphoma-2 (BCL-2), and BCL-2-associated X (BAX) were determined by Western blotting technique. ResultsFlap survival rate significantly improved and neutrophil recruitment and release were enhanced after treatment with the compound. Angiogenesis was promoted. L-borneol protected against oxidative stress by increasing superoxide dismutase activity and decreasing malondialdehyde content. It downregulated the hypoxia-inducible factor nuclear transcription factor-κB pathway, leading to the inhibition of several inflammatory factors. Simultaneously, it facilitated the expression of vascular endothelial growth factor and BCL-2. ConclusionThe study shows that L-borneol may promote skin flap survival by inhibiting HIF-1α/NF-κB pathway.

KLF2 Regulates NLRP3 Inflammasome Priming and Activation in Murine Myeloid Cells

Introduction: Krüppel-like factor-2 (KLF2) is a critical nodal transcription factor and a tonic repressor of myeloid cell activation. Loss of KLF2 is associated with a pro-inflammatory and pro-thrombotic state. Mice deficient in myeloid-KLF2 (MK2KO) experience significantly higher mortality from lipopolysaccharide (LPS), and have increased serum and tissue levels of pro-inflammatory cytokines such as IL-1β. IL-1β is a product of NLRP3 inflammasome priming and activation, which has been implicated in numerous inflammatory and immune-mediated diseases. Neutrophils are the first responders of the innate immune system and are responsible for bacterial antigen detection. We thus aimed to elucidate the role of KLF2 in the regulation of NLRP3 inflammasome activation and subsequent IL-1β release in the neutrophil compartment. Methods: 8-week old adult MK2KO ( Lyz2Cre/ Klf2fl/fl) and control ( Lyz2Cre) mice were used for obtaining neutrophils from the bone marrow. Neutrophils were plated in RPMI-1640 media and stimulated with 10 ng/mL of LPS for 1 hour (h) and 2 h, followed by nigericin for 30 minutes. LPS acts as a priming agent for NLRP3 inflammasome assembly, and nigericin is a potassium ionophore which enables the activation step and subsequent IL-1β release. Supernatant was used for IL-1β ELISA and cell lysate was used for protein extraction and Western blotting to quantify NLRP3 protein expression. GraphPad Prism was used for statistical analyses. Results: Neutrophils from both MK2KO and Cre mice have significantly increased production of IL-1β when stimulated with LPS followed by nigericin (Figure 1), and loss of KLF2 leads to significantly increased IL-1B release from neutrophils. MK2KO neutrophils also have significantly more NLRP3 protein expression as compared to Cre neutrophils at baseline, and in response to LPS and nigericin (Figure 2). This increase in NLRP3 protein expression was time dependent and dose-dependent (Figure 2). Conclusion: KLF2 negatively regulates the transcriptional expression of NLRP3 and loss of KLF2 leads to increased NLRP3 inflammasome priming, at baseline and in response to LPS. Loss of KLF2 also affects the second step of the NLRP3 inflammasome pathway - the activation step, leading to increased IL-1β release in neutrophils in adult mice. Ongoing studies are examining the mechanisms underlying KLF2 control of NLRP3 transcription and other aspects of inflammasome activation. Inflammasome activation has been implicated in sepsis-related mortality due to the pro-inflammatory cytokine storm, and thus modulating KLF2 might help in developing novel targets to reduce this burden of mortality.

Caffeic acid modulates activation of neutrophils and attenuates sepsis-induced organ injury by inhibiting 5-LOX/LTB4 pathway

BackgroundSepsis is a critical systemic inflammatory syndrome which usually leads to multiple organ dysfunction. Caffeic acid (CA), a phenolic compound derived from various plants, has been proved to be essential in neuroprotection, but its role in septic organ damage is unclear. This research aimed to investigate whether CA protects against organ injury in a mouse model of cecal ligation and puncture (CLP). MethodsCA (30 mg/kg) or vehicle was administered by intraperitoneal injection immediately after CLP. The samples of blood, lungs, and livers were collected 24 h later. Organ injury was assessed by histopathological examination (HE staining), neutrophil infiltration (myeloperoxidase fluorescence), oxidative stress levels (MDA, SOD, HO-1), and inflammatory cytokines (TNF-α, IL-1β, and IL-6) release in lung and liver tissues. Neutrophil extracellular trap (NET) formation was analyzed by immunofluorescence. In vitro experiments were performed to investigate the potential mechanisms of CA using small interfering RNA (siRNA) techniques in neutrophils, and the effect of CA on neutrophil apoptosis was analyzed by flow cytometry. ResultsResults showed that CA treatment improved the 7-day survival rate and attenuated the histopathological injury in the lung and liver of CLP mice. CA significantly reduced neutrophil infiltration in the lungs and livers of CLP mice. TNF-α, IL-1β, IL-6 and LTB4 were reduced in serum, lung, and liver of CA-treated CLP mice, and phosphorylation of MAPK (p38, ERK, JNK) and p65 NF-κB was inhibited in lungs and livers. CA treatment further increased HO-1 levels and enhanced superoxide dismutase (SOD) activity, but reduced malondialdehyde (MDA) levels and NET formation. Similarly, in vitro experiments showed that CA treatment and 5-LOX siRNA interference inhibited inflammatory activation and NET release in neutrophils, suppressed MAPK and NF-κB phosphorylation in LPS-treated neutrophils, and decreased LTB4 and cfDNA levels. Flow cytometric analysis revealed that CA treatment reversed LPS-mediated delayed apoptosis in human neutrophils, and Western blot also indicated that CA treatment inhibited Bcl-2 expression but increased Bax expression. CA treatment did not induce further changes in neutrophil apoptosis, inflammatory activation, and NET release when 5-LOX was knocked down by siRNA interference. ConclusionsCA has a protective effect on lung and liver injury in a murine model of sepsis, which may be related to inhibition of the 5-LOX/LTB4 pathway.