Polymorphonuclear Neutrophil Function Research Articles

Cellular Response of Immune Cells in the Upper Respiratory Tract After Treatment with Cold Atmospheric Plasma In Vitro.

Cold atmospheric plasma (CAP) has antimicrobial properties and is also known to stimulate the immune system. These properties could be useful for the development of a novel therapeutic or preventive strategy against respiratory infections in the upper respiratory tract (URT) such as ventilator-associated pneumonia (VAP) without inducing an immune overreaction. This study investigated the cellular responses of polymorphonuclear neutrophils (PMNs) after exposure to CAP in a three-dimensional (3D) model of the URT. In vitro experiments were conducted using PMNs isolated from human blood to assess cell migration, intracellular production of reactive oxygen species (ROS), NETosis, surface marker expression (CD11b, CD62L, and CD66b), and cell death with live cell imaging and flow cytometry. CAP was applied for 5 min using two distinct modalities: pressurized air plasma with a plasma intensive care (PIC) device and nebulized air plasma (NP) with a new humidity resistent surface microdischarge (SMD) plasma source, both developed by Terraplasma Medical GmbH. There were no significant signs of cell damage or overstimulation with either device under the conditions tested. However, the NP device caused milder effects on PMN functionality compared to the PIC device, but also demonstrated reduced antibacterial efficacy and reactive oxygen/nitrogen species (RONS) production, as analyzed with colorimetric/fluorimetric assay kits. These findings highlight a trade-off between the two CAP modalities, each with distinct advantages and limitations. Further studies are necessary to investigate these effects in the clinical setting and evaluate the long-term safety and efficacy of CAP treatment in the URT.

In-situ nanoplatform with synergistic neutrophil intervention and chemotherapy to prevent postoperative tumor recurrence and metastasis

In addition to residual tumor cells, surgery-induced inflammation significantly contributes to tumor recurrence and metastasis by recruiting polymorphonuclear neutrophils (PMNs) and promoting their involvement in tumor cell proliferation, invasion and immune evasion. Efficiently eliminating residual tumor cells while concurrently intervening in PMN function represents a promising approach for enhanced postoperative cancer treatment. Here, a chitosan/polyethylene oxide electrospun fibrous scaffold co-delivering celecoxib (CEL) and doxorubicin-loaded tumor cell-derived microparticles (DOX-MPs) is developed for postoperative in-situ treatment in breast cancer. This implant (CEL/DOX-MPs@CP) ensures prolonged drug retention and sustained release within the surgical tumor cavity. The released DOX-MPs effectively eliminate residual tumor cells, while the released CEL inhibits the function of inflammatory PMNs, suppressing their promotion of residual tumor cell proliferation, migration and invasion, as well as remodeling the tumor immune microenvironment. Importantly, the strategy is closely associated with interference in neutrophil extracellular trap (NET) released from inflammatory PMNs, leading to a substantial reduction in postoperative tumor recurrence and metastasis. Our results demonstrate that CEL/DOX-MPs@CP holds great promise as an implant to enhance the prognosis of breast cancer patients following surgery.

C/EBPε and its acetylation in PMN enhance the tolerance to trauma.

Severe trauma can lead to numerous serious complications, threating the well-being and vitality of the afflicted. The quantity and functionality of PMNs undergo rapid transformations in response to severe trauma, playing a pivotal role in the trauma response. The absence of CCAAT/enhancer-binding protein ε (C/EBPε) profoundly impairs the functionality of polymorphonuclear neutrophils (PMNs), a function of paramount importance in trauma. In this study, by generating mice with C/EBPε knocked out or overexpressed, we substantiate that C/EBPε ensures the restoration of PMN function, enhancing the expression of antimicrobial proteins and thereby promoting trauma recovery. Furthermore, diminished expression of C/EBPε is observed in trauma patients, with levels displaying a negative correlation with ISS and APACHE II scores, suggesting its potential as a prognostic indicator for clinical treatment. Mechanistically, we uncover the upregulation of SIRT1 and the inhibition of P300 participating in the suppression of C/EBPε acetylation, consequently reducing the resilience of mice to trauma. As therapeutic interventions, whether through the sole administration of PMN, NAM treatment, or their combination, all result in an increased survival rate in traumatic mice. In conclusion, our study elucidates the role of C/EBPε in enhancing the resilience to trauma and identifies C/EBPε acetylation as a critical regulatory mechanism, offering potential therapeutic approaches involving PMN transfusion and NAM treatment.

Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction

BackgroundThe function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals.ResultsBacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging.ConclusionsOur findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

Highly Motile Neutrophil Cytoplast ameliorate Bacterial Pneumonia

Abstract Polymorphonuclear neutrophils (PMN) function as innate immune cell through rapid mobilization at site of infection and prompt phagocytosis and bactericidal activity. We addressed in vivo in mouse lungs the function of cytoplasts (PMNcyt), the PMN generated following release of extracellular traps (NETs). We found ~50% of PMN lost their nucleus post-NETosis following pneumonia induced by Pseudomonas aeruginosa (PA), and become PMNcyt. Interestingly, , PMNcyt in lung microvessels showed >2 greater velocity and > 3 greater deformability as compared to PMN. PMNcyt were also 9x more efficient in killing airway PA as compared to PMN. Using the protein arginine deiminase 4 (PAD4) inhibitor (Cl-amidine) to prevent NET formation in mice, we prevented the generation of PMNcyt demonstrating that PMNcyt were derived post-vital NETosis. We also showed PMNcyt retained the ability to phagocytose PA. Moreover, the inhibition of PMNcyt generation by PAD4 blocker induced severe lung injury in the pneumonia model, indicating an important anti-bacterial protective role of PMNcyt. Adoptive transfer of PMNcyt in PA infected mice also significantly reduced bacterial load and prevented lung tissue injury in mice. The results suggest a crucial host-defense function of PMNcyt generated post vital NETosis and their usefulness as cell-based therapy in inflammatory diseases such as pneumonia.

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.

Impaired polymorphonuclear neutrophil functions in diabetics

Background and objectives: Polymorphonuclear neutrophils (PMN) are the first line of host resistance against infections. Diabetics are prone to both bacterial and fungal infections. The present study evaluated the phagocytic and killing activity of PMN in diabetics. Material and methods: Females aged 30 to 50 years with and without diabetes mellitus were enrolled. Functions of PMN were assessed by determining the phagocytic rate, phagocytic index and killing of C. albicans by PMN. Results: A total of 36 diabetic patients and 15 age matched non-diabetic healthy individuals were enrolled. Phagocytosis and killing of C. albicans by PMN were significantly (p<0.05) lower in patients with diabetes mellitus compared to non-diabetic healthy individuals (86.5±14.6 vs. 94.5±4.2; 56.7±23.8 vs. 81.5±24.2). Conclusion: Phagocytic and killing functions of PMN were significantly reduced in patients with diabetes mellitus. IMC J Med Sci. 2024; 18(1):008. DOI: https://doi.org/10.55010/imcjms.18.008 *Correspondence:Tanzinah Nasrin, Microbiologist, Quality Control Laboratory, Department of Fisheries, Ministry of Fisheries and Livestock, Dhaka, Bangladesh. Email: tanzinahn8@gmail.com

Obesity Correlates with Chronic Inflammation of the Innate Immune System in Preeclampsia and HELLP Syndrome during Pregnancy.

HELLP syndrome is characterized by hemolysis, elevated liver enzymes, and a low platelet count and poses an increased risk to the pregnant woman and the unborn child. Individual risk factors such as obesity may alter immunocompetence and influence the course of preeclampsia (PE) or HELLP syndrome. Blood samples were collected from 21 pregnant women (7 healthy, 6 with PE, and 8 with HELLP syndrome) and polymorphonuclear neutrophils (PMNs) were subsequently isolated. Production of radical oxygen species (ROS), cell movement, and NETosis were assessed by live-cell imaging. Surface protein expression and oxidative burst were analyzed by flow cytometry. PE and HELLP patients had significantly higher BMI compared to the healthy control group. Depending on the expression of CD11b, CD62L, and CD66b on PMNs, a surface protein activation sum scale (SPASS) was calculated. PMNs from patients with high SPASS values showed prolonged and more targeted migration with delayed ROS production and NETosis. Obesity is associated with a chronic inflammatory state, which in combination with immunological triggers during pregnancy could modulate PMN functions. Pregnant women with higher BMI tend to have higher SPASS values, indicating activation of the innate immune system that could co-trigger PE or HELLP syndrome.

Phagocyte population in the inflammatory pattern of the bronchi of asthma patients regulated by interleukin-17A and interferon-gamma during the airway response to a cold stimulus

Introduction. Based on the common effector functions of polymorphonuclear neutrophils and macrophages as phagocytic cells, their role in the formation of an acute reaction of the respiratory tract to a cold stimulus in patients with asthma is of concern.Aim. The study of the concentration of phagocytes, IL-17A and IFN-γ in the inflammatory pattern of the bronchi of asthma patients depending on the airway reaction to a cold stimulus.Materials and methods. 129 patients with asthma were examined. The design of the study included questioning patients using a validated questionnaire Asthma Control Test (ACT, Quality Metric Inc., 2002), collection of induced and spontaneously produced sputum, exhaled breath condensate (EBC), bronchoprovocation test with a 3-minute isocapnic hyperventilation with cold (-20ºС) air (IHCA) with an assessment of the airway response (ΔFEV1) by spirometry.Results. Group 1 (n=55) included individuals with ΔFEV1 -10% and below, group 2 (n=74) – with ΔFEV1 above -10%: -15 (-21; -11) and -3.7 (-6.1; -0.38)%, respectively (p=0.0002). According to the level of ACT (17 [13; 21.5] and 19 [14; 22] points) and indicators of lung function (FEV1 [93.0±2.4 and 97.1±2.4%] and FEF25-75 [63.5±3.5 and 72.0±3.7%]), the patients had no significant intergroup differences. The pattern of bronchial inflammation in group 1 was mixed (neutrophils ≥40%), in group 2 – eosinophilic. In response to the IHCA test, the number of neutrophils significantly increased in the sputum of patients in group 1, the number of macrophages and the number of structurally intact epithelial cells decreased, in proportion to this, the level of IFN-γ and IFN-γ-inducible protein IP-10 (CXCL10) increased in the EBC in relation to patients of the 2nd group. A direct relationship was found between baseline concentrations of IP-10 and IFN-γ (Rs=0.7; p<0.01) in EBC.Conclusion. The airway response to a cold stimulus of patients with asthma is accompanied by functional activation of phagocytic cells with an escalation of neutrophilic inflammation and a decrease in the number of macrophages infiltrating the bronchi associated with an increase in the concentration of IFN-γ, which stimulates the processes of respiratory burst and triggers cell destruction and cytolysis.

The Effect of Local Anesthetics on Neutrophils in the Context of Different Isolation Techniques.

Various functions of polymorphonuclear neutrophils (PMNs) are related to diseases and postoperative plasma changes. The influence of some local anesthetics (LAs) on PMNs obtained by conventional isolation methods and their functions has already been demonstrated. This study investigates the effect of selected LAs on PMNs, comparing a new isolation method with conventional ones. To obtain the PMNs, we performed either gelafundin sedimentation, hypotonic lysis or density gradient centrifugation. Subsequently, PMNs were mixed with different concentrations of bupivacaine, levobupivacaine, lidocaine or ropivacaine. Live cell imaging and flow cytometry were performed to quantify the migration, ROS production, NETosis and antigen expression of PMNs. We found the inhibition of chemotaxis and ROS production by LAs. PMNs showed a strong reduction in time to half maximal NETosis in response to bupivacaine and lidocaine, but not to levobupivacaine and ropivacaine. We also found distinct differences in survival time and migration duration between the isolation methods. This suggests that the careful selection of LAs has a short-term impact on in vitro PMNs.

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.

Recombinant GM-CSF enhances the bactericidal ability of PMNs by increasing intracellular IL-1β and improves the prognosis of secondary Pseudomonas aeruginosa pneumonia in sepsis.

This study tested the hypothesis that recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances polymorphonuclear neutrophils (PMNs) via interleukin (IL)-1β to improve the prognosis of secondary infection in sepsis. The latter stage of sepsis is prone to induce immunosuppression, resulting in secondary fatal infections. Recombinant GM-CSF has become a way for sepsis-induced immunosuppression due to its immunomodulatory effect. However, the functional impact of GM-CSF on PMNs in sepsis remains obscure. This study aimed to study the role of recombinant GM-CSF on the bactericidal ability of PMNs in septic mice, assessing its effect on the prognosis of secondary pneumonia, and explore the mechanism of recombinant GM-CSF by intervening PMNs in patients with sepsis. The C57BL/6J sepsis mouse model was induced by cecal ligation and puncture. Recombinant murine GM-CSF (rmGM-CSF) was used in vivo when mice developed immunosuppression, which was characterized by abnormal bactericidal function of PMNs in peripheral blood. rmGM-CSF improved the prognosis of secondary pneumonia and reversed the function of PMNs. PMNs isolated by Percoll from septic patients were treated by recombinant human GM-CSF (rhGM-CSF) in vitro. The expression of CD11b, reactive oxygen species, phagocytosis, and neutrophil extracellular trap release in PMNs were enhanced by rhGM-CSF treatments. Whole-transcriptomic sequencing of mouse PMNs indicated that recombinant GM-CSF increased the expression of Il1b gene in PMNs. Blocking and inhibiting IL-1β release effectively counteracted the enhancing effect of GM-CSF on the bactericidal function of PMNs. rmGM-CSF enhances the bactericidal function of PMNs in vivo and improves the prognosis of secondary pneumonia in septic mice, and recombinant GM-CSF increases IL-1β precursor reserves, which, if stimulated, can rapidly enhance the bactericidal capacity of PMNs.

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.

NO-producing Polymorphonuclear Neutrophils Confer Protection against Chlamydia psittaci in Mouse Lung Infection.

Whether polymorphonuclear neutrophils (PMN) exert a protective role upon chlamydial infection by expressing inducible nitric oxide (NO) synthase (iNOS) and producing NO remains unclear. This issue was addressed using BALB/c mice infected with C. psittaci (Cps) 6BC strain. Methods mainly included flow cytometry, immunofluorescence, qRT-PCR, and Western blot. The number of PMN was significantly increased during Cps infection, which was accompanied by the increased iNOS expression and NO production in the mouse lungs. PMN were the major source of NO during pulmonary Cps infection and inhibited Cps multiplication in an iNOS/NO-dependent manner. Depletion of PMN aggravated Cps-induced disease and increased Cps burden. Mechanically, NF-κB and STAT1 signaling pathways, but not MAPK signaling pathways, were required for the induction of iNOS expression and NO production in PMN by Cps infection. Thus, our findings highlight the protective role of NO-producing PMN in Cps infection. NO-producing PMN confer a protective role during pulmonary Cps infection in mice, and thus our study sheds new light on PMN function during Chlamydia infection.

κ-Opioid Receptors Improve Vascular Endothelial Dysfunction in Salt-Sensitive Hypertension via PI3K/Akt/eNOS Signaling Pathway.

κ-Opioid receptors (κ-OR) are widely used to regulate the activity of the cardiovascular system. To explore the effect and mechanism of κ-OR on salt-sensitive hypertensive endothelial dysfunction, we used Dah1 rats to construct a rat model of salt-sensitive hypertension on a high-salt (HS) diet. Then, the rats were treated with κ-OR activators U50,488H (1.25 mg/kg) and inhibitor nor-BNI (2.0 mg/kg) for 4 weeks, respectively. The rat aortas were collected to detect the contents of NO, ET-1, AngII, NOS, T-AOC, SO, and NT. Protein expression was determined for NOS, Akt, and Caveolin-1. In addition, the vascular endothelial cells were extracted, and the levels of NO, TNF-α, IL-1, IL-6, IL-8, IL-10, p-Akt, and p-eNOS in cell supernatants were detected. In vivo results showed that compared with the HS group, treated with U50,488H promoted rats' vasodilation by increasing the NO content and decreasing ET-1 and AngII contents. U50,488H reduced endothelial cell apoptosis and attenuated vascular, smooth muscle cell and endothelial cell injury. U50,488H also enhanced the rats' response to oxidative stress by increasing the NOS and T-AOC contents. Moreover, U50,488H increased the eNOS, p-eNOS, Akt, and p-AKT expression and decreased the iNOS and Caveolin-1 expression. In vitro results showed that U50,488H promoted NO, IL-10, p-Akt, and p-eNOS levels in endothelial cell supernatants versus the HS group. And U50,488H reduced the adhesion of peripheral blood mononuclear cells and polymorphonuclear neutrophils to endothelial cells and the migration function of polymorphonuclear neutrophils. Our study suggested that κ-OR activation may improve vascular endothelial dysfunction in salt-sensitive hypertensive rats through the PI3K/Akt/eNOS signaling pathway. This may be a potential therapeutic approach in the treatment of hypertension.

Optimized flow cytometry assays to monitor neutrophil activation in human and mouse whole blood samples

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples.Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions.Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively.Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.

Recombinant Mouse GM-CSF Enhances the Bactericidal Ability of Pmns and Improves the Prognosis of Secondary Pseudomonas Aeruginosa Pneumonia By Intracellular IL-1β

Objective: Sepsis is one of the most common clinical emergency and critical illness. The later stage of sepsis is prone to induce immunosuppression and results in secondary fatal infections. Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes the proliferation, differentiation and functional maturation of bone marrow hematopoietic stem cells, and has the function of enhancing immunity, so recombinant GM-CSF becomes an important direction for the treatment of sepsis-induced immunosuppression. However, the effect of recombinant GM-CSF on abnormal polymorphonuclear neutrophils (PMNs) in sepsis remains elusive. This study aimed to study the role of recombinant GM-CSF on the bactericidal ability of PMNs in septic mice, assessing its effect on the prognosis of secondary pneumonia. Methods: The C57BL/6J septic mouse model was induced by cecal ligation and puncture (CLP). Recombinant mouse GM-CSF (rmGM-CSF) was used in vivo when mice developed immunosuppression, then PMNs CD11b expression, reactive oxygen species (ROS), phagocytosis and neutrophil extracellular trap release (NETs) were detected by flow cytometry and fluoresce microplate reader. Pseudomonas aeruginosa pneumonia was induced by intratracheal injection after rmGM-CSF intervention in CLP mice, and the survival of mice were monitored. Meanwhile, PMNs from mouse peripheral blood were isolated for whole-transcriptomic sequencing. Combining with the results of bioinformatics analysis, anti-GM-CSF antibody and Caspase-1 inhibitor (Ac-YVAD-cmk) were treated into peripheral blood PMNs from septic patients in vitro, and then bactericidal ability of PMNs and intracellular IL-1β concentration were evaluated by flow cytometry and ELISA. Results: 1. The septic mice were developed 5 days after CLP, which manifested as decreased lymphocytes in bone marrow, spleen and peripheral blood. The septic mice showed declined expression of MHC-II molecules and abnormal bactericidal function of PMNs in peripheral blood. 2. The expression of MHC-II in monocytes and the bactericidal function of PMNs were improved when septic mice were treated with rmGM-CSF in vivo. 3. RmGM-CSF improved the prognosis of secondary pneumonia in septic mice. 4. Percoll-isolated PMNs from septic patients were treated with rhGM-CSF in vitro. The expression of CD11b, ROS, phagocytosis and NETs release in PMNs were enhanced compared with those without rhGM-CSF treatments. 5. The whole-transcriptomic sequencing of mouse PMNs indicated that recombinant GM-CSF increased the expression of IL-1β in PMNs. 6. Caspase-1 is an important enzyme for the cleavage of pro form IL-1β into mature form. Ac-YVAD-cmk effectively inhibited the activation of IL-1β. treatments reduced the ROS, phagocytosis and release of NETs of PMNs, suggesting that rmGM-CSF enhanced the bactericidal ability of PMNs by IL-1β. Conclusion: RmGM-CSF enhances the bactericidal function of PMNs in vivo, and improves the prognosis of secondary pneumonia in septic mice. In vitro experimental data showed rhGM-CSF strengthen PMNs bactericidal ability by stimulating active IL-1β expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Salivary Gene Expression of RANK, RANKL, and OPG in Type 1 Diabetes Mellitus and Periodontal Disease Patients.

The relationship between type 1 diabetes mellitus (T1DM) and periodontal disease may exhibit by the alteration of bone metabolism. However, evidence for this relationship is scarce and inconclusive. Thus, the aims of the present study were to investigate salivary receptor activator of nuclear factor kappa-β (RANK), receptor activator of nuclear factor kappa-β ligand (RANKL), osteoprotegerin (OPG) gene expression and the RANKL:OPG ratio in T1DM and non-T1DM. Secondary objective was to determine the relationships of RANK, RANKL and OPG gene expression to clinical parameters of T1DM and periodontal disease. Twenty patients with T1DM and twenty age-matched non-T1DM were recruited. Clinical periodontal parameters were measured. Total RNA was isolated from non-stimulated saliva, and the relative gene expressions of RANK, RANKL, OPG and RANKL:OPG ratio were determined by quantitative real-time polymerase chain reaction. The T1DM group had significantly higher mean periodontal parameters than the non-T1DM group, while the mean plaque scores of both groups were not significantly different. There was a trend of higher relative gene expression of RANK, RANKL, and the RANKL:OPG ratio and lower expression of OPG in T1DM group but no statistic significant different when compared to non-T1DM. In the T1DM group, RANKL:OPG correlated with the percentage of bleeding sites, whereas RANK, RANKL, and HbA1c levels correlated with pocket depth. Bone metabolisms demonstrating by decreased OPG gene expression and upregulated of RANK, RANKL, RANKL:OPG with higher pocket depth and bleeding in T1DM may play an important role in periodontal destruction in T1DM.

Supplementation with N-carbamoylglutamate during the transition period improves the function of neutrophils and reduces inflammation and oxidative stress in dairy cows

The aim of this study was to investigate the effects of N-carbamoylglutamate (NCG) supplementation during the transition period on the functions of blood polymorphonuclear neutrophils (PMN), inflammation, and oxidative stress in dairy cows. Thirty multiparous Chinese Holstein dairy cows at wk 4 before parturition were blocked into 2 groups by parity, body weight, and milk yield of previous lactation, and randomly allocated to 2 dietary treatments of basal diet supplemented without (control, n = 15) or with 20 g/d per cow of NCG (NCG, n = 15). The supplementation was carried out from d -21 to 21 relative to calving. Health incidents (mastitis, retained placenta, and lameness) were recorded, and blood samples were collected at d -21, -7, 0 (the calving date), 7, and 21 relative to parturition and analyzed for variables related to inflammation and oxidative stress. In addition, whole blood was collected at d 7 to isolate PMN and used for analysis of the expression of functional genes and from d -21 to 21 for determination of weekly hematological parameters. The number of lymphocytes was greater at d 7 in the blood of NCG cows. The plasma level of malondialdehyde was lower in the NCG group, and blood reactive oxygen species were lower at d 7, whereas total antioxidant capacity tended to be greater in the NCG group and glutathione peroxidase tended to be higher at d 21 in cows fed NCG, suggesting that NCG supplementation improved antioxidation in cows. In addition, the concentration of serum amyloid A was lower in NCG-fed animals during the postpartum stage. Blood concentrations of IL6 and tumor necrosis factor-α were lower and tended to be lower in NCG-fed animals at d 7, respectively. Meanwhile, the concentrations of IL6 tended to be lower in NCG-fed animals at d 21. Furthermore, the expression of S100A9 and MMP9 in the PMN was lower and tended to be lower, respectively, whereas the expression of ITGB2, XBP1 tended to be higher and expression of CLEC6A was higher in NCG-fed cows. Overall, our results indicated that supplementation with NCG during the transition period showed the beneficial effects on animal health, by improving PMN functions and alleviating inflammation status and oxidative stress in dairy cows.

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.