PMN Function Research Articles

Abstract We060: Hypoxia Inducible Factor-2α Enhances Neutrophil Survival and Promotes Cardiac Injury Following Myocardial Infarction via cIAP-1 Signaling

Rationale: Heart failure is a major cause of mortality following myocardial infarction (MI). Neutrophils (PMNs) are among the first immune cells to accumulate in the infarcted region. While some long-term beneficial functions of PMN in heart injury are now appreciated, en mase PMN accumulation in injured hearts is still associated with worsened resolution of inflammation and disease outcomes. MI causes extensive hypoxic injury to heart tissue, where hypoxia-inducible factors (HIFs) play critical roles in cellular functions. While HIF1α regulation of cell survival, migration and metabolism have been well-studied, the role of HIF-2α in these processes is not well-understood. Even less is known of HIF-2α contributions to PMN effector functions, especially in the context of MI. Objectives: The current study aimed to 1. Mechanistically explore how HIF-2α regulates survival of tissue infiltrating PMNs post-MI and 2. Determine how HIF-2α activity in PMNs impacts heart injury resolution post-MI, and whether specific HIF-2α deletion in PMNs can alleviate MI-induced tissue injury. Methods & Results: Infarct size, cardiac functions monitored by echocardiography, and scar tissue formation after left anterior descending coronary ligation injury model were compared between littermate control and specific PMN HIF-2α depletion (Ly6G-cre +/- HIF-2α fl/fl ) mice. Myocardial infarction size, cardiac dysfunction, scare tissue deposit and survival of tissue infiltrating PMNs post-MI were substantially attenuated in mice with PMN HIF-2α deletion. A significantly reduced lifespan of HIF-2α knockout PMNs was further confirmed by fate-mapping EdU pulse chase experiments. Mechanistically, molecular studies revealed that HIF-2α transcriptionally regulates expression of the pro-survival factor cIAP1 in PMNs binding to an HRE located in the promoter region of BIRC2 , in a hypoxia-inducible manner. Ex-vivo pharmacological inhibition of cIAP1 expression in PMNs using Birinapant resulted in dramatic cell death, establishing the critical role of cIAP1 in PMN survival. Finally, our data demonstrate that the protective effect of HIF-2α deletion in PMNs on MI outcomes was due to elevated recruitment of reparative macrophages into infarcted hearts. Conclusion: HIF-2α, through cIAP1 regulation, promotes PMN survival and tissue retention post-MI, leading to worsen cardiac functions and scar formation.

Decreased neutrophil function in newly diagnosed multiple myeloma patients is restored with lenalidomide therapy.

Bacterial infections are common and a major cause of morbidity and mortality in multiple myeloma (MM). We have investigated the function of polymorphonuclear leukocyte (PMN), the immune system's first line of defense against bacteria, in peripheral blood (PB) and bone marrow (BM) samples from patients with newly diagnosed MM (NDMM), smoldering MM (SMM), monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Phagocytosis and oxidative burst in PMN cells from patients and healthy donors were investigated using PhagoTest and PhagoBurst assay. PMN from NDMM, SMM, and MGUS patients had reduced phagocytosis and oxidative burst ability compared with healthy controls. The dysfunction was most prominent in BM samples from MM, SMM, and MGUS patients. Importantly the reduced phagocytosis in MM patients was restored in patients on lenalidomide therapy. Consistently the ability of Escherichia coli stimulated oxidative burst in BM was reduced for the MM, SMM, and MGUS cohort in contrast to the healthy controls and the patients on lenalidomide treatment. Our results show that MM patients have neutrophil dysfunction that could contribute to susceptibility for bacterial infections and that lenalidomide therapy was associated with restored PMN function.

Effect of type of anticoagulant, transportation time, and glucose in the culture media on neutrophil viability and function test results in dairy cattle.

In dairy cattle research, in vitro assessment of innate immune function is commonly evaluated by flow cytometry via the quantitative analysis of circulating polymorphonuclear leukocytes (PMN) functionalities specifically focusing on the capacities for phagocytosis (PC) and oxidative burst (OB). Variations in these PMN functions, however, may not only be influenced by the health status of the animals but also by technical, non-animal related factors. Our objectives were to assess the PMN viability, PC and OB capacities from blood samples collected in tubes coated with different anticoagulants (acid citrate dextrose (ACD) and ethylenediaminetetraacetic acid (EDTA)) and stored for 0, 3, 6, 9, and 12 h at 4°C (to mimic transportation timeframe). Furthermore, we evaluated the PMN functionalities (PC and OB) in samples incubated in culture medium with glucose (7.2 mM) versus no glucose. Over five replicates, coccygeal blood samples were collected from three nulliparous Holstein heifers (5 ACD and 5 EDTA per heifer) and allocated in a refrigerated container (4°C) for 0, 3, 6, 9, and 12 h. At each time point, PMN were isolated using gradient centrifugation. Immunolabeled PMN (CH138A) were subjected to a tricolor fluorescent staining to evaluate their viability (viable, apoptotic, and necrotic PMN). Phagocytosis and OB were assessed by incubating PMN with fluorescent beads and by phorbol 12-myristate 13-acetate stimulation, respectively. The effects of anticoagulant type, storage time, and presence of glucose in the culture medium on PMN viability and function parameters were fitted in mixed linear regression models. The proportion of viable PMN at 0 h was similar for ACD and EDTA (92 ± 4.6% and 93 ± 4.6%, respectively) but it decreased to 78 ± 4.6% for ACD and 79 ± 4.6% for EDTA after 6 h of storage. The proportion of viable PMN was not different between ACD and EDTA at any time point. The proportion of PMN that engulfed beads (PC percentage) and the PC median fluorescence intensity (MFI) reached their highest value after 3 h of storage compared with the other time points. However, the anticoagulant type (ACD versus EDTA) and the presence of glucose in the culture medium did not influence these PC parameters. Oxidative burst MFI was higher in PMN incubated in glucose-supplemented culture medium versus no glucose. We demonstrated that technical factors interfere with the evaluation of PMN viability and functionality, which can potentially lead to bias in the findings of a research hypothesis. To conclude, the present study showed that the optimal timeframe for performing PMN function analyses is within 3 hours after blood sampling. Furthermore, the presence of 7.2 mM glucose in the culture medium, a common concentration in formulation of cell culture medium, increases the in vitro OB capacity, potentially masking any impairments in in vivo PMN dysfunctionality.

Effect of exogenous melatonin on the cellular response of Holstein heifer calves during vaccination.

Despite rigorous vaccination protocols, calf morbidity is the primary contributor to economic loss in the calf sector of the dairy industry. Melatonin has modulated immune response in other mature animal species. We hypothesized that exogenous melatonin may improve the cellular response to vaccination in dairy calves. Our objective was to evaluate the effect of exogenous melatonin on polymorphonuclear leukocyte (PMN) function in Holstein heifer calves during immunization. Sixty neonatal Holstein heifers were enrolled by birth cohort (block) and randomized to one of four treatments: control (CON), vaccination of 0.5mg ovalbumin on days 0 and 21 (VAC), implantation of 24mg melatonin on day 0 (MEL), or both melatonin and vaccine treatments (MVAC). Jugular blood was collected on days 0, 21, 42, and 63 to measure circulating melatonin, anti-ovalbumin immunoglobulin-G, and PMN function. Calves implanted with melatonin had greater circulating melatonin than non-implanted on day 21 (P < 0.01). Anti-ovalbumin IgG was greater for vaccinated than non-vaccinated calves (P < 0.01). Anti-ovalbumin IgG was greater for MVAC than VAC calves on day 63. Percent of cells and mean florescence intensity of cells performing oxidative burst decreased from day 0 to day 63 (P < 0.01) but were not affected by treatment (P ≥ 0.26). There was a tendency (P = 0.10) for an interaction of melatonin, vaccination, and day for the mean florescence intensity of cells performing phagocytosis where MVAC was greater than all other treatments on d 42. Exogenous melatonin may alter PMN function of calves during vaccination. Further research is needed to define the effect of melatonin on development of antigen-specific IgG during programmed vaccination protocols.

The intriguing strategies of Tannerella forsythia's host interaction.

Tannerella forsythia, a member of the "red complex" bacteria implicated in severe periodontitis, employs various survival strategies and virulence factors to interact with the host. It thrives as a late colonizer in the oral biofilm, relying on its unique adaptation mechanisms for persistence. Essential to its survival are the type 9 protein secretion system and O-glycosylation of proteins, crucial for host interaction and immune evasion. Virulence factors of T. forsythia, including sialidase and proteases, facilitate its pathogenicity by degrading host glycoproteins and proteins, respectively. Moreover, cell surface glycoproteins like the S-layer and BspA modulate host responses and bacterial adherence, influencing colonization and tissue invasion. Outer membrane vesicles and lipopolysaccharides further induce inflammatory responses, contributing to periodontal tissue destruction. Interactions with specific host cell types, including epithelial cells, polymorphonuclear leukocytes macrophages, and mesenchymal stromal cells, highlight the multifaceted nature of T. forsythia's pathogenicity. Notably, it can invade epithelial cells and impair PMN function, promoting dysregulated inflammation and bacterial survival. Comparative studies with periodontitis-associated Porphyromonas gingivalis reveal differences in protease activity and immune modulation, suggesting distinct roles in disease progression. T. forsythia's potential to influence oral antimicrobial defense through protease-mediated degradation and interactions with other bacteria underscores its significance in periodontal disease pathogenesis. However, understanding T. forsythia's precise role in host-microbiome interactions and its classification as a keystone pathogen requires further investigation. Challenges in translating research data stem from the complexity of the oral microbiome and biofilm dynamics, necessitating comprehensive studies to elucidate its clinical relevance and therapeutic implications in periodontitis management.

Trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and invivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

Neisseria gonorrhoeae Coinfection during Chlamydia muridarum Genital Latency Does Not Modulate Murine Vaginal Bacterial Shedding.

Chlamydia trachomatis and Neisseria gonorrhoeae are the most frequently reported agents of bacterial sexually transmitted disease worldwide. Nonetheless, C. trachomatis/N. gonorrhoeae coinfection remains understudied. C. trachomatis/N. gonorrhoeae coinfections are more common than expected by chance, suggesting C. trachomatis/N. gonorrhoeae interaction, and N. gonorrhoeae infection may reactivate genital chlamydial shedding in women with latent (quiescent) chlamydial infection. We hypothesized that N. gonorrhoeae would reactivate latent genital Chlamydia muridarum infection in mice. Two groups of C. muridarum-infected mice were allowed to transition into genital latency. One group was then vaginally inoculated with N. gonorrhoeae; a third group received N. gonorrhoeae alone. C. muridarum and N. gonorrhoeae vaginal shedding was measured over time in the coinfected and singly infected groups. Viable C. muridarum was absent from vaginal swabs but detected in rectal swabs, confirming C. muridarum genital latency and consistent with the intestinal tract as a C. muridarum reservoir. C. muridarum inclusions were observed in large intestinal, but not genital, tissues during latency. Oviduct dilation was associated with C. muridarum infection, as expected. Contradicting our hypothesis, N. gonorrhoeae coinfection did not reactivate latent C. muridarum vaginal shedding. In addition, latent C. muridarum infection did not modulate recovery of vaginal viable N. gonorrhoeae. Evidence for N. gonorrhoeae-dependent increased C. muridarum infectivity has thus not been demonstrated in murine coinfection, and the ability of C. muridarum coinfection to potentiate N. gonorrhoeae infectivity may depend on actively replicating vaginal C. muridarum. The proportion of mice with increased vaginal neutrophils (PMNs) was higher in N. gonorrhoeae-infected than in C. muridarum-infected mice, as expected, while that of C. muridarum/N. gonorrhoeae-coinfected mice was intermediate to the singly infected groups, suggesting latent C. muridarum murine infection may limit PMN response to subsequent N. gonorrhoeae infection. IMPORTANCE Our work builds upon the limited understanding of C. muridarum/N. gonorrhoeae coinfection. Previously, N. gonorrhoeae infection of mice with acute (actively replicating) vaginal C. muridarum infection was shown to increase recovery of viable vaginal N. gonorrhoeae and vaginal PMNs, with no effect on C. muridarum vaginal shedding (R. A. Vonck et al., Infect Immun 79:1566-1577, 2011). It has also been shown that chlamydial infection of human and murine PMNs prevents normal PMN responses, including the response to N. gonorrhoeae (K. Rajeeve et al., Nat Microbiol 3:824-835, 2018). Our findings show no effect of latent genital C. muridarum infection on the recovery of viable N. gonorrhoeae, in contrast to the previously reported effect of acute C. muridarum infection, and suggesting that acute versus latent C. muridarum infection may have distinct effects on PMN function in mice. Together, these studies to date provide evidence that Chlamydia/N. gonorrhoeae synergistic interactions may depend on the presence of replicating Chlamydia in the genital tract, while chlamydial effects on vaginal PMNs may extend beyond acute infection.

Mitochondrial ROS production by neutrophils is required for host antimicrobial function against Streptococcus pneumoniae and is controlled by A2B adenosine receptor signaling.

Polymorphonuclear cells (PMNs) control Streptococcus pneumoniae (pneumococcus) infection through various antimicrobial activities. We previously found that reactive oxygen species (ROS) were required for optimal antibacterial function, however, the NADPH oxidase is known to be dispensable for the ability of PMNs to kill pneumococci. In this study, we explored the role of ROS produced by the mitochondria in PMN antimicrobial defense against pneumococci. We found that the mitochondria are an important source of overall intracellular ROS produced by murine PMNs in response to infection. We investigated the host and bacterial factors involved and found that mitochondrial ROS (MitROS) are produced independent of bacterial capsule or pneumolysin but presence of live bacteria that are in direct contact with PMNs enhanced the response. We further found that MyD88-/- PMNs produced less MitROS in response to pneumococcal infection suggesting that released bacterial products acting as TLR ligands are sufficient for inducing MitROS production in PMNs. To test the role of MitROS in PMN function, we used an opsonophagocytic killing assay and found that MitROS were required for the ability of PMNs to kill pneumococci. We then investigated the role of MitROS in host resistance and found that MitROS are produced by PMNs in response to pneumococcal infection. Importantly, treatment of mice with a MitROS scavenger prior to systemic challenge resulted in reduced survival of infected hosts. In exploring host pathways that control MitROS, we focused on extracellular adenosine, which is known to control PMN anti-pneumococcal activity, and found that signaling through the A2B adenosine receptor inhibits MitROS production by PMNs. A2BR-/- mice produced more MitROS and were significantly more resistant to infection. Finally, we verified the clinical relevance of our findings using human PMNs. In summary, we identified a novel pathway that controls MitROS production by PMNs, shaping host resistance against S. pneumoniae.

Hydroxycarboxylic acid receptor 2 (HCA2) agonists induce NET formation and MMP-9 release from bovine polymorphonuclear leukocytes

Periparturient cows are commonly fed diets supplemented with Niacin (nicotinic acid, NA) because of its anti-lipolytic properties. NA confers its anti-lipolytic effects by activating the hydroxycarboxylic acid 2 receptor (HCA2). HCA2 is also activated by the ketone body beta-hydroxybutyrate (BHB) and circulating BHB levels are elevated in postpartum dairy cows. The HCA2 receptor is highly expressed in bovine polymorphonuclear leukocytes (PMN) and could link metabolic and innate immune responses in cattle. We investigated how HCA2 agonists affected bovine PMN function in vitro. We studied different PMN responses, such as granule release, surface expression of CD11b and CD47, generation of neutrophil extracellular traps (NETs), and apoptosis. NA, BHB, and 4,4aR,5,5aR-tetrahydro-1H-cyclopropa [4,5] cyclopenta [1,2-c] pyrazole-3-carboxylic acid (MK-1903) treatment triggered the release of matrix metalloproteinase 9 (MMP-9), a component of the tertiary granule, from neutrophils. Additionally, all HCA2 agonists induced NETs formation but did not affect surface expression of CD11b and CD47. Finally, none of the HCA2 agonists triggered apoptosis in bovine PMN. This information will give new insights into the potential role of the HCA2 receptor in the bovine innate immune response.

Evaluation of peripheral blood polymorphonuclear cell functions after an oral carbohydrate overload in obese and insulin dysregulated horses

Obesity and insulin dysregulation (ID) are increasingly prevalent conditions in equid populations worldwide. Immune impairment is well described in humans with metabolic dysfunction and is reported but still incompletely understood in horses. This study evaluated the effect of acute induced transient hyperglycemia on apoptosis, phagocytosis and oxidative burst activity of peripheral blood polymorphonuclear cells (PMN) of lean and obese adult horses with or without insulin dysregulation. Seventeen adult horses were allocated into three groups based on their body condition score (BCS) and metabolic status: lean-insulin sensitive (lean-IS), obese-insulin sensitive (obese-IS) and obese-insulin dysregulated (obese-ID). ID was determined by insulin tolerance testing (ITT). Blood glucose elevation was induced through an infeed-oral glucose test (in-feed OGT), and all assessments of PMN functions (apoptosis, phagocytosis and oxidative burst) were done in vitro after isolation from peripheral blood before and 120 min after carbohydrate overload. Results were analyzed using a repeated measures linear mixed model with significance defined at P < 0.05. No differences in apoptosis were observed between experimental groups at any time point. Phagocytic capacity was significantly lower at baseline in the obese-ID group but increased in response to glucose administration when compared to the other two groups. Basal reactive oxygen species production in the obese-IS group differed significantly from the lean-IS and obese-ID groups and decreased significantly in response to glucose administration. Results from this study showed that both metabolic status itself, and oral glucose administration, seem to be factors that alter PMN functionality in horses, specifically phagocytosis and oxidative burst.

The Age-Driven Decline in Neutrophil Function Contributes to the Reduced Efficacy of the Pneumococcal Conjugate Vaccine in Old Hosts.

Despite the availability of vaccines, Streptococcus pneumoniae (pneumococcus) remains a serious cause of infections in the elderly. The efficacy of anti-pneumococcal vaccines declines with age. While age-driven changes in antibody responses are well defined, less is known about the role of innate immune cells such as polymorphonuclear leukocytes (PMNs) in the reduced vaccine protection seen in aging. Here we explored the role of PMNs in protection against S. pneumoniae in vaccinated hosts. We found that depletion of PMNs in pneumococcal conjugate vaccine (PCV) treated young mice prior to pulmonary challenge with S. pneumoniae resulted in dramatic loss of host protection against infection. Immunization boosted the ability of PMNs to kill S. pneumoniae and this was dependent on bacterial opsonization by antibodies. Bacterial opsonization with immune sera increased several PMN anti-microbial activities including bacterial uptake, degranulation and ROS production. As expected, PCV failed to protect old mice against S. pneumoniae. In probing the role of PMNs in this impaired protection, we found that aging was accompanied by an intrinsic decline in PMN function. PMNs from old mice failed to effectively kill S. pneumoniae even when the bacteria were opsonized with immune sera from young controls. In exploring mechanisms, we found that PMNs from old mice produced less of the antimicrobial peptide CRAMP and failed to efficiently kill engulfed pneumococci. Importantly, adoptive transfer of PMNs from young mice reversed the susceptibility of vaccinated old mice to pneumococcal infection. Overall, this study demonstrates that the age-driven decline in PMN function impairs vaccine-mediated protection against Streptococcus pneumoniae.

6 Effect of Exogenous Melatonin on the Cellular and Humoral Response of Holstein Heifer Calves During Vaccination

Abstract Despite rigorous vaccination protocols, calf morbidity is the primary contributor to economic loss in beef and dairy industries. Melatonin has been used as a vaccine adjuvant in many species, yet there are few studies in cattle. It was hypothesized implantation of slow-release (90-day) exogenous melatonin during vaccination could bolster cellular and humoral immune function. The objective was to evaluate the effect of exogenous melatonin on polymorphonuclear leukocyte (PMN) function and adaptive immune response in dairy calves vaccinated against a model antigen (ovalbumin). Total serum protein (TSP) was measured in Holstein heifer calves (n=60; &amp;lt; 3 d old) which were randomly assigned to one of four treatments: either no treatment (CON), a two-series vaccination of 0.5 mg ovalbumin on d 0 and 21 (VAC), an implantation of 24 mg melatonin on d 0 (MEL), or both melatonin and vaccine treatments (MVAC). Jugular blood was collected on days 0, 21, 42, 63, and 84 to measure PMN function and anti-ovalbumin immunoglobulin-G (IgG). Experimental design was a RCBD with repeated measures (day), a two-by-two factorial arrangement of treatments, and block was enrollment cohort. TSP was not correlated with any measurements on day 0 (r2 ≤ 0.16,P ≤ 0.23). There was a treatment by day interaction for median florescent intensity of phagocytosis where MVAC was greater than other treatments on day 42 (P =0.01). Across treatments, neutrophil oxidative burst intensity decreased after day 0 (P &amp;lt; 0.01) and phagocytosis intensity increased toward day 42 and decreased toward day 84 (P &amp;lt; 0.01). As expected, there was a treatment by day interaction (P &amp;lt; 0.01) were vaccinated calves developed greater anti-ovalbumin IgG over time than non-vaccinated calves, and MVAC tended (P = 0.08) to be greater than VAC on d 63. Exogenous melatonin may influence PMN function and potentially IgG response during vaccination.

Neutrophils-From Bone Marrow to First-Line Defense of the Innate Immune System.

Neutrophils (polymorphonuclear cells; PMNs) form a first line of defense against pathogens and are therefore an important component of the innate immune response. As a result of poorly controlled activation, however, PMNs can also mediate tissue damage in numerous diseases, often by increasing tissue inflammation and injury. According to current knowledge, PMNs are not only part of the pathogenesis of infectious and autoimmune diseases but also of conditions with disturbed tissue homeostasis such as trauma and shock. Scientific advances in the past two decades have changed the role of neutrophils from that of solely immune defense cells to cells that are responsible for the general integrity of the body, even in the absence of pathogens. To better understand PMN function in the human organism, our review outlines the role of PMNs within the innate immune system. This review provides an overview of the migration of PMNs from the vascular compartment to the target tissue as well as their chemotactic processes and illuminates crucial neutrophil immune properties at the site of the lesion. The review is focused on the formation of chemotactic gradients in interaction with the extracellular matrix (ECM) and the influence of the ECM on PMN function. In addition, our review summarizes current knowledge about the phenomenon of bidirectional and reverse PMN migration, neutrophil microtubules, and the microtubule organizing center in PMN migration. As a conclusive feature, we review and discuss new findings about neutrophil behavior in cancer environment and tumor tissue.

Isolation of Human Neutrophils from Whole Blood and Buffy Coats.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

Flow Cytometric Assessment of the Viability and Functionality of Uterine Polymorphonuclear Leukocytes in Postpartum Dairy Cows.

Simple SummaryDairy cows experience immune suppression around calving, which can result in the development of uterine diseases. White blood cells, and more specifically polymorphonuclear leukocytes (PMN), are important components of the immune system. Although it is regarded as a common research approach to study PMN isolated from blood, it would be interesting to know more about the viability and functionality of uterine PMN. We developed a method to isolate PMN from the bovine postpartum uterus to perform viability and functionality tests. We also evaluated whether we could identify uterine PMN using a specific antibody. Uterine cells were recovered using an adapted medical brush, and PMN were successfully isolated and identified. The percentage of viable intra-uterine PMN in postpartum cows (9–37 days in milk) roughly ranged from 10 to 80%, indicating that the viability of the uterine PMN is highly dynamic. We could also identify PMN that ingested labeled particles, which lets us conclude that uterine PMN are functional. Using the presently described methods, further research can be performed to unravel the role of uterine PMN viability and functionality in bovine uterine health.Postpartum dairy cows experience impaired peripheral polymorphonuclear leukocyte (PMN) functionality, which has been associated with reproductive tract inflammatory diseases. However, it has not been elucidated yet whether endometrial PMN functionality is (equally) impaired. We developed a method for endometrial PMN isolation and flow cytometric assessment of their viability and functionality. We also evaluated PMN immunolabeling, using a specific bovine granulocyte marker, CH138A. Blood and endometrial cytobrush samples were collected in duplicate from seventeen clinically healthy Holstein-Friesian cows between 9 and 37 days in milk. The proportion of viable, apoptotic, and necrotic PMN in endometrial samples roughly ranged from 10 to 80%, indicating highly dynamic endometrial PMN populations in the postpartum uteri. Endometrial PMN functionality testing revealed that PMN immunolabeling increased the accuracy, although this protocol might influence the median fluorescence intensity of the sample. Phagocytosis seemed the most stable and reliable endometrial PMN function and could be assessed satisfactorily without prior CH138A immunolabeling. However, the interpretation of oxidative burst and intracellular proteolysis tests remains challenging. The correlation between peripheral and endometrial PMN functionality was poor. Further research is warranted to unravel the role of uterine PMN viability and functionality in bovine uterine health.

Evaluation of Immunostimulant Activity and Inhibition of Cytokine Storm Activity of Proprietary Herbal Formulation Virulina®

Immunostimulation is the strengthening of the immune system through the induction of non-specific immune responses. Synthetic drugs have harmful side effects, enormous costs, and are ineffectual in controlling several pathological disorders. Therefore, nowadays, herbal immunostimulants have been considered as a superior and less toxic opportunity for the management of disorders as well as their complications. The objective of the current investigation is the formulation of Virulina®-Natural Solutions (VL®-NS) polyherbal formulation and its evaluation for immunostimulant and cytokine storm inhibition activity. The immunostimulant &amp; anti-inflammatory potential of Virulina® were determined by using in-vitro phagocytosis through polymorphonuclear analysis, indicating the invasion of leukocytes. In-vitro cytokine storm inhibitory activity was also performed using mouse macrophage cells (RAW264.7) treated with polyinosinic-polycytidylic acid to test the ability of Virulina® to attenuate the immune responses. The inflammatory responses were evaluated by the endotoxin-lipopolysaccharide (LPS) model following the endotoxin challenges of aggravating the inflammatory mediators (cytokines); thereby, the LPS derived from E. coli as antigen at a dose of 10 mgkg-1, i.p was used for the systemic inflammation. VL®-NS exhibited enhanced phagocytic efficacy at 100 mgml-1 in PMN function test. VL®-NS significantly decreases the expression of a cytokine such as interleukin-6, tumor necrosis factor-α &amp; vascular endothelial growth factor in RAW264.7 cell culture. The increase in LPS-induced cytokine levels in rat serum was dose-dependently &amp; significantly (p&lt;0.05) inhibited by VL®-NS treatment. VL®-NS tend to be potent immunostimulant therapy as a superior and less toxic opportunity for the strengthening of the immune system for the management of several inflammatory disorders.

Effects of oral calcium bolus supplementation on intracellular polymorphonuclear leukocyte calcium levels and functionality in primiparous and multiparous dairy cows

The objectives of this study were (1) to characterize Ca levels and polymorphonuclear leukocyte (PMN) function in primiparous and multiparous animals following oral Ca bolus supplementation, and (2) to determine differential responses of boluses containing a lower dose of Ca than traditionally used in primiparous animals on Ca levels and PMN function. Jersey × Holstein crossbred animals (n = 104) were enrolled within 24 h of parturition. All animals were blocked by time relative to calving and randomly assigned to treatment. The Ca boluses were composed of a mixture of Ca chloride, Ca sulfate, and Ca propionate. For objective 1, animals were assigned to control (CON; no Ca supplementation), or a series of 2 Ca boluses given 24 h apart for a total of 50 g of Ca. Objective 2 treatments included control (CON; no Ca supplementation), a series of 2 Ca boluses given 24 h apart containing 50 g of Ca, or a series of 2 Ca boluses given 24 h apart containing 25 g of Ca. Blood samples were collected on d 1 (<24 h), 2, 3, 5, and 7 relative to parturition. Total serum Ca, serum haptoglobin, PMN intracellular Ca, PMN intracellular Ca after stimulation with an environmental Escherichia coli, PMN L-selectin surface expression, and PMN phagocytic and oxidative burst activities were analyzed. For objective 1 a tendency was detected for a treatment difference on basal intracellular PMN Ca and a treatment difference on E. coli-stimulated intracellular PMN Ca. We detected a parity × DIM effect for PMN oxidative burst intensity. However, no other interactions or parity effects on other functional PMN variables were detectable. In primiparous animals, we found a treatment difference for E. coli-stimulated intracellular PMN Ca among animals given 50 g of Ca but no treatment difference on basal intracellular PMN Ca. The 50 g of Ca treatment increased both PMN phagocytosis and oxidative burst intensities. Supplementing animals with 50 g of oral Ca increased intracellular PMN Ca and influenced PMN function.

Wound healing and PMN function at hydrophilic‐surface titanium‐zirconium implants in diabetic and healthy patients

Wound healing and PMN function at hydrophilic‐surface titanium‐zirconium implants in diabetic and healthy patients

Polymorphonuclear Cell Chemotaxis and Suicidal NETosis: Simultaneous Observation Using fMLP, PMA, H7, and Live Cell Imaging.

Chemotaxis and the formation of suicidal neutrophil extracellular traps (suicidal NETosis) are key functions of polymorphonuclear cells (PMNs). Neutrophil extracellular traps in particular are known to be significantly involved in the severity of inflammatory and immunological disorders such as rheumatoid arthritis and Crohn's disease. Therefore, detailed knowledge of PMNs is essential for analyzing the mechanisms involved in, and developing new therapies for, such diseases. To date, no standard method to analyze these cell activities has been established. This study used in vitro live cell imaging to simultaneously observe and analyze PMN functions. To demonstrate this, the effects of phorbol-12-myristat-13-acetat (PMA, 0.1-10 nM), N-formylmethionine-leucyl-phenylalanine (fMLP, 10 nM), and protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) on PMN chemotaxis and suicidal NETosis were studied. PMA (1 nM-10 nM) resulted in significant concentration-dependent behavior in chemotaxis and an earlier onset of maximum oxidative burst and NET formation of up to 44%. When adding H7, PMA-triggered PMN functions were reduced, demonstrating that all three functions rely mostly on protein kinase C (PKC) activity, while PKC is not essential for fMLP-induced PMN activity. Thus, the method here described can be used to objectively quantify PMN functions and, especially through the regulation of the PKC pathway, could be useful in further clinical studies of immunological disorders.