Number Of Polymorphonuclear Neutrophils Research Articles

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

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

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.

КЛИНИЧЕСКОЕ ЗНАЧЕНИЕ ПОЛИМОРФНОЯДЕРНЫХ НЕЙТРОФИЛОВ В СТРАТИФИКАЦИИ ДЕТЕЙ С ОСТРОЙ БОЛЬЮ В ЖИВОТЕ

Background. Acute abdominal pain in children is defined as acute pain of non-traumatic genesis lasting up to 5-7 days, and presents a serious diagnostic and tactical problem. Many aspects of diagnosis and treatment remain controversial. Aim. To determine the clinical significance of polymorphonuclear neutrophil count in the stratification of children with acute abdominal pain. Material and Methods. A prospective randomized blind clinical trial was performed. The results of examination and treatment of 633 children admitted to Grodno Regional Children's Clinical Hospital with acute abdominal pain were analyzed. The number of polymorphonuclear neutrophils was analyzed in relative (PNRV) and absolute (PNAV) values. Statistics included evidence-based criteria, ROC and precision-recall curve analysis, Kullback's measure of informativeness (Jxi), Bayes' theorem. Results. Separation points were determined for the PNRV and PNAV tests. It was 65% for the PNRV test and 8.952×109/L for the PNAV test. For PNRV values less than 65%, the probability of acute appendicitis (AA) as a cause of acute abdominal pain was 1.8% (CI 0.2 to 3.4); the overall informative value was 2.94. With PNAV test values less than 6.5×109/L, the probability of AA was 1.2% (CI 0.03 to 2.4%); the overall informative value was 5.24. It was demonstrated that an increase in test values above the indicated values did not significantly increase the probability of AA as a cause of acute abdominal pain in children. Conclusion. The tests studied are highly informative and can be used to stratify patients with acute abdominal pain. It is necessary to validate the results obtained taking into account the specific capabilities of a given medical institution and the type of equipment used.

Methanol Extract of Katuk (Sauropus androgynus) Leaves as an Anti-inflammatory Agent: Animal Study in Carrageenan-induced Rat Models of Inflammation

Background: Inflammation is a response in the human body to survive during infection, injury and tissue damage. Acute inflammation causes edema and polymorphonuclear neutrophils (PMNs) release. Sauropus androgynus leaves which contain flavonoids, tannins, saponins, steroids and triterpenoids may have anti-inflammatory properties. These compounds can be extracted with methanol. This research aimed to determine the anti-inflammatory effect of S. androgynus leaves methanol extract (SALME) on carrageenan induced-rats. Materials and methods: True experimental study was conducted using 12 Wistar rats. Rats were induced with carrageenan subcutaneously on the plantar pedis. Carrageenan-induced rats were treated with/without various doses of SALME. Edema volume was measured with a plethysmometer. The plantar pedis tissues were collected and stained with haematoxylin- eosin (HE) staining, then PMNs were observed and counted under a light microscope. All data were analyzed by one-way ANOVA, Bonferroni post hoc test, Pearson correlation and linear regression. Results: SALME had significant effects on the volume of edema (p=0.000) and the number of PMNs (p=0.000). The most effective dose to reduce the edema volume and decrease the PMNs cell number was 37.80 mg/200 g body weight (BW). SALME doses may affect 91.0% of edema volume and 89.2% of PMNs cell number. Edema volume had a significant, robust correlation (92.3%) with PMNs cell number. Conclusion: SALME is confirmed to have an anti-inflammatory activity by reducing the edema volume and decreasing the PMNs cell number. Keywords: Sauropus androgynus, methanol extract, anti-inflamamatory, carrageenan, plantar pedis, rats

Implication of Neutrophils Extracellular Traps in the Pathogenesis of SARS-CoV-2 pneumonia.

Peripheral blood polymorphonuclear neutrophils (PMNs) forming extracellular traps (NETs), as well as endothelial- and platelet-derived parameters, have been analyzed in patients with SARS-CoV-2 pneumonia, and their prognostic role has been evaluated. Eighty-seven consecutive patients hospitalized with SARS-CoV-2 pneumonia were prospectively selected. A sample of 30 healthy individuals served as the control group. Clinical and oxygenation (oxygen saturation to fraction of inspired oxygen ratio—SpO2/FiO2) characteristics and PMNs forming NETs, serum levels of myeloperoxidase, E-selectin, vascular cell adhesion molecule 1—VCAM1—vascular endothelial growth factor, P-selectin, platelet factor 4 and plasma concentrations of D-dimer were evaluated at hospital admission, at discharge and 14 days after discharge. Intensive care unit admission or death was the primary composite endpoint. Patients showed a higher number of PMNs forming NETs than healthy controls. The absolute number of PMNs forming NETs was inversely correlated with oxygen status (SpO2/FiO2) and positively with inflammatory (C-reactive protein, ferritin) markers and VCAM1. A decrease in, but not a normalization of NETs and endothelial-derived parameters was observed in patients who survived. In conclusion, the formation of NETs runs parallel to that of other inflammatory and endothelial activation markers, and is inverse to the oxygenation parameters, supporting a pathogenic role for PMNs in this entity.

Présentation de deux modèles expérimentaux pour l’étude de la vitalité des plaies en pathologie médico-légale : une étude clinique prospective non interventionnelle et un modèle animal

In forensic pathology, wound age evaluation allows to determine if a wound was inflicted before or after death, and to date wounds of different ages. This dating is performed in conventional histopathology by observing inflammatory cells and hemorrhage at the wound site. However, these criteria seem to show low sensitivity and/or specificity. The aim of our study was to compare two models of wound vitality evaluation: a human surgical model, and a porcine experimental model; using these histological criteria. In the two human (n=38) and porcine (n=11) models, three wounds were performed at regular time-lapse before devascularization/sacrifice, and a control wound after devascularization/sacrifice. The main evaluation criteria were the presence of interstitial hemorrhage and the number of interstitial polymorphonuclear neutrophils at 10 high power fields. In the two models, the number of polymorphonuclears neutrophils was significantly higher in vital wounds compared to the post-devascularization/sacrifice wounds (P<0.001), with a very low sensitivity (human model: 4.3%; porcine: 47%). Hemorrhagic infiltration was more frequent in vital wounds (human: P<0.001; porcine: P=0.01), with a low specificity (human: 48%; porcine: 54%). This first study confirms, in the two models, the limitations of conventional histopathology in wound vitality evaluation. The next step will be testing several immunohistochemical markers in the two models.

Allogenic mesenchymal stem cell-conditioned medium does not affect sperm parameters and mitigates early endometrial inflammatory responses in mares

This study aimed to evaluate the effects of mesenchymal stem cell-conditioned medium (MSC-CM) on sperm parameters, intrauterine polymorphonuclear neutrophils (PMN), intrauterine fluid accumulation (IUF), and fertility in mares. In experiment 1, two ejaculates from ten stallions were extended to 50 million sperm/mL using a milk-based extender. Thereafter, 20 mL of extended semen was added of MSC-CM as follows: 0, 5, 10, 15, and 20 mL. Sperm kinetics and plasma membrane integrity were evaluated immediately after dilution (T0) and 2 h post-incubation at 37 °C (T2). In experiment 2, mares characterized as resistant (n = 13) or susceptible (n = 7) to endometritis were inseminated with fresh semen 24 h post-induction of ovulation in two (Control and CM-1) and three (Control, CM-1, and CM-2) cycles in a crossover, as follows: control, no pharmacological interference; CM-1, supplementation of semen insemination dose at 3:4 (v:v, MSC-CM:semen); CM-2, 30 mL of MSC-CM was infused into the uterus 24 h before insemination. Endometrial cytology and uterine fluid were collected 6 and 24 h after insemination to evaluate the number of PMNs and concentrations of interleukins IL6, IL10, and TNFα. IUF was determined by ultrasonography 24 and 48 h after insemination. Pregnancy status was diagnosed 14 days after ovulation. The addition of MSC-CM to semen did not influence sperm parameters at T0 and T2 (P > 0.05) and reduced (CM-1; P < 0.05) the number of PMNs at 6 h post-insemination in resistant mares. In susceptible mares, PMNs at 6 and 24 h post-insemination, as well as IUF were reduced (P < 0.05) in both treated cycles (CM-1 and CM-2). In addition, MSC-CM downregulated IL6 and upregulated IL10 concentrations in the uterus of susceptible mares after insemination. There were no differences in fertility rates among groups both in resistant (Control, 77%, 10/13; CM-1, 62%, 8/13) and susceptible mares (Control, 42.8%, 3/7; CM-1, 57.1%, 4/7; CM-2, 85.7%. 6/7). In conclusion, MSC-CM did not affect sperm parameters when mixed with diluted semen, and reduced post-insemination inflammatory responses in mares.

Mine-site derived particulate matter exposure exacerbates neurological and pulmonary inflammatory outcomes in an autoimmune mouse model

ABSTRACT The Southwestern United States has a legacy of industrial mining due to the presence of rich mineral ore deposits. The relationship between environmental inhaled particulate matter (PM) exposures and neurological outcomes within an autoimmune context is understudied. The aim of this study was to compare two regionally-relevant dusts from high-priority abandoned mine-sites, Claim 28 PM, from Blue Gap Tachee, AZ and St. Anthony mine PM, from the Pueblo of Laguna, NM and to expose autoimmune-prone mice (NZBWF1/J). Mice were randomly assigned to one of three groups (n = 8/group): DM (dispersion media, control), Claim 28 PM, or St. Anthony PM, subjected to oropharyngeal aspiration of (100 µg/50 µl), once per week for a total of 4 consecutive doses. A battery of immunological and neurological endpoints was assessed at 24 weeks of age including: bronchoalveolar lavage cell counts, lung gene expression, brain immunohistochemistry, behavioral tasks and serum autoimmune biomarkers. Bronchoalveolar lavage results demonstrated a significant increase in number of polymorphonuclear neutrophils following Claim 28 and St. Anthony mine PM aspiration. Lung mRNA expression showed significant upregulation in CCL-2 and IL-1ß following St. Anthony mine PM aspiration. In addition, neuroinflammation was present in both Claim 28 and St. Anthony mine-site derived PM exposure groups. Behavioral tasks resulted in significant deficits as determined by Y-maze new arm frequency following Claim 28 aspiration. Neutrophil elastase was significantly upregulated in the St. Anthony mine exposure group. Interestingly, there were no significant changes in serum autoantigens suggesting systemic inflammatory effects may be mediated through other molecular mechanisms following low-dose PM exposures.

Sperm transport and endometrial inflammatory response in mares after artificial insemination with cryopreserved spermatozoa

This study aimed to determine whether the insemination site and dose with cryopreserved sperm of reproductively normal mares affect the sperm population in uterine tubes and the intensity of endometrial inflammatory response. Experimental subjects were estrous mares inseminated, in the mid-uterine body (Body) or the tip of the uterine horn (Tip), ipsilateral to the dominant follicle, with one 0.5 mL straw with 50 × 106 sperm (50) or with eight straws with 50 × 106 sperm/straw (400). Mares were slaughtered 2 h, 4 h and 12 h after artificial insemination (AI) and randomly assigned to following groups: Body 50 (n = 19) (2 h, 4 h or 12 h); Tip 50 (n = 29) (2 h, 4 h, or 12 h); Body 400 (n = 24) (2 h, 4 h, or 12 h); Tip 400 (n = 21) (2 h, 4 h, or 12 h). A Control group (n = 16) was not inseminated. After slaughter, uterine tubes were separated from uterus, and uteri and tubes flushed with phosphate-buffered saline (PBS). After flushing, an endometrial sample was collected from ipsilateral and contralateral horns and mid-uterus body for further histopathological examination. A sample of each uterine tube flushing was examined for sperm count, and a sample of each uterine flushing was used for polymorphonuclear neutrophils (PMNs) count. Data were analyzed using PROC GLM from SASv9.4. Insemination time, site, sperm dose, and their interactions were considered independent variables and sperm and PMNs numbers dependent variables. Deep horn insemination increased ipsilateral uterine tube sperm number without an increase in the inflammatory reaction compared with the uterine body insemination. The higher the insemination dose, the higher the uterine tubes’ sperm number and inflammatory reaction, with a quicker resolution. In conclusion, the insemination site and dose affected sperm in the uterine tubes, while post-insemination time and dose influenced the inflammatory reaction.

Blood collection technique, anticoagulants and storing temperature have minor effects on the isolation of polymorphonuclear neutrophils

In the isolation of polymorphonuclear neutrophils (PMNs) the technique and other external factors can have great influence on the quality and quantity of isolated neutrophils. To elucidate the influence of the blood collection technique, anticoagulants and storing temperature on isolated PMNs healthy volunteers provided blood samples with different needles and collection techniques, anticoagulants (EDTA, heparin, citrate) and storing temperatures (4, 22, 37 °C). From each blood sample PMNs were isolated and compared regarding number of PMNs and oxidative burst. The blood collection technique, anticoagulants and storing temperature had minor impact on isolated PMNs. All three tested cannulas and anticoagulants can be used to obtain blood samples for PMN isolation. For storing temperatures 37 °C should be preferred. Regarding time between the PMN isolation and the actual experiments, a time span of maximum 1 h should be targeted.

Substance P blockade effects on peripheral blood leukocytic reaction in secondary chronic inflammation

In an experimental prospective controlled study the leukocyte response of peripheral blood on the background of blockade of receptors of substance P was studied on the carrageenan model of secondary chronic inflammation in 132 rats. Compared with the natural course of inflammation, at 6 hours there was a significant decrease in total white blood cells count by 1.28 (p&lt;0.05), as well as a significant decrease in the number of polymorphonuclear neutrophils by 4 times (p&lt;0.05) and the number of eosinophils by 3 times (p&lt;0.05) by 10 days. On day 28, there was a significant increase in the number of monocytes in 2 times (p&lt;0,01). The results of the study indicate that in natural conditions of inflammation, substance P stimulates blood leukocytes, as well as prolongs the inflammatory response, and its blocking helps to reduce the inflammatory response.

Identification of the key differentially expressed genes and pathways involved in neutrophilia.

Polymorphonuclear neutrophils (PMNs) are the most important determinants in the acute inflammatory response. Pathologically increased numbers of PMNs in the circulation or specific tissues (or both) lead to neutrophilia. However, the genes expressed and pathways involved in neutrophilia have yet to be elucidated. By analysis of three public microarray datasets related to neutrophilia (GSE64457, GSE54644, and GSE94923) and evaluation by gene ontology, pathway enrichment, protein–protein interaction networks, and hub genes analysis using multiple methods (DAVID, PATHER, Reactome, STRING, Reactome FI Plugin, and CytoHubba in Cytoscape), we identified the commonly up-regulated and down-regulated different expressed genes. We also discovered that multiple signaling pathways (IL-mediated, LPS-mediated, TNF-α, TLR cascades, MAPK, and PI3K-Akt) were involved in PMN regulation. Our findings suggest that the commonly expressed genes involved in regulation of multiple pathways were the underlying molecular mechanisms in the development of inflammatory, autoimmune, and hematologic diseases that share the common phenotypic characteristics of increased numbers of PMNs. Taken together, these data suggest that these genes are involved in the regulation of neutrophilia and that the corresponding gene products could serve as potential biomarkers and/or therapeutic targets for neutrophilia.

The Gut Microbiome Regulates Psychological Stress-Induced Inflammation in Sickle Cell Disease

Psychological stress (PS) is reported to precipitate vaso-occlusive episides (VOE) in sickle cell disease (SCD), but the mechanisms by which it does, are largely unknown. We subjected humanized (Berkeley) SCD mice to restraint stress and analyzed VOE using intravital microscopy. We found that stress exacerbated VOE as shown by reduced venular blood flow rates (control: 494 ± 30 × 103 pL/s; stress: 380 ± 20 × 103 pL/s; n = 25 - 43; p = 0.009) and reduced survival time (median survival time in control and stress groups: 394 min and 175 min, respectively; p = 0.02, Log-rank test). As previous studies show that adherent polymorphonuclear neutrophils (PMNs), especially aged PMNs marked by low CD62L and high CXCR4 expression, contribute to VOEs, we evaluated the behavior of leukocytes in live mice and observed a robust PMN response manifested by significantly increased adhesion and interactions with sickle RBC in stressed SCD animals. Flow cytometry analysis revealed a striking increase in aged PMNs in stressed SCD mice (control: 0.7 ± 0.1 × 106/ml; stress: 2.9 ± 0.3 × 106/ml; n = 3 - 4; p = 0.001), suggesting that the aged PMN expansion induced by stress may lead to the exacerbation of VOE in stressed SCD mice. Since aged PMN numbers are regulated by microbiota under steady state, we investigated whether response to PS also required the presence of the microbiota. Depletion of microbiota with broad-spectrum antibiotics (ABX) in SCD mice significantly prevented stress-induced aged PMN expansion (control: 4.8 ± 1.2 × 106/ml; ABX: 1.2 ± 0.3× 106/ml; n = 4 - 5; p = 0.0006), which was further confirmed using C57BL/6 germ-free (GF) mice (control: 1.1 ± 0.3 × 106/ml; GF: 0.2 ± 0.1 × 106/ml; n = 4; p = 0.003). Interestingly, analysis of inflammatory cytokines revealed a microbiota-dependent augmentation of IL-17A in the stressed mice. To investigate the role of IL-17A in the stress response, we neutralized IL-17A using an antibody (Ab) in C57BL/6 mice and found that IL-17A inhibition significantly dampened the aged PMN expansion induced by stress (isotype control Ab: 0.6 ± 0.1 × 106/ml; anti-IL-17A: 0.1 ± 0.0 × 106/ml; n = 4 - 5; p = 0.0004), which was also confirmed using IL-17A-/- mice. Further analyses revealed that Th17 lymphocytes were the major IL-17A-producing cells contributing to the stress response. In addition, depletion of microbiota with ABX or neutralization of IL-17A significantly alleviated stress-exacerbated VOE as measured by reduced PMN activation, improved blood flow rate and prolonged SCD mouse survival. As segmented filamentous bacteria (SFB) commensals were previously found to induce a Th17 response, we next tested the hypothesis that SFB was the driving bacteria in the stress response. Indeed, we found that PS-induced aged PMN expansion was greater in SFB+ mice (from Taconic) than in SFB- mice (from Jackson lab) (Taconic: 0.64 ± 0.11× 106/ml; Jax: 0.13 ± 0.02 × 106/ml; n = 4 - 5; p = 0.005). Depletion of SFB using vancomycin, a drug that eliminates Gram+ bacteria (including SFB), significantly reduced stress-induced aged PMN expansion. Furthermore, colonization of GF mice with SFB augmented aged PMN expansion after stress compared to stress control GF mice (control GF: 0.08 ± 0.02 × 106/ml; GF + SFB: 0.34 ± 0.10× 106/ml; n = 4 - 5; p = 0.04), suggesting that SFB was the key bacteria promoting aged PMN expansion during stress. The stress mechanisms involved the hypothalamic-pituitary-adrenal (HPA) axis since the blockade of glucocorticoid (GC) production using metyrapone or following adrenalectomy significantly prevented aged PMN expansion in stressed mice (PBS: 0.5 ± 0.1 × 106/ml; metyrapone: 0.1 ± 0.1× 106/ml; n = 5; p = 0.02. sham: 1.2 ± 0.2 × 106/ml; adrenalectomy: 0.4 ± 0.2 × 106/ml; n = 4 - 5; p = 0.01). Moreover, PS and dexamethasone greatly increased the gut permeability. Blockade of GC synthesis using metyrapone significantly alleviated stress-induced exacerbation of VOE manifested by reduced inflammation, improved the blood flow rate and prolonged SCD mice survival (median survival time in control and metyrapone groups: 155 min and 200 min, respectively; p = 0.02, Log-rank test). Overall, our results suggest that stress activation of the HPA axis increases the intestinal permeability, thereby promoting exposure to commensals that stimulate Th17 cells to produce IL-17A, which enables the expansion of aged polymorphonuclear neutrophils driving vaso-occlusion in SCD. Disclosures Frenette: Albert Einstein College of Medicine, Inc: Patents &amp; Royalties; Ironwood Pharmaceuticals: Research Funding; Cygnal Therapeutics: Equity Ownership; Pfizer: Consultancy.

Periovulatory administration of firocoxib did not alter ovulation rates and mitigated post-breeding inflammatory response in mares

Non-steroidal anti-inflammatory drugs (NSAIDs) are a therapeutic option for the treatment of inflammation. However, negative effects of non-selective NSAIDs for treatment of mares with endometritis have been described, including delayed uterine clearance and impairment of ovulations. Firocoxib is a specific cyclooxygenase-2 (COX-2) inhibitor and has the ability to act in the uterus of mares. We investigated the effects of firocoxib on ovulation rate, numbers of polymorphonuclear neutrophils (PMNs), and COX-2 protein levels in the endometrial tissue of susceptible mares after insemination. Two experiments were conducted. In experiment 1, twenty mares were evaluated in two consecutive estrous cycles broken into the following groups: Control - no pharmacological interference; Treatment - mares were treated with 0.2 mg/kg of firocoxib orally. The treatment began on the day of ovulation induction, and firocoxib was administered until one day after artificial insemination (AI). Ovulation was induced with 1 mg of deslorelin acetate and the mares were inseminated 24 h after the injection. Ovulation was confirmed 48 h after induction, and embryos were collected eight days after ovulation. Experiment 2: Nine mares susceptible to persistent mating-induced endometritis (PMIE) were artificially inseminated. The mares were examined with ultrasound and inseminated with fresh semen in two consecutive cycles, control and treated, in a cross-over study design. The amount of intrauterine fluid was measured, and endometrial samples were collected 24 h after AI. The number of PMNs was determined by endometrial cytology and biopsy, and COX-2 labeling in endometrial samples was evaluated by immunohistochemistry. Firocoxib treatment did not induce ovulatory failure or affect embryo recovery rate in Experiment 1. In Experiment 2, firocoxib treatment reduced inflammation after AI in mares as evidenced with results regarding PMN numbers/percentage and endometrial COX-2 staining. In conclusion, the proposed treatment with firocoxib reduced endometrial inflammation in mares susceptible to PMIE after breeding, with no adverse effects.

Impaired Peritoneal Macrophage Function and Delayed Neutrophil Recruitment are Secondary to a Leaky Gut in JAM‐A Deficient Mice

Junctional adhesion molecule‐A (JAM‐A) is a transmembrane glycoprotein expressed on leukocytes, endothelia and epithelia that regulates endothelial/epithelial barrier function as well as leukocyte transendothelial/transepithelial migration. While biological roles of JAM‐A have been extensively studied with JAM‐A knockout mice (Jam‐a KO), tissue‐specific JAM‐A deficient animals remain poorly documented. Therefore, we generated mice with tissue selective deletion of JAM‐A in myelomonocytic cells (LysM‐cre; Jam‐a fl/fl) and the intestinal epithelium (Villin‐cre; Jam‐a fl/fl). We then used an acute peritonitis model induced by intraperitoneal injection of zymosan or lipopolysaccharide (LPS) to study the role of tissue selective expression of JAM‐A in recruitment of polymorphonuclear neutrophils (PMN). Consistent with previous reports, infiltration of PMN into the peritoneum in response to zymosan or LPS was significantly reduced in Jam‐a KO versus Jam‐a WT mice. Surprisingly, in LysM‐cre; Jam‐a fl/fl, the number of PMN recruited into the peritoneum was not affected in response to zymosan nor LPS compared to control littermates, indicating that JAM‐A on PMN is not required for infiltration under such conditions. Importantly, intestinal epithelial‐selective loss of JAM‐A resulted in significant reduction of PMN infiltration in Villin‐cre; Jam‐a fl/fl mice, confirming that extrinsic factors, independent on JAM‐A expression on PMN are necessary for leukocyte recruitment to peritoneum under the conditions studied. We also observed that in response to zymosan or LPS, peritoneal macrophages from Jam‐a KO or Villin‐cre; Jam‐a fl/fl mice produced less of the chemokine CXCL1 and had reduced activity of the transcription factor NFkB. Conversely, peritoneal macrophages from LysM‐cre; Jam‐a fl/fl mice were unaffected compared to Jam‐a fl/fl mice. Given that loss of JAM‐A on intestinal epithelial cells resulted in increased intestinal permeability, we hypothesized that bacterial derived products may impair peritoneal macrophage function. Therefore, experiments were conducted with germ free Villin‐cre; Jam‐a fl/fl mice. We observed that macrophage responses as well as peritoneal recruitment of PMN in response to zymosan or LPS was not affected compared to control mice. These findings suggest that a leaky intestinal barrier and intestinal microbes are critical in regulating systemic neutrophil recruitment through effects on macrophage release of inflammatory mediators. Such observations highlight the functional link between the intestinal barrier, microbiota and the regulation of innate immune responses.Support or Funding InformationNIH grants 1R01DK097256 to TLD, DK59888, DK55679 to AN and DK072564, DK061379, and DK079392 to CP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

Polymorphonuclear Neutrophils Play a Decisive Role for Brain Injury and Neurological Recovery Poststroke.

Pro-inflammatory agents released by pathogens, dying host cells, and neutrophils act synergistically to destroy host tissues: a working hypothesis.

We postulate that the extensive cell and tissue damage inflicted by many infectious, inflammatory and post-inflammatory episodes is an enled result of a synergism among the invading microbial agents, host neutrophils and dead and dying cells in the nidus. Microbial toxins and other metabolites along with the plethora of pro-inflammatory agents released from activated neutrophils massively recruited to the infectious sites and high levels of cationic histones, other cationic peptides, proteinases and Th1 cytokines released from activated polymorphonuclear neutrophils (PMNs) and from necrotized tissues may act in concert (synergism) to bring about cell killing and tissue destruction. Multiple, diverse interactions among the many potential pro-inflammatory moieties have been described in these complex lesions. Such infections are often seen in the skin and aerodigestive tract where the tissue is exposed to the environment, but can occur in any tissue. Commonly, the tissue-destructive infections are caused by group A streptococci, pneumococci, Staphylococcus aureus, meningococci, Escherichia coli and Shigella, although many other microbial species are seen on occasion. All these microbial agents are characterized by their ability to recruit large numbers of PMNs. Given the complex nature of the disease process, it is proposed that, to treat these multifactorial disorders, a “cocktail” of anti-inflammatory agents combined with non-bacteriolytic antibiotics and measures to counteract the critical toxic role of cationic moieties might prove more effective than a strategy based on attacking the bacteria alone.

CD11b Regulates Fungal Outgrowth but Not Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis

Background and Aims: In immunosuppressed individuals Aspergillus (A.) fumigatus is a frequent cause of invasive pulmonary aspergillosis (IPA) which is highly associated with relevant morbidity and mortality. Moreover, it often occurs in patients suffering from leukocyte-adhesion deficiency type 1 (LAD1) which is triggered by a functional loss of CD18 in ß2 integrin receptors as these receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit. ß2 integrin receptors are differentially expressed by leukocytes, and are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Here, we asked for the importance of CD11b/CD18 also termed MAC-1 which is required for phagocytosis of opsonized A. fumigatus conidia by polymorphonuclear neutrophils (PMN) for control of pulmonary A. fumigatus infection.Methods: We used a murine IPA model (C57BL/6) and challenged CD11b deficient (CD11b-/-) or wild type (WT) mice with A. fumigatus conidia intratracheally. Afterwards, some mice were sacrificed 24h after infection. In these mice PMN recruitment and cytokine patterns were examined by analyzing bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) using flow cytometry, cytospin analysis, and cytometric bead array. Additionally, pulmonary fungal clearance and inflammation in murine lungs were analyzed by fungal culture assays and histopathologic examination using a scoring system. Furthermore, survival was studied with neutropenic animals serving as positive controls. To determine PMNs phagocytic activity and fungal killing capacity ex vivo, PMN were purified from bone marrow of CD11b-/- or WT mice by magnetic cell sorting using Ly6G specific antibodies. Afterwards, isolated PMN were stimulated with pacific blue-labeled tomato red-fluorescent modified A. fumigatus conidia and analyzed by flow cytometry.Results: We found that lung homogenates from CD11b-/- mice obtained 24h after infection showed an enhanced fungal burden as compared to lungs from WT mice. In contrast, lung tissue from infected CD11b-/- mice displayed impaired pulmonary inflammation as assessed by Hematoxilin-Eosin (HE) staining. Furthermore, the number of mucus-producing cells in bronchi of A. fumigatus infected CD11b-/- mice was decreased compared to cells in bronchi of WT mice. However, we observed markedly higher numbers of PMN in BALF of infected CD11b-/- mice compared to corresponding WT mice samples. In contrast, BALF derived from infected CD11b-/- mice contained lower levels of three different proinflammatory cytokines (TNF-α, IL-1α, IL-1β) compared to BALF from WT mice while levels of other cytokines and chemokines (IL-5, IL-6, IL-10, GM-CSF, CXCL1, CCL2) were comparable. In contrast, we measured higher levels of the chemokine CCL5 known as a relevant chemoattractant in innate and adaptive immune cells in BALF obtained from CD11b-/- mice. Interestingly, numbers of PMN, lymphocytes, and monocytes in the peripheral blood of A. fumigatus infected mice did not differ in a genotype-dependent manner. However, CD11b-/- mice PMN showed lower phagocytic activity than WT PMN, indicating an impaired capability to clear A. fumigatus. Nevertheless, CD11b-/- mice were finally characterized by similar long-term survival as WT mice after IPA induction while the PMN-depleted control mice died as expected.Conclusions: Our study demonstrates, that CD11b deficiency on myeloid cells affects the early course of IPA. This may be due to the importance of MAC-1 for PMN effector functions, and their interplay with DC and other leukocytes. Further work is necessary to elucidate the long-term course of IPA in CD11b-/- mice with regard to the interplay of PMN with dendritic cells, the efficacy of adaptive immune responses, and the potential pulmonary overexpression of CCL5. Beyond patients with LAD1 syndrome, we suggest our findings as being clinically relevant in other immunocompromised patients suffering from severe opportunistic infections. DisclosuresRadsak:Celgene: Honoraria, Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant; TEVA: Consultancy; Gilead: Other: Travel grant; Takeda: Consultancy; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Honoraria, Other: Travel grant.

IL-13 attenuates early local CXCL2-dependent neutrophil recruitment for Candida albicans clearance during a severe murine systemic infection

To investigate the role of IL-13 during a severe systemic Candida albicans infection, BALB/c control and IL-13−/− mice were examined for colony forming units (CFU) in the kidneys and survival days after intravenous infection. Proinflammatory mediators and cell recruitment into the tissue were measured by quantitative real-time PCR, a multiple ELISA system, and morphological cell differentiation. The IL-13-/- group exhibited a lower CFU number in the kidneys at 4 days and survived longer than the control mice, which was accompanied by significantly higher expression of C-X-C motif ligand 2 (CXCL2), IFN-γ, and polymorphonuclear neutrophils (PMNs) in the infected kidneys. By contrast, the expression of transforming growth factor β (TGF-β) and IL-17 A on day 10 were significantly higher in the control mice than in the IL-13-/- group. When using an intratracheal infection model, the IL-13-/- group recruited a greater number of PMNs in 6 h, with rapidly increased CXCL2 in the alveolar space. In vitro testing with cultured bone-marrow-derived cells demonstrated rapid CXCL2 mRNA upregulation at 3 h after contact with C. albicans, which decreased with recombinant IL-13 pretreatment, whereas rIL-13 retained TGF-β upregulation. In a murine model of Candida systemic infection, preexistent IL-13 limits both the rapid CXCL2 elevation and PMN aggregation in the target organ to suppress inflammatory mediators, which also attenuates local pathogen clearance within four days.

Can Sperm Selection, Inseminating Dose, and Artificial Insemination Technique Influence Endometrial Inflammatory Response in Mares?

The aim of this study was to investigate the influence of sperm concentration, sperm quality, and semen deposition site on uterine inflammatory response in mares. Twenty-five mares with healthy reproductive tracts were used for artificial inseminations (AIs) using frozen-thawed semen and divided into 5 experimental groups: Control—insemination with 800 × 106 total spermatozoa in the body of the uterus (n = 5); G2—insemination with 40 × 106 total motile sperm (TMS) into uterine body (n = 5); G3—insemination with 40 × 106 TMS at the uterotubal junction by hysteroscopy (HI) (n = 5); G4—insemination with 40 × 106 TMS at the uterotubal junction by HI, with frozen-thawed semen selected by Percoll gradient (n = 5); G5—insemination with 40 × 106 total motility sperm into uterine body, using frozen-thawed semen selected by Percoll gradient (n = 5). Number of polymorphonuclear neutrophils in uterine cytology was observed at 8 and 24 hours after AI. A reduced inflammatory endometrial reaction (P < .05) was observed, both at 8 and 24 hours, in mares inseminated with low sperm dose (40 × 106) compared with those inseminated with regular semen dose (800 × 106), with the exception of G4 that did not differ from the control group. Sperm selection by Percoll gradient did not influence the intensity of endometrial inflammatory response (P > .05) regardless of the type of insemination (conventional or hysteroscopic). In addition, mares inseminated by hysteroscopy showed a greater local inflammatory reaction (P < .05) in relation to those inseminated into uterine body. In conclusion, reduction of sperm insemination dose may be an alternative method to decrease endometrial inflammatory response in mares. However, hysteroscopic insemination and the current protocols used for sperm selection by Percoll did not show efficient results to reduce endometrial inflammation in mares. Further studies about sperm selection and HI insemination techniques are necessary to determine the benefits and the appropriate protocol to be performed in barren mares.