Polymorphonuclear Leukocyte Function Research Articles

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 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.

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.

Effect of Hydrogen Sulfide on Essential Functions of Polymorphonuclear Leukocytes.

Impaired polymorphonuclear leukocyte (PMNL) functions contribute to increased infections and cardiovascular diseases in chronic kidney disease (CKD). Uremic toxins reduce hydrogen sulfide (H2S) levels and the anti-oxidant and anti-inflammatory effects of H2S. Its biosynthesis occurs as a side process of transsulfuration and in the disposal of adenosylhomocysteine, a transmethylation inhibitor and proposed uremic toxin. PMNL chemotaxis was measured by the under-agarose method, phagocytosis, and oxidative burst by flow cytometry in whole blood and apoptosis by determining DNA content by flow cytometry and morphological features by fluorescence microscopy. Sodium hydrogen sulfide (NaHS), diallyl trisulphide (DATS) and diallyl disulphide (DADS), cysteine, and GYY4137 were used as H2S-producing substances. Increased H2S concentrations did not affect chemotaxis and phagocytosis. NaHS primed PMNL oxidative burst activated by phorbol 12-myristate 13-acetate (PMA) or E. coli. Both DATS and cysteine significantly decreased E. coli-activated oxidative burst but had no effect on PMA stimulation. While NaHS, DADS, and cysteine attenuated PMNL apoptosis, GYY4137 decreased their viability. Experiments with signal transduction inhibitors suggest that the intrinsic apoptosis pathway is mainly involved in GYY4137-induced PMNL apoptosis and that GYY4137 and cysteine target signaling downstream of phosphoinositide 3-kinase.

Impacts of Various Nutraceutical Milk Replacer Supplements on the Health and Performance of High-Risk Holstein Calves

The objectives of this study were to determine the impacts of supplementing various nutraceuticals in milk replacers, including a blend of probiotics, β-glucan extract, mannanoligosaccharide extract, or a non-immunoglobulin rich extract, from colostrum on the performance and health of high-risk calves and to determine carry-over effects into the immediate post-weaned period. One hundred bull calves were acquired from a local calf ranch within 24 h of birth and randomly assigned to one of five dietary treatments added to milk replacer only: (1) Control (CON), no additive; (2) Immu-PRIME (ImmPr), 1.5 g/d ImmPr first 3 d only (Sterling Technology, Brookings, SD, USA); (3) Beta glucan (BG), 1 g/d BG extract (ImmuOligo, Irvine, CA, USA); (4) Mannanoligosaccharide + Bacillus subtilis (MOS+Bs), 3 g/d CEREVIDA EXCELL-M + 4 x 109 CFU/d Bacillus subtilis (MB Nutritional Sciences, Lubbock, TX, USA); and (5) PROVIDA CALF (PRO), proprietary blend of 2 x 109 CFU/d of a Lactobacillus casei and an Enterococcus faecium + 2 x 109 CFU/d Saccharomyces cerevisiae (MB Nutritional Sciences, Lubbock, TX, USA). Calves were weaned at d 56, comingled, and treatment carry-over effects evaluated through d 84. Starter intake was measured daily and BW weekly. Peripheral blood samples were collected and analyzed for hematology and serum was analyzed for haptoglobin concentrations. Polymorphonuclear leukocyte (PMNL) function was assessed through surface L-selectin expression, phagocytic, and oxidative burst activities against Escherichia coli. Data were analyzed using Proc Mixed in SAS (SAS 9.4, Cary, NC). The BG calves consumed the most starter from d 1 to 28 and pre-weaned average daily gain was greater for both the PRO and BG when compared to the CON group. There was a tendency for BG, MOS+Bs, and PRO to have reduced serum haptoglobin throughout the study. Total leukocyte, neutrophil, and lymphocyte counts were reduced among MOS+Bs calves, whereas BG calves tended to have the greatest neutrophil:lymphocyte ratio. Oxidative burst function for PMNL was reduced among BG and PRO treatments, suggesting a decreased need for neutrophil function. Supplementing BG, MOS+Bs, and PRO all influenced the performance and health of high-risk calves, but mechanistically appear to be different.

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.

Effect of injectable trace mineral supplementation on peripheral polymorphonuclear leukocyte function, antioxidant enzymes, health, and performance in dairy cows in semi-arid conditions

The objective of this study was to evaluate the effect of subcutaneous injections of 15 mg/mL Cu, 5 mg/mL Se, 60 mg/mL Zn, and 10 mg/mL Mn on health, performance, polymorphonuclear leukocyte (PMNL) function, circulating glutathione peroxidase (GPx) and superoxide dismutase (SOD) concentrations, and inflammation of dairy cows undergoing the transition period in high temperature-humidity index. A total of 923 multiparous cows from 2 commercial dairy farms were randomly allocated into 1 of 2 treatment groups as follows: control and injectable trace mineral supplementation (ITMS). Cows in the ITMS group received 7 mL of subcutaneous injections at dry-off (208 ± 3 d of gestation), 260 ± 3 d of gestation, and at 35 ± 3 d in milk (DIM). Data regarding health traits, reproductive performance, milk yield, and survivability were extracted from farm database software, and animals were followed-up until 300 DIM. For a subset of 142 cows from one herd, blood samples were collected at enrollment, and at 3 ± 1, 7 ± 1, 10 ± 1, and 35 ± 3 DIM to evaluate hematology, PMNL function, GPx and SOD concentrations, and circulating haptoglobin. Logistic regression was used to assess health and pregnancy per artificial insemination at first service. Cox proportional hazards models were used to evaluate hazard of pregnancy and culling. Mixed linear regression models accounting for repeated measures were used to assess all continuous variables collected over time. Parity, twinning, and previous gestation length were considered as potential confounders. Farm was included as a random effect. The ITMS cows tended to have lower incidence of metritis and stillbirth compared with control group. However, ITMS treatment did not influence the incidence of other diseases (e.g., mastitis, retained placenta), milk yield, reproductive performance, culling, and leukocyte count. Neutrophil-to-lymphocyte ratio, PMNL phagocytosis, and oxidative burst as well as intensity of the oxidative burst were greater for ITMS-treated cows in comparison to control cows. The ITMS cows had decreased expression of the adhesion molecule L-selectin on PMNL surface. The serum concentration of GPx and SOD were not affected by ITMS treatment. In conclusion, ITMS tended to reduce the incidence of metritis and stillbirth parturition, improved PMNL function, and improved the inflammatory status of dairy cows undergoing the transition period in high temperature-humidity index conditions. However, these findings did not translate into improved milk yield, reproductive performance, and survivability.

Prophylactic use of an ultra-diluted complex on polymorphonuclear leukocyte function and respiratory scores of weaned Holstein calves immediately after grouping

The aim of the study was to evaluate the effect of an ultra-diluted complex supplementation as a prophylactic strategy on immunity, performance, and respiratory scores of weaned Holstein calves immediately after grouping. Thirty-six weaned Holstein female calves (80.4±1.3 days old; 105.6±10.4 kg) were allocated to 6 pens (n=6 per pen) in a completely randomized design experiment in a double-blind, placebo-controlled trial. During a 28 days period, animals received a total mixed ration and were enrolled into two different groups (n=18 per group): 1) Control (basal diet + calcium carbonate, top-dressed at 30 g/animal/d – ultra-diluted placebo vehicle), or 2) ultra-diluted complex (basal diet + TopVita™-Real H, top-dressed at 30 g/animal/d – Sulphur:10-60 + Viola tricolor:10-14 + Caladium seguinum:10-30 + Zincum oxydatum:10-30 + Phosphorus:10-60 + Cardus marianus:10-60 + Colibacillinum:10-30 + Podophyllum:10-30 + Vehicle: calcium carbonate; q.s. 1kg). Blood samples were collected from each heifer at enrollment and 28 days later to assess polymorphonuclear leukocyte (PMNL) function and blood cell count. Body weight was assessed at enrollment and 28 days later at the end of the study. Regarding respiratory-screening process, a calf scoring system modified for calves in group pens was used. There was no effect of prophylactic ultra-diluted treatment on PMNL, nor it affected lymphocytes count and its ratio. Besides, the ultra-diluted product did not affect body weight and ADG. Further, no effect was observed in respiratory scores throughout the study period. In conclusion, the ultra-diluted complex did not improve blood cells count and PMNL function, nor it had impact on the performance of weaned Holstein calves after grouping.

Effect of High-Density Lipoprotein from Healthy Subjects and Chronic Kidney Disease Patients on the CD14 Expression on Polymorphonuclear Leukocytes.

In uremic patients, high-density lipoprotein (HDL) loses its anti-inflammatory features and can even become pro-inflammatory due to an altered protein composition. In chronic kidney disease (CKD), impaired functions of polymorphonuclear leukocytes (PMNLs) contribute to inflammation and an increased risk of cardiovascular disease. This study investigated the effect of HDL from CKD and hemodialysis (HD) patients on the CD14 expression on PMNLs. HDL was isolated using a one-step density gradient centrifugation. Isolation of PMNLs was carried out by discontinuous Ficoll-Hypaque density gradient centrifugation. CD14 surface expression was quantified by flow cytometry. The activity of the small GTPase Rac1 was determined by means of an activation pull-down assay. HDL increased the CD14 surface expression on PMNLs. This effect was more pronounced for HDL isolated from uremic patients. The acute phase protein serum amyloid A (SAA) caused higher CD14 expression, while SAA as part of an HDL particle did not. Lipid raft disruption with methyl-β-cyclodextrin led to a reduced CD14 expression in the absence and presence of HDL. HDL from healthy subjects but not from HD patients decreased the activity of Rac1. Considering the known anti-inflammatory effects of HDL, the finding that even HDL from healthy subjects increased the CD14 expression was unexpected. The pathophysiological relevance of this result needs further investigation.

Predicting the risk of retained fetal membranes and metritis in dairy cows according to prepartum hemogram and immune and metabolic status

Uterine diseases affect a significant proportion of dairy cows, causing significant economic losses. Immune and metabolic statuses are associated with the risk of retained fetal membranes (RFM) and metritis. The hypothesis of this study was that it is possible to use such responses to predict the risk of RFM and metritis. Data from cows (Jersey = 143, Holstein = 116) previously used in four experiments were used. Cow factors [parity, BCS change from −28 to 0 d relative to calving, calf sex, calving problems (twins, stillbirth, dystocia)] were evaluated for their association with the risk of RFM and metritis. Blood samples collected in the last week of gestation were used to measure polymorphonuclear leukocyte phagocytosis, oxidative burst, and expression of CD18 and CD62L, total blood count, haptoglobin optical density, and concentrations of glucose, non-esterified fatty acids, and β-hydroxybutyrate. Cows were treated with egg ovalbumin at −21, −7, and 7 d relative to calving and blood samples were used to determine the anti-ovalbumin IgG optical density. Univariable analyses were carried out to identify variables associated with the risk of RFM and metritis. The significant (P ≤ 0.15) variables were included in multivariable models from which variables with P > 0.15 were removed in a backward stepwise fashion. Risk of RFM was reduced in the absence of calving problems [adjusted odds ratio (AOR) 95 % confidence interval (CI) = 0.13 (0.02, 0.86)], when intensity of phagocytosis [AOR (95 % CI) = 0.50 (0.25, 1.04)] and expression of CD62L [AOR (95 % CI) = 0.71 (0.46, 1.09)] by polymorphonuclear leukocyte were greater, and when prepartum non-esterified fatty acid concentration was lower [AOR (95 % CI) = 8.71 (0.49, 153.84)]. Calf sex [AOR (95 % CI) = 0.35 (0.10, 1.25)], calving problem [AOR (95 % CI) = 0.14 (0.02, 1.08)], PMNL phagocytosis intensity [AOR (95 % CI) = 0.72 (0.47, 1.11)], polymorphonuclear leukocyte intensity of expression CD18 [AOR (95 % CI) = 0.78 (0.60, 1.00)] and CD62L [AOR (95 % CI) = 0.77 (0.60, 0.98)], and haptoglobin optical density [AOR (95 % CI) = 1.16 (0.97, 1.39)] were associated with metritis. Indexes generated from the estimates of the multivariable analyses to predict the risk of RFM [area under the curve = 0.77 (95 % CI = 0.70 to 0.84)] and metritis [area under the curve = 0.76 (95 % CI = 0.70, 0.81)] demonstrated that polymorphonuclear leukocyte function, non-esterified fatty acid concentration, and haptoglobin optical density prepartum may be used as predictors of uterine diseases.

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.

Glycolysis and mitochondrial function regulate the radical oxygen species production induced by platelet-activating factor in bovine polymorphonuclear leukocytes

Dairy cows undergo metabolic disturbances in the peripartum period, during which infectious inflammatory diseases and detrimental polymorphonuclear leukocytes (PMN) functions, such as radical oxygen species (ROS) production, are observed. Platelet-activating factor (PAF) is a key pro-inflammatory mediator that increases PMN ROS production. To date, the role of glycolysis and mitochondria in PAF-induced ROS production in bovine PMN has not been known. The aim of this study was to assess whether inhibition of glycolysis and disruption of mitochondrial function alter the oxidative response induced by PAF. We isolated PMN from non-pregnant Holstein Friesian heifers and pre-incubated them with 2-deoxy-d-glucose (2-DG; 2 mM, 30 min), carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 5 μM, 5 min), oligomycin (10 μM, 30 min) or rotenone (10 μM, 30 min). Respiratory burst was measured by luminol-chemiluminescence assay, while mitochondrial ROS (mtROS) were evaluated by MitoSOX probe and flow cytometry. Also, we detected the presence of mitochondria by MitoTracker Deep Red FM probe and changes in mitochondrial membrane potential (Δψm) were assessed by JC-1 probe and flow cytometry. We observed that all inhibitors separately were able to reduce PAF-induced ROS production. Presence of mitochondria was detected and PAF increased the Δψm, while CCCP reduced it. 2-DG and rotenone reduced the mtROS production induced by PAF. CCCP did not alter the mtROS and oligomycin administered independently increased mtROS production. We concluded that PAF-induced ROS production is glycolysis- and mitochondria-dependent. Bovine PMN have a functional mitochondrion and PAF induced mtROS via glycolysis and mitochondrial complex-I activity. Our results highlight an important modulation of cellular metabolism in the oxidative response induced by proinflammatory agents, which could contribute to PMN disfunction during peripartum in cattle.

The effect of prepartum negative dietary cation-anion difference and serum calcium concentration on blood neutrophil function in the transition period of healthy dairy cows

Our objectives were to assess the effects of a diet with a negative dietary cation-anion difference (DCAD) before calving on phagocytosis (Pc) and oxidative burst (OB) function of circulating neutrophils, and to determine the associations of serum ionized (iCa) and total calcium (tCa) concentrations with Pc and OB in transition dairy cows. We hypothesized that multiparous cows fed a negative DCAD diet prepartum would have greater iCa and tCa, and thus improved Pc and OB. From 3 wk before expected parturition until calving, 38 healthy multiparous cows from 3 farms were assigned to negative DCAD treatment (TRT; -100 mEq/kg of diet dry matter; n = 21) or a control (CON; 95 mEq/kg of dry matter; n = 17) diet. Each farm was on one treatment or the other at a time, but all farms contributed cows to both groups. Urine pH was measured weekly and in TRT was 6.1 ± 0.8 with 80% of 50 samples <7 and 74% ≤ 6.5. Phagocytosis, OB, iCa, and tCa were measured at d -7, 1, and 4 relative to calving. Median fluorescence intensity for Pc (MFIP) and OB (MFIOB), and the shift of percentage of cells active for Pc (PPc) and OB (POB) were measured in isolated, stimulated neutrophils via flow cytometry. Outcomes were assessed with mixed linear regression models accounting for repeated measures. There were no differences between treatments in the 4 neutrophil function outcomes. Although MFIOB varied over time, there were no interactions of treatment with time for any outcome. Serum ionized and tCa did not differ between TRT and CON. The least squares means ± standard deviation for iCa were: d -7, 1.23 ± 0.12 vs. 1.21 ± 0.12; d 1, 1.07 ± 0.12 vs. 1.02 ± 0.12; d 4, 1.16 ± 0.12 vs. 1.17 ± 0.12 mmol/L for TRT and CON, respectively; and for tCa: d -7 2.39 ± 0.25 vs 2.44 ± 0.31; d 1, 2.01 ± 0.25 vs 1.97 ± 0.31; d 4, 2.33 ± 0.25 vs 2.32 ± 0.31 mmol/L, respectively. The proportion of blood samples with tCa <2.15mmol/L at d -7, 1 and 4 was 5, 76, and 13%, respectively, with no differences between TRT and CON. Correlations of iCa or tCa with each of the 4 polymorphonuclear leukocyte (PMN) function outcomes were weak (r < |0.3|). We did not observe the hypothesized differences in aspects of innate immunity in clinically healthy multiparous cows fed a negative DCAD. We underline that cows that experienced clinical disease were excluded from this study, which is important for interpretation of the results.

Immune profile of hyaluronic acid hydrogel polyethylene glycol crosslinked: An in vitro evaluation in human polymorphonuclear leukocytes.

Neauvia hydrogel (N-Gel) is a hyaluronic acid-based dermal filler, cross-linked with polyethylene glycol. This filler contains sodium hyaluronate at different concentrations, poly(ethylene glycol) diglycidyl ether cross-linked, glycine, and l-prolyne. Assessing any effects of N-Gel on immunity and inflammation is of crucial importance. The aim of the study was to characterize the ability of N-Gel to modulate human polymorphonuclear leukocyte (PMN) functions, including migration, oxidative metabolism, and production of proinflammatory mediators. N-Gel was tested on isolated human PMN. Spontaneous and N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated migration were examined using the Boyden Chamber technique, whereas the oxidative metabolism was assessed through spectrofluorometric measurement of reactive oxygen species (ROS) production under resting conditions and after stimulation with fMLP. Tumor necrosis factor (TNF)-α and interleukin (IL)-8 mRNA levels were measured by real-time PCR after stimulation with fMLP or Escherichia coli lipopolysaccharide. This study showed that N-Gel reduced fMLP-induced migration and ROS production without affecting these functions in resting cells. In addition, incubation of PMN with N-Gel effectively reduced both TNF-α and IL-8 mRNA levels. N-Gel modulates critical functions of human PMN such as migration and oxidative metabolism, indicating its potential as an anti-inflammatory agent.

Associations between circulating levels of natural antibodies, total serum immunoglobulins, and polymorphonuclear leukocyte function in early postpartum dairy cows

The objective of this study was to investigate the associations between natural antibodies (NAbs) and total serum immunoglobulins (Igs) concentrations (both from IgG and IgM isotypes), with variables of polymorphonuclear leukocytes (PMN) function of dairy cows in the early postpartum period. Seventy-five healthy postpartum dairy cows at 2 ± 1 days in milk were enrolled in a cross-sectional study. Natural and total antibodies in serum samples were measured by ELISA. Flow cytometry was used to determine the phagocytosis and oxidative burst capacities of PMN and the quantification of the adhesion molecule l-selectin. Leukocyte count and differentials, and serum haptoglobin were also measured. A positive correlation between NAbsIgM and total serum IgM and between NAbsIgM and NAbsIgG were found. Before performing the associations between circulating Igs concentrations and PMN function variables, cows were categorized into having low, medium or high circulating antibodies based on their NAbs and total Igs serum concentrations. None of the PMN variables assessed differed between low, medium, and high cows for both NAbsIgM and NAbsIgG. While associations between PMNs function and total IgG were not observed, some associations between total IgM concentrations and PMN activity were found. Cows with high serum IgM had greater phagocytic activity compared to cows in the low IgM group. Finally, the proportion of PMN that performed oxidative burst and PMN surface expression of l-selectin intensity was greater in high total IgM group when compared to medium and low groups. In conclusion, association between NAbs, and PMN activity variables were not observed, but total serum IgM was associated to some PMN function variables in early post-partum dairy cows.

PSVI-5 Rumen-protected methionine supplementation during the peripartal period reduces the expression of LGALS-1, -3 and -4 in polymorphonuclear leukocytes (PMNL) and secretion of Gal-2 and Gal-12 in plasma of Holstein cows

Abstract The objective of this study was to evaluate the effect of methionine(Met) supplementation on genes encoding Galectins (LGALS) and secretion in plasma during the periparturient period. Dairy cows are susceptible to increased incidence and severity of disease during the periparturient period. Impaired polymorphonuclear leukocyte (PMNL) functions such as phagocytosis and pathogen killing capacity contribute to immune dysfunction and disease. Galectins (GALS) are an evolutionarily conserved family of animal lectins important in disease and homeostasis. They behave like pathogen-associated molecular patterns and cell-surface receptors whose activation often results in signaling cascades to activate cells such as PMNL. Fourteen Holstein cows supplemented with either rumen-protected Met (n = 7) or no Met (CON; n = 7) were used. Diets were fed from −21 ± 2 before expected calving date and continued until 30 days postpartum. To evaluate LGALS expression and secretion of Gal, PMNL were isolated from blood and plasma was harvested at -10, +7, and +30 d relative to expected calving. The expression of LGALS1 (P = 0.09), LGALS3 (P = 0.02), and LGALS4 (P = 0.02) was lower in response to Met, but there was no effect on LGALS8, LGALS9, and LGALS12. Met supplementation reduced Gal-2 and Gal-12 secretion in plasma at -10, +7, and +30 d (P &amp;lt; 0.0005 and P &amp;lt; 0.0001 respectively). Methionine supplementation modulates the expression of GAL involved in inflammation and lipolysis in cow blood. Modulation of GAL in PMNL and plasma in response to Met may have implications for the use of dietary interventions to target gene expression in PMNL for improved function and needs further study.

NADPH Oxidase Controls Neutrophil Infiltration in the Lung in Early Response to Fungal Cell Walls Via LTB4

The leukocyte NADPH oxidase generates superoxide, the precursor to reactive oxygen species important for host defense and immunoregulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD), characterized by serious bacterial and fungal infections and aberrant inflammation. Aspergillus pneumonia is frequent, and associated with pyogranulomatous inflammation. Of note, even sterile fungal cell walls elicit increased neutrophilic inflammation in CGD mice, demonstrating that that NADPH oxidase can limit inflammation independent of its antimicrobial effects. Leukotriene B4 (LTB4) is a potent inflammatory mediator, acting as a chemoattractant and activating other polymorphonuclear leukocyte (PMN) functions. PMN themselves are prominent source of LTB4, and sense LTB4 through its receptor BLT1, which stimulates additional LTB4 synthesis by autocrine and paracrine routes. This leads to feed forward amplification of LTB4 production locally and formation of PMN clusters. Ca2+ acts as an important second messenger in PMN, including regulation of LTB4 synthesis. Activated human CGD PMN have increased intracellular Ca2+ levels compared to PMN from healthy donors due to electrogenic effects of the NADPH oxidase (Geiszt M. et al JBC 1997). Intracellular Ca2+ overload in PMN is speculated to contribute to increased PMN pro-inflammatory activity, but specific pathways and in vivo relevance are not well defined. Therefore, we therefore investigated the production of LTB4 by murine CGD PMN and the role of LTB4 in PMN recruitment and lung hyperinflammation following pulmonary challenge with zymosan, a sterile fungal cell wall preparation. Zymosan-stimulated PMN from Cybb-null mice, a model for X-linked CGD, produced higher LTB4 in vitro compared to wild type (WT), even in the presence of SOD and catalase to counteract oxidative degradation. Higher LTB4 production correlated with higher levels of intracellular Ca2+ in stimulated X-CGD PMN. The increased LTB4 produced by X-CGD PMN was dependent on cell density and the LTB4 receptor BLT1, consistent with a feed-forward loop that amplified LTB4 production. Zymosan-stimulated X-CGD PMN also formed larger and more numerous clusters in vitro compared to WT PMN. Cluster formation was abrogated by inhibiting LTB4 synthesis with the 5-lipoxygenase (5-LO) inhibitor zileuton or by the BLT1 receptor antagonist U75302, demonstrating that cluster formation was LTB4 and BLT1 dependent. We next examined whether LTB4 regulated the response to zymosan in the lung. Following intranasal administration of zymosan, X-CGD mice had higher PMN numbers in bronchoalveolar lavage (BAL), larger PMN foci by lung histology, and increased lung LTB4 as compared to WT mice. These differences were evident by 8 hr and progressed over the first 24 hr post-challenge, in contrast to WT mice where inflammation plateaued at 8 hr. Treatment with zileuton or U75302 just prior to zymosan significantly reduced lung PMN numbers for both WT and X-CGD mice at 8 hr post challenge, and lung PMN remained substantially reduced in X-CGD mice at 24 hrs. Moreover, delaying administration of zileuton to 8 hr after zymosan challenge also significantly decreased PMN inflammation in X-CGD but not WT mice at 24 hrs, indicating that ongoing synthesis of LTB4 otherwise continued to promote PMN recruitment in CGD mice. These results demonstrate that PMN production of LTB4 in response to fungal cell walls is limited by NADPH oxidase via its effects on intracellular Ca2+. In oxidase-deficient CGD mice, LTB4 plays a major role in driving excessive PMN recruitment to the lung in the early response to zymosan. This study is the first to implicate LTB4 in promoting neutrophilic lung inflammation in response to fungal cell walls in CGD, likely by an amplified feed-forward loop involving increased production of LTB4 by CGD PMN. LTB4 could be a potential therapeutic target to ameliorate CGD hyperinflammation. Disclosures No relevant conflicts of interest to declare.

Club cell protein 16 in sera from trauma patients modulates neutrophil migration and functionality via CXCR1 and CXCR2

BackgroundClub Cell protein (CC)16 correlates with lung injury and respiratory complications, which are in part triggered by polymorphonuclear leukocytes (PMNL) in severely traumatized patients (TP). CC16 exerts anti-inflammatory and immunosuppressive effects, however, its influence on PMNL functions after trauma is unknown. Here, we evaluated whether CC16 present in sera from TP could modify the biological functions of PMNL.MethodsSera from 16 severely injured TP without pneumonia (no P, n = 8) or with pneumonia (P, n = 8) were collected at admission to emergency department (ED) and 1 day prior pneumonia and pre-incubated with or without anti-CC16 antibody for CC16 neutralization. Samples from the equal post-injury days in the corresponding no P group were used. Neutrophils were isolated from healthy volunteers (HV, n = 5) and incubated with 20% of the serum medium from TP, respectively. In PMNL, CD62L, CD11b/CD18 and CD31 expression, migratory capacity, phagocytosis rate, oxidative burst and apoptosis were investigated. In isolated PMNL, CXCR1 and CXCR2 were neutralized before stimulation with CC16, and oxidative burst, phagocytosis and apoptosis were analyzed in neutrophils and their subsets.ResultsSerum from the P group enhanced significantly PMNL migration compared to no P group, while CC16-neutralization further increased the migratory rate of PMNL in both groups. CC16-neutralization increased significantly the expression of CD62L in the P group at ED. Oxidative burst was significantly increased in the P group vs. no P during the study period. CC16 seemed to have no influence on oxidative burst and phagocytosis in TP. However, in a more controlled study design, CC16 induced a significant increase of oxidative burst and a decrease of apoptosis of CD16+ granulocytes. These effects were markedly observed in mature CD16brightCD62Lbright and immune suppressive CD16brightCD62Ldim neutrophils. In mature subset, CXCR1 and CXCR2 neutralization diminished CC16-induced effects.ConclusionsCC16 in sera from multiply traumatized patients, notably of those with pneumonia, has significant effects on PMNL. The results suggest an association of CC16 with CXCR1 and CXCR2. Our data suggest that CC16 reduces the migratory capacity of PMNL and thus modulates their function in patients with respiratory complications after trauma.

Targeted Protein Delivery: Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions (Adv. Healthcare Mater. 17/2019)

Targeted Protein Delivery: Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions (Adv. Healthcare Mater. 17/2019)

Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions.

The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury.