Response Of Polymorphonuclear Leukocytes Research Articles

Dual species transcriptomics reveals conserved metabolic and immunologic processes in interactions between human neutrophils and Neisseria gonorrhoeae.

Neisseria gonorrhoeae (the gonococcus, Gc) causes the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe lifelong sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs, neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing was used to define Gc and PMN transcriptional profiles alone and after infection. Core host and bacterial responses were assessed for two strains of Gc and three human donors' PMNs. Comparative analysis of Gc transcripts revealed overlap between Gc responses to PMNs, iron, and hydrogen peroxide; 98 transcripts were differentially expressed across both Gc strains in response to PMN co-culture, including iron-responsive and oxidative stress response genes. We experimentally determined that the iron-dependent TbpB is suppressed by PMN co-culture, and iron-limited Gc have a survival advantage when cultured with PMNs. Analysis of PMN transcripts modulated by Gc infection revealed differential expression of genes driving cell adhesion, migration, inflammatory responses, and inflammation resolution pathways. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and prostaglandin PGE2 were induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of two isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease.

Peptides Derived from Soybean β-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes.

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein β-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of β-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean β-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

Blood polymorphonuclear leukocyte responses against Staphylococcus aureus in primiparous and pluriparous Lacaune and Santa Inês ewes

• This study provides novel insights into PMNLs’ functionality in primiparous and pluriparous ewes. • A difference was observed in the intensity of the phagocytic response to S. aureus in pluriparous ewes. • Antibody-mediated opsonization of S. aureus was associated with a lower intensity of phagocytosis. In this study, we evaluated the viability, intracellular production of reactive oxygen species (ROS) and phagocytosis of Staphylococcus aureus by blood polymorphonuclear leukocytes (PMNLs) in primiparous and pluriparous Santa Inês and Lacaune ewes. Blood was collected from 14 Santa Inês ewes (seven primiparous and seven pluriparous) and 14 Lacaune ewes (seven primiparous and seven pluriparous). The viability of PMNLs, intracellular ROS production, and S. aureus phagocytosis were evaluated using flow cytometry. No parity effect on blood’s PMNLs functionality was found. However, comparing primiparous and pluriparous ewes, we noticed a difference in the opsonized phagocytic response to S. aureus , suggesting that pluriparous ewes could be better protected against S. aureus , although further field studies are needed. Antibody-mediated opsonization of S. aureus was associated with a lower intensity of S. aureus phagocytosis in both primiparous and pluriparous ewes, while it had no effect on the percentage of S. aureus phagocytosis. The percentage and geometric mean fluorescence intensity (GMFI) of S. aureus phagocytosis behaved differently in Lacaune and Santa Inês ewes, which could be explained by breed and farm effects. The intensity of intracellular ROS production by PMNLs when stimulated with S. aureus was higher than that of unstimulated basal PMNLs in both primiparous and pluriparous ewes, while no effect on the percentage of PMNLs that produced ROS was observed. Thus, our study provides novel insights into PMNLs’ functionality in primiparous and pluriparous ewes, which could impact their health, although further research is needed to extrapolate conclusions to the field.

The Extracellular Polysaccharide Matrix of Pseudomonas aeruginosa Biofilms Is a Determinant of Polymorphonuclear Leukocyte Responses.

Bacterial biofilms may cause chronic infections due to their ability to evade clearance by the immune system and antibiotics. The persistent biofilms induce a hyperinflammatory state that damages the surrounding host tissue. Knowledge about the components of biofilms that are responsible for provoking the harmful but inefficient immune response is limited. Flagella are known to stimulate the response of polymorphonuclear leukocytes (PMNs) to planktonic solitary bacteria. However, we provide evidence that flagella are not a prerequisite for the response of PMNs to Pseudomonas aeruginosa biofilms. Instead, we found that extracellular matrix polysaccharides in P. aeruginosa biofilms play a role in the response of PMNs toward biofilms. Using a set of P. aeruginosa mutants with the ability to produce a subset of matrix exopolysaccharides, we found that P. aeruginosa biofilms with distinct exopolysaccharide matrix components elicit distinct PMN responses. In particular, the PMNs respond aggressively toward a biofilm matrix consisting of both Psl and alginate exopolysaccharides. These findings are relevant for therapeutic strategies aimed at dampening the collateral damage associated with biofilm-based infections.

The protective roles of tea tree oil extracts in bovine mammary epithelial cells and polymorphonuclear leukocytes

BackgroundTea tree oil (TTO) plays an important role in antibacterial activity and alleviating the inflammatory responses. Bovine mammary epithelium and polymorphonuclear leukocytes (PMNL) can actively respond to bovine mastitis infection. However, regulatory effects of TTO extracts on the innate immune response of bovine mammary epithelial cells (BMECs) and PMNL remain not reported. Therefore, aim of the study was to evaluate the effects of TTO extracts on the mRNA levels of the genes involved in the innate immune response of BMECs and PMNL.ResultsOur results demonstrated that addition of 0.025% and 0.05% TTO increased the proliferation of BMECs, and significantly enhanced (P < 0.05) the viability of BMECs exposed to Staphylococcus aureus (S. aureus). An inhibitory effect was observed against the growth of S. aureus by TTO incubation. The 0.05% TTO reduced S. aureus biofilm formation, association and invasion of S. aureus to BMECs, and changed the morphological and structural features of S. aureus. The proinflammatory cytokines IL-1β, IL-6, and TNF-α were decreased (P < 0.001) by the incubation of TTO. Interestingly, the expression of IL-8 known for PMNL chemotactic function was elevated (P < 0.05) by 0.05% TTO treatment. Consistently, 0.05% TTO increased the migration of PMNL in S. aureus-exposed BMECs when compared with S. aureus treatment alone (P < 0.05). In addition, PMNL incubated with 0.05% TTO decreased the levels of NFKB inhibitor alpha (NFKBIA) and TNF-α.ConclusionsOur results indicate that use of TTO can relieve the BMECs pro-inflammatory response caused by S. aureus and promote the migration of PMNL to mount the innate immune responses, and it may be novel strategy for the treatment of bovine mastitis caused by S. aureus.

IL-17A induced hematopoietic reprogramming produces both PMN and MDSC at the post-acute stage of inflammation.

Abstract Our previous study suggests that polymorphonuclear leukocyte (PMN) response is enhanced at the post-acute stage of murine colitis due to changes in bone marrow (BM) myelopoiesis. Herein, we report that hematopoietic reprogramming during the post-acute phase of inflammation produces myeloid derived suppressor cells (MDSC) that co-infiltrate the intestine with PMN. Employing Percoll density gradients, we analyzed dynamic changes to the myeloid compartment by separating BM leukocytes from DSS colitis-to-recovery mice into four density-increasing bands with myeloid leukocytes enriched in bands III (50–60%) and IV (60–70%). Band III was comprised of monocytes and low-density granulocytes; both confirmed to be M-MDSCs and G-MDSCs, respectively, by displaying potent inhibition of T-cell proliferation. Despite significant decreases in the PMN population, presumably caused by mobilization into inflamed tissues, MDSC numbers significantly increase beginning at the post-acute stage of colitis. Furthermore, MDSC numbers do not return to baseline until late in the recovery stage. The expanded G-MDSCs also showed increased CXCR2 expression, which guides egress out of BM, and produced ROS upon PMA activation. Adoptive transfer assays demonstrated that functionally enhanced PMN infiltrated the intestine at the post-acute stage to clear the existing inflammatory reaction, while MDSC accumulated in the intestine during recovery suggesting a role in tissue repair. IL-17A signaling at the post-acute stage of inflammation likely induced hematopoietic reprogramming of the myeloid compartment, as its neutralization blocked expansion and infiltration of both PMN and MDSC.

Prepartal Energy Intake Alters Blood Polymorphonuclear Leukocyte Transcriptome During the Peripartal Period in Holstein Cows.

In the dairy industry, cow health and farmer profits depend on the balance between diet (ie, nutrient composition, daily intake) and metabolism. This is especially true during the transition period, where dramatic physiological changes foster vulnerability to immunosuppression, negative energy balance, and clinical and subclinical disorders. Using an Agilent microarray platform, this study examined changes in the transcriptome of bovine polymorphonuclear leukocytes (PMNLs) due to prepartal dietary intake. Holstein cows were fed a high-straw, control-energy diet (CON; NEL = 1.34 Mcal/kg) or overfed a moderate-energy diet (OVE; NEL = 1.62 Mcal/kg) during the dry period. Blood for PMNL isolation and metabolite analysis was collected at −14 and +7 days relative to parturition. At an analysis of variance false discovery rate <0.05, energy intake (OVE vs CON) influenced 1806 genes. Dynamic Impact Approach bioinformatics analysis classified treatment effects on Kyoto Encyclopedia of Genes and Genomes pathways, including activated oxidative phosphorylation and biosynthesis of unsaturated fatty acids and inhibited RNA polymerase, proteasome, and toll-like receptor signaling pathway. This analysis indicates that processes critical for energy metabolism and cellular and immune function were affected with mixed results. However, overall interpretation of the transcriptome data agreed in part with literature documenting a potentially detrimental, chronic activation of PMNL in response to overfeeding. The widespread, transcriptome-level changes captured here confirm the importance of dietary energy adjustments around calving on the immune system.

The Effect of Rosmarinic Acid on Immunological and Neurological Systems: A Basic Science and Clinical Review

Rosmarinic acid (RosA) is an ester of caffeic acid and 3,4-dihydroxyphenylactic acid. RosA has a number of beneficial biological activities, including antiviral, antibacterial, anti-inflammatory, and antioxidant properties. Oxidative stress is recognized as a common factor in many neurodegenerative diseases and is a proposed mechanism for age-related degenerative processes as a whole. RosA has shown antioxidative action, acting as a scavenger of free radicals. RosA protects neurons from oxidative stress, significantly attenuated hydrogen peroxide-induced reactive oxygen species generation, and apoptotic cell death and could contribute at least in part to neuroprotective effects. RosA has also been shown to suppress immunoglobulin responses and inflammatory responses of polymorphonuclear leukocytes which may explain its effectiveness at treating seasonal allergic rhinoconjuctivitis in human clinical trials. The anti-inflammatory effects of RosA along with its immunomodulatory effects may also make it a novel agent for the treatment of auto-immune disorders. This review is focused on the effect of RosA on both immunological and neurological systems and its potential as a novel therapeutic agent for the treatment of associated conditions, especially those in which oxidative stress is a factor.

Functional activity of neutrophilic polymorphonuclear leukocytes in the first five days postpartum.

To assess the chemotactic activity and phagocytic response of neutrophilic polymorphonuclear leukocytes among women in the first five days postpartum. A prospective, cross-sectional clinical-laboratory study was conducted. Data of 31 postpartum women during the first five days after vaginal delivery were compared with those of 24 healthy non-pregnant non-postpartum women matched for age. The inclusion criteria were postpartum, clinically and obstetrically healthy women; vaginal delivery, singleton pregnancy carried to term; non-hypertensive, hyperglycemic, allergic, malnourished or with autoimmune or neoplastic diseases; not having received vaccines or blood products in the last three months. The Control Group was chosen according to the same inclusion criteria but involving non-pregnant non-postpartum women. The chemotactic activity of neutrophilic polymorphonuclear leukocytes was assessed by determining the distance from directed migration to bacterial lipopolysaccharide, in three Boyden chamber assays. The phagocytic response was identified by assessing the Zymosan particles' ingestion in three assays carried out in Leighton tubes. The Student's t-test was used in the statistical analysis, adopting a 5% level of significance. The chemotactic activity of neutrophilic polymorphonuclear leukocytes from postpartum women in the presence of homologous (73.2 ± 6.9) and autologous (78.6 ± 13.9) sera showed a significant increase compared to the values observed in the Control Group (64.1 ± 4.1 and 66.6 ± 5.4). Both chemotactic response and phagocytosis ingestion phase of neutrophilic polymorphonuclear leukocytes were significantly increased (p < 0.05) in postpartum women compared to healthy non-pregnant and non-postpartum women. There was an increase in the chemotactic activity and phagocytic response of neutrophilic polymorphonuclear leukocytes during the first five days after vaginal delivery in women.

Glucose supplementation has minimal effects on blood neutrophil function and gene expression in vitro

During early lactation, glucose availability is low and the effect of glucose supply on bovine polymorphonuclear leukocyte (PMNL) function is poorly understood. The objective of this study was to determine the effect of glucose supplementation on the function and transcriptomic inflammatory response of PMNL from cows in early and mid-lactation in vitro. Twenty Holstein cows in early (n=10; days in milk=17±3.1) and mid-lactation (n=10; days in milk=168±14.8) were used for this study. Jugular blood was analyzed for serum concentrations of nonesterified fatty acids, β-hydroxybutyrate, and glucose. Polymorphonuclear leukocytes were isolated and diluted using RPMI (basal glucose concentration was 7.2 mM) to different concentrations of PMNL/mL for phagocytosis, chemotaxis, gene expression, and medium analyses. Working solutions of glucose (0 or 4 mM of d-glucose) and lipopolysaccharide (0 or 50μg/mL) were added and tubes were incubated for 120 min at 37°C. Media were analyzed for concentrations of glucose and tumor necrosis factor-α (TNF-α). Data were analyzed in a randomized block (stage of lactation) design. Challenge with lipopolysaccharide increased the expression of the genes encoding for nuclear factor kappa B (NFKB1), IL-10 (IL10), IL1B, IL6, IL8, TNF-α (TNFA), glucose transporter 3 (SLC2A3), and the concentration of TNF-α in medium (147.3 vs. 72.5 pg/mL for lipopolysaccharide and control, respectively). Main effect of stage of lactation was minimal where the expression of IL10 increased for cows in early compared with cows in mid-lactation. After lipopolysaccharide challenge, cows in early lactation experienced more marked increases in the expression of IL6, TNFA, and IL8 when compared with cows in mid-lactation. Glucose supplementation had minimal effects on gene expression where glucose supplementation increased the expression of lysozyme (LYZ). Glucose supplementation increased PMNL phagocytosis but did not alter chemotaxis, morphology, or concentration of TNF-α in the medium. Under the conditions of the experiment, stage of lactation had minimal effects on PMNL response to glucose supply where only the expression of NFKB1 and the production of TNF-α were greater for cows in mid-lactation when compared with early lactation. Metabolic profiles for cows in early lactation did not parallel those for cows during the early postpartum period and may partly explain results for this study. Future studies investigating the effect of glucose supply on bovine PMNL function in vivo and how this may be altered by stage of lactation are warranted.

Neisseria gonorrhoeae elicits extracellular traps in primary neutrophil culture while suppressing the oxidative burst.

ABSTRACT Neisseria gonorrhoeae (the gonococcus) causes gonorrhea and is uniquely adapted to survive within the human reproductive tract. Gonococci evade host immune surveillance in part by varying their pili and opacity-associated proteins. These variable surface antigens influence interactions with host epithelial and immune cells. A potent polymorphonuclear leukocyte (PMN) response is a hallmark of symptomatic gonococcal infection, with vast numbers of PMNs recruited to the site of infection. A large body of literature describes gonococcus-PMN interactions, but the factors driving the outcome of infection are not fully understood. Gonococci have been described to both induce and suppress the PMN oxidative burst, but we determined that gonococci differentially affect induction of the PMN oxidative burst depending on the multiplicity of infection (MOI). Infecting PMN at an MOI of <20 gonococci elicits an oxidative burst, while an MOI of >20 suppresses the burst. Oxidative burst in response to gonococci is enhanced by, but does not require, expression of pili or opacity proteins. Neutrophil extracellular traps (NETs) were observed in gonococcus-infected PMNs, a process which requires an oxidative burst, yet gonococci induced NETs under suppressing conditions. The NETs were unable to kill gonococci despite killing the common vaginal bacterium Lactobacillus crispatus. Thus, gonococci influence PMN biology to promote their own survival by suppressing the oxidative burst of PMNs and stimulating the formation of NETs, which do not effectively kill gonococci, illustrating how N. gonorrhoeae has evolved to modulate PMN responses to promote infection.

Rosmarinic Acid as a Novel Agent in the Treatment of Allergies and Asthma*

Rosmarinic acid, a caffeic acid ester and a component of several members of the Lamiaceae family including Rosmarinus officinalis, Perilla spp., and&nbsp;Salvia officinialis, has antioxidant and anti-inflammatory effects and is used for the treatment of asthma and reactive airway diseases, allergic disorders such as allergic rhinitis, otitis media, chemical sensitivity and multiple allergen reactivity. Rosmarinic acid can be used alone or in combination with other agents such as antioxidants, essential fatty acid supplements and other plant derived products. Rosmarinic acid can be administered orally and no adverse drug interactions have been reported. Nausea or mild stomach upset has been reported in sensitive patients in association with its oral intake and it is therefore recommended that Rosmarinic acid be ingested with food. Rosmarinic acid has shown free radical scavenging ability and suppression of allergic immunoglobulin and inflammatory responses of polymorphonuclear leukocytes, which may underlie its effectiveness in the treatment of allergic disorders, as demonstrated in clinical trials. Rosmarinic acid is a valuable agent for the treatment of allergic conditions, which is of importance considering the recent increase in the incidence of allergies, asthma and lung diseases associated with airborne pollutants.

Proefschriften 25 jaar na dato 35. Parodontale afbraak bij syndroom van Down: een immunologisch probleem

In Down syndrome the prevalence of periodontal disease is high. Twentyfive years ago in a series of controlled experiments, based on an experimental gingivitis model, clinical, histological and immunological characteristics of a group children with Down syndrome and matched control children were evaluated. In the Down syndrome children the gingival inflammation occurred earlier and was more extensive. On the tissue level the early response was characterized by a polymorphonuclear leucocytes response. Chemotaxis assays were performed to rule out impaired function. It was found that random migration for the peripheral blood-polymorphonuclear leucocytes and chemotaxis in both groups of children were comparable; hence such a factor cannot be responsible the early polymorphonuclear leucocytes' response in the children with Down syndrome. The most striking feature in the group with Down syndrome was the delayed and impaired response of lymphocytes during plaque development compared to the controls. This impaired lymphocyte function was also observed in a pilot study on 1 child with Down syndrome. It showed a less pronounced T cell suppressor function and a lack of immune regulation. The high level of gingival inflammation in children with Down syndrome must therefore be related to their impaired adaptive immunity.

Urethral inflammation in male chimpanzees caused by ureaplasmas and Chlamydia trachomatis

Specimens from men with acute non-gonococcal urethritis were tested to determine their microbial content and then given intra-urethrally to male chimpanzees. Two animals received ureaplasmas only and one became infected. The second did so when given a different strain. Both developed a polymorphonuclear leukocyte (PMNL) response. Two chimpanzees received a mixture of ureaplasmas and Chlamydia trachomatis and there was a suggestion that the ureaplasmas delayed or suppressed the chlamydial response. The latter, that is urethral infection with a pronounced PMNL response, was most clearly seen in a chimpanzee given C. trachomatis only. No inflammation was detected in two chimpanzees acting as controls. Three of five chimpanzees given ureaplasmas genitally, and one that had them endogenously, had them transiently in the oropharynx about 2 weeks later. The occurrence of ureaplasmas in the conjunctiva of three chimpanzees inoculated at this site was also transient and without inflammation. The possibility that Mycoplasma genitalium might have been in the inocula and caused urethral inflammation was discounted largely because no animal had antibody to this mycoplasma.

Effect of parenteral l-alanyl-l-glutamine administration on phagocytic responses of polymorphonuclear neutrophilic leukocytes in dogs undergoing high-dose methylprednisolone sodium succinate treatment

To determine whether parenteral l-alanyl-l-glutamine (Ala-Gln) administration modulated phagocytic responses of polymorphonuclear neutrophilic leukocytes (PMNs) from dogs undergoing high-dose methylprednisolone sodium succinate (MPSS) treatment. 15 healthy Beagles. Dogs were randomly assigned to 3 treatment groups (n = 5/group): 38-hour IV infusion of saline (0.9% NaCl) solution (control group), saline solution with 8.5% amino acids (2.3 g/kg/d), or saline solution with 8.5% amino acids (1.8 g/kg/d) and 20% l-alanyl-l-glutamine (Ala-Gln; 0.5 g/kg/d). High-dose MPSS treatment was initiated at the same time that IV infusions began, such that a total dose of 85 mg of MPSS/kg was administered through multiple IV injections over a 26-hour period. The infusions were maintained until 12 hours after the last MPSS injection. Blood samples collected before MPSS injections began and 2, 12, and 24 hours after injections ceased were used to evaluate PMN function. MPSS injections resulted in an increase in the total number of circulating leukocytes and increases in neutrophil and monocyte counts but did not affect lymphocyte, eosinophil, or basophil counts. Lymphocyte counts in the Ala-Gln group were higher than in the control group 12 hours after MPSS injections finished. Relative to preinfusion values, phagocytic capacity, oxidative burst activity, and filamentous actin polymerization of PMNs were suppressed in all dogs except those that received Ala-Gln. Parenteral Ala-Gln administration in dogs resulted in an increase in PMN phagocytic responses that were suppressed by high-dose MPSS treatment.

Estrogen negatively regulates epithelial wound healing and protective lipid mediator circuits in the cornea

Estrogen receptors (ERs) are expressed in leukocytes and in every ocular tissue. However, sex-specific differences and the role of estradiol in ocular inflammatory-reparative responses are not well understood. We found that female mice exhibited delayed corneal epithelial wound closure and attenuated polymorphonuclear (PMN) leukocyte responses, a phenotype recapitulated by estradiol treatment both in vivo (topically in male mice) and in vitro (corneal epithelial cell wound healing). The cornea expresses 15-lipoxygenase (15-LOX) and receptors for lipoxin A(4) (LXA(4)), which have been implicated in an intrinsic lipid circuit that regulates corneal inflammation and wound healing. Delayed epithelial wound healing correlated with lower expression of 15-LOX in the regenerated epithelium of female mice. Estradiol in vitro and in vivo down-regulated epithelial 15-LOX expression and LXA(4) formation, while estradiol abrogation of epithelial wound healing was completely reversed by treatment with LXA(4). More important, ERβ and ERα selectively regulated epithelial wound healing, PMN cell recruitment, and activity of the intrinsic 15-LOX/LXA(4) circuit. Our results demonstrate for the first time a sex-specific difference in the corneal reparative response, which is mediated by ERβ and ERα selective regulation of the epithelial and PMN 15-LOX/LXA(4) circuit. These findings may provide novel insights into the etiology of sex-specific ocular inflammatory diseases.

Bacterial toll-like receptor agonists induce sequential NF-κB-mediated leukotriene B4 and prostaglandin E2 production in chicken heterophils

Studies of the response of the primary avian polymorphonuclear leukocyte, the heterophil, to microbe associated molecular patterns (MAMPs) through toll-like receptors (TLR) has concentrated on the activation of the respiratory burst, release of intracellular granules, and the induction of cytokine and chemokine expression. Virtually no studies have been described on the role of lipid mediators, leukotrienes and prostaglandins, as effectors of the avian inflammatory response. We have previously shown that flagellin (FLG), the bacterial lipoprotein mimic palmitoly-3-cysteine–serine–lysine-4 (PAM), and unmethylated CpG motifs of bacteria DNA (CpG) are all potent activators of the avian innate immune system. In the present studies, we hypothesized that FLG, PAM, and CpG are also capable of eliciting the production of these lipid mediators of inflammation by avian heterophils. Compared to non-stimulated control heterophils, all three TLR agonists were potent inducers (3–5-fold increase) of a rapid production (30min) of leukotriene B4 (LTB4) followed by a later release (60–120min) of prostaglandin (PGE2) by the heterophils. LTB4 and PGE2 production were derived from lipoxygenase-5 (5-LO) and cyclooxygenase-2 (COX-2) enzymatic activities, respectively, as the selective 5-LO (caffeic acid) and COX-2 (NS-398) inhibitors eliminated LTB4 and PGE2 production from the MAMP-stimulated heterophils. These results demonstrate that both the lipoxygenase and cycloxygenase pathways are operational in avian heterophils in response to bacterial MAMPs. Treatment of heterophils with either FLG, PAM, or CpG also induced a significant increase in DNA binding by NF-κB family members’ p50, c-Rel, and RelB. Additionally, the production of LTB4 and PGE2 were inhibited following treatment of heterophils with the specific pharmacologic inhibitor of NF-κB (Bay 11–7086), thus suggesting that TLR pathway activation of NF-κB controls LTB4 and PGE2 production. This the first report of the production of lipid mediators of inflammation by avian heterophils in response to PAMPs. Since FLG, lipoproteins, and bacterial CpG DNA are abundant during bacterial infections, these data support their role in the inflammatory response mediated by avian heterophils.

Membrane cholesterol modulates the fluid shear stress response of polymorphonuclear leukocytes via its effects on membrane fluidity

Continuous exposure of polymorphonuclear leukocytes (PMNLs) to circulatory hemodynamics points to fluid flow as a biophysical regulator of their activity. Specifically, fluid flow-derived shear stresses deactivate leukocytes via actions on the conformational activities of proteins on the cell surface. Because membrane properties affect activities of membrane-bound proteins, we hypothesized that changes in the physical properties of cell membranes influence PMNL sensitivity to fluid shear stress. For this purpose, we modified PMNL membranes and showed that the cellular mechanosensitivity to shear was impaired whether we increased, reduced, or disrupted the organization of cholesterol within the lipid bilayer. Notably, PMNLs with enriched membrane cholesterol exhibited attenuated pseudopod retraction responses to shear that were recovered by select concentrations of benzyl alcohol (a membrane fluidizer). In fact, PMNL responses to shear positively correlated (R(2) = 0.96; P < 0.0001) with cholesterol-related membrane fluidity. Moreover, in low-density lipoprotein receptor-deficient (LDLr(-/-)) mice fed a high-fat diet (a hypercholesterolemia model), PMNL shear-responses correlated (R(2) = 0.5; P < 0.01) with blood concentrations of unesterified (i.e., free) cholesterol. In this regard, the shear-responses of PMNLs gradually diminished and eventually reversed as free cholesterol levels in blood increased during 8 wk of the high-fat diet. Collectively, our results provided evidence that cholesterol is an important component of the PMNL mechanotransducing capacity and elevated membrane cholesterol impairs PMNL shear-responses at least partially through its impact on membrane fluidity. This cholesterol-linked perturbation may contribute to dysregulated PMNL activity (e.g., chronic inflammation) related to hypercholesterolemia and causal for cardiovascular pathologies (e.g., atherosclerosis).

Nasal PMN response to repeated challenge with endotoxin in healthy volunteers

Rationale: We have employed nasal challenge with lipopolysaccharide (LPS) followed by nasal lavage (NL) to experimentally induce and examine upper airway inflammation in human volunteers. It is unclear however whether adaptation within individuals occurs following repeated nasal challenge. This was a pilot study to determine if repeated nasal LPS challenge yields attenuation of markers of inflammation (primarily neutrophil response) in the NL fluid of healthy humans.Methods: We employed a 3-day nasal LPS challenge protocol with NL using a “split nose” design. The control and LPS nares received two consecutive day saline (0.9% saline/day) and LPS (2 µg LPS/day) challenges, respectively followed by an LPS (2 µg/day) challenge to each nare on Day 3. NL was performed immediately pre Day 1 challenges and 6-h post nasal LPS challenges on both Days 1 and 3. Markers of inflammation (PMNs/mg, cytokines) were assessed in NL and the inflammatory response to LPS (measured as the difference between pre and post challenge) was evaluated in both nares on Day 3 and compared to Day 1.Results: Significant (p < 0.05) blunting of the LPS-induced polymorphonuclear leukocyte (PMN) response was observed in the nare that received repeated LPS challenges as compared to the control nare (67.60 ± 22.39 vs. 157.8 ± 76.04 PMN/mg) and initial LPS challenge on Day 1 (121 ± 32 PMN/mg). Decreased soluble CD14 and significantly decreased interleukin-8 were also found in the repeat LPS-treated nare. In the LPS-treated nare, the blunted PMN response on Day 3 correlated well with the observed PMN response on Day1 (r = 0.58, p = 0.02).Conclusions: We show attenuation of PMN response to repeated LPS in the nasal airways in healthy humans. Effect of repeat endotoxin exposure prior to allergen delivery on local airway inflammation in both healthy and atopic subjects can be studied.

Exogenous procalcitonin evokes a pro-inflammatory cytokine response

Procalcitonin (ProCT) is increased in serum of septic patients and those with systemic inflammation. Endogenous levels of ProCT might influence the response of polymorphonuclear leukocytes (PMNs), independently of endotoxin, in clinical disease. Healthy human volunteers. Recombinant human ProCT (rhProCT). Whole blood and PMNs were exposed in vitro to exogenous rhProCT. Interleukin (IL)-6, IL-8, IL-10, IL-13, tumor necrosis factor-alpha (TNFα), IL-1β, and macrophage inflammatory protein (MIP)-1β (pg/ml) were measured by multiplex suspension bead-array immunoassay, and migration and phagocytosis were measured in PMNs. In a whole-blood model, a dose-dependent increase in IL-6, TNFα, and IL-1β of the cell-free supernatant was noted. Pre-incubation with ProCT, at doses consistent with clinical sepsis, resulted in a decrease in PMN migration without alteration in phagocytosis of Staphylococcus aureus or indirect measurements of bacterial killing. Clinically relevant levels of ProCT influence immunologic responses that may contribute to systemic inflammatory response and septic shock.