Adjunctive Immunotherapeutic Efficacy of N-Formylated Internal Peptide of Mycobacterial Glutamine Synthetase in Mouse Model of Tuberculosis.

BACKGROUND: Host-directed therapies are a comparatively new and promising method for the treatment of tuberculosis. A variety of host pathways, vaccines and drugs have the potential to provide novel adjunctive therapies for the treatment of tuberculosis. In this connection, we have earlier reported the immunotherapeutic potential of N-formylated N-terminal peptide of glutamine synthetase of Mycobacterim tuberculosis H37Rv (Mir SA and Sharma S, 2014). Now in the present study, we investigated the immunotherapeutic effect of N-terminally formylated internal-peptide 'f- MLLLPD' of mycobacterial glutamine synthetase (Rv2220) in mouse model of tuberculosis. METHODS: The N-terminally formylated peptide, f-MLLLPD was tested for its potential to generate Reactive Oxygen Species (ROS) in murine neutrophils. Further, its therapeutic effect alone or in combination with anti-tubercular drugs was evaluated in mouse model of tuberculosis. RESULTS: The f-MLLLPD peptide treatment alone and in combination with ATDs reduced the bacterial load (indicated as colony forming units) in lungs of infected mice by 0.58 (p<0.01) and 2.92 (p<0.001) log10 units respectively and in their spleens by 0.46 (p<0.05) and 2.46 (p<0.001) log10 units respectively. In addition, the observed histopathological results correlated well with the CFU data. CONCLUSION: The results of the current study show that f-MLLLPD peptide confers an additional therapeutic efficacy to the anti-tuberculosis drugs.

Downregulation of Microparticle Release and Pro-Inflammatory Properties of Activated Human Polymorphonuclear Neutrophils by LMW Fucoidan.

Exposition of neutrophils (polymorphonuclear neutrophils, PMNs) to bacterial products triggers exacerbated activation of these cells, increasing their harmful effects on host tissues. We evaluated the possibility of interfering with the classic immune innate responses of human PMNs exposed to bacterial endotoxin (lipopolysaccharide, LPS), and further stimulated with bacterial formyl peptide (N-formyl-methionine-leucine-phenylalanine, fMLP). We showed that the low- molecular-weight fucoidan (LMW-Fuc), a polysaccharide extracted from brown algae, attenuated the exacerbated activation induced by fMLP on LPS-primed PMNs, in vitro, impairing chemotaxis, NET formation, and the pro-survival and pro-oxidative effects. LMW-Fuc also inhibited the activation of canonical signaling pathways, AKT, bad, p47phox and MLC, activated by the exposition of PMN to bacterial products. The activation of PMN by sequential exposure to LPS and fMLP induced the release of L-selectin+ microparticles, which were able to trigger extracellular reactive oxygen species production by fresh PMNs and macrophages. Furthermore, we observed that LMW-Fuc inhibited microparticle release from activated PMN. In vivo experiments showed that circulating PMN-derived microparticles could be detected in mice exposed to bacterial products (LPS/fMLP), being downregulated in animals treated with LMW-Fuc. The data highlight the autocrine and paracrine role of pro-inflammatory microparticles derived from activated PMN and demonstrate the anti-inflammatory effects of LMW-Fuc on these cells.

Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor.

N-formyl-Met-Leu-Phe (fMLF) is a model PAMP/DAMP driving human PMN to sites of injury/infection utilizing the GPCR, FPR1. We examined a microtiter plate format for measurement of FPR1 phosphorylation in adherent PMN at high densities and found that a new phosphosensitive FPR1 fragment, 25K-FPR1, accumulates in SDS-PAGE extracts. 25K-FPR1 is fully inhibited by diisopropylfluorophosphate PMN pretreatment but is not physiologic, as its formation failed to be significantly perturbed by ATP depletion, time and temperature of adherence, or adherence mechanism. 25K-FPR1 was minimized by extracting fMLF-exposed PMN in lithium dodecylsulfate at 4°C prior to reduction/alkylation. After exposure of adherent PMN to a 5 log range of PAF before or after fMLF, unlike in suspension PMN, no inhibition of fMLF-induced FPR1 phosphorylation was observed. However, PAF induced the release of 40% of PMN lactate dehydrogenase, implying significant cell lysis. We infer that PAF-induced inhibition of fMLF-dependent FPR1 phosphorylation observed in suspension PMN does not occur in the unlysed adherent PMN. We speculate that although the conditions of the assay may induce PAF-stimulated necrosis, the cell densities on the plates may approach levels observed in inflamed tissues and provide for an explanation of PAF's divergent effects on FPR1 phosphorylation as well as PMN function.

Vasoactive intestinal peptide dampens formyl-peptide-induced ROS production and inflammation by targeting a MAPK-p47phox phosphorylation pathway in monocytes.

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47phox, p67phox, p40phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal-regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the "MAPK-p47phox phosphorylation-NOX2 activation" axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.

Neutrophil Receptor Response to Bacterial N-formyl Peptides is Similar in Term Newborn Infants and Adults in Contrast to IL-8.

We have previously observed that neutrophils from neonates exhibit different migratory responses to intermediate and end-target chemoattractants compared to adults. The aim of this study was to investigate the effect of the chemoattractants IL-8 (intermediate) and formyl-methionyl-leucyl-phenylalanine (fMLP; end-target) on cell surface receptor expression involved in adhesion, migration and granule release of neutrophils from term newborn infants and adults. Heparinized cord blood from 16 healthy term newborn infants delivered by caesarean section and peripheral blood from 17 healthy adults were incubated with 1 µm IL-8 or 0.1 µm fMLP, previously defined as optimal inducers of neutrophil migration. The leukocytes were labelled with antibodies to cell surface receptors (CD11b, CD15S, CD18, CD35, CD44, CD64, CD65, CD88, CD162, CD181 and CD182). Receptor expression was quantified by flow cytometry analysis. Upregulation of CD11b and downregulation of CD88 and CD182 after stimulation with IL-8 were more pronounced in adults than in neonates (P < 0.05, P < 0.05 and P ≤ 0.001, respectively), whereas fMLP induced changes in receptor expression that were of the same magnitude in neutrophils from neonates as from adults. We observed similar expression of receptors that mediate adhesion, migration, granule activation and phagocytosis induced by fMLP in neutrophils from neonates and adults. In contrast, differences between neonates and adults, induced by IL-8, suggest that the neutrophil response to intermediate chemoattractants might lead to a compromised infectious response in newborn infants.

An iminosugar-based heparanase inhibitor heparastatin (SF4) suppresses infiltration of neutrophils and monocytes into inflamed dorsal air pouches.

Local infiltration of inflammatory cells is regulated by a number of biological steps during which the cells likely penetrate through subendothelial basement membranes that contain heparan sulfate proteoglycans. In the present study, we examined whether administration of heparastatin (SF4), an iminosugar-based inhibitor of heparanase, could suppress local inflammation and degradation of heparan sulfate proteoglycans in basement membranes. In a carrageenan- or formyl peptide-induced dorsal air pouch inflammation model, the number of infiltrated neutrophils and monocytes was significantly lower in mice after topical administration of heparastatin (SF4). The concentration of chemokines MIP-2 and KC in pouch exudates of drug-treated mice was similar to control. In a zymosan-induced peritonitis model, the number of infiltrated cells was not altered in drug-treated mice. To further test how heparastatin (SF4) influences transmigration of inflammatory neutrophils, its suppressive effect on migration and matrix degradation was examined in vitro. In the presence of heparastatin (SF4), the number of neutrophils that infiltrated across a Matrigel-coated polycarbonate membrane was significantly lower, while the number of neutrophils passing through an uncoated membrane was not altered. Lysate of bone marrow-derived neutrophils released sulfate-radiolabeled macromolecules from basement membrane-like extracellular matrix, which was suppressed by heparastatin (SF4). Heparan sulfate degradation activity was almost completely abolished after incubation of lysate with protein G-conjugated anti-heparanase monoclonal antibody, strongly suggesting that the activity was due to heparanase-mediated degradation. Taken together, in a dorsal air pouch inflammation model heparastatin (SF4) potentially suppresses extravasation of inflammatory cells by impairing the degradation of basement membrane heparan sulfate.

The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation.

Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton's tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation.

In Vitro Evaluation of the Allergic Potential of Antibacterial Peptides: Camel and Citropin.

Peptide-based drugs are promising group of compounds which are characterized by specificity to their in vivo targets and high potency of action (antineoplastic, immunoregulatory, antibacterial). The peptides, however, involve a relatively high risk of allergic reactions that are not predictable on the basis of their sequence and chemical properties. In this study, peripheral blood was obtained from 53 patients including 38 hypersensitive patients and 15 control patients. Basophil activation stimulated by two antibacterial peptides (camel, citropin 1.1), and acetylsalicylic acid was assessed by means of BAT (basophil activation test). Basophil activation stimulated by camel occurred in 7 of 38 patients with hypersensitivity (18.42%) as well as in 2 of 15 control patients (13.33%). Basophils were activated by citropin 1.1 in 7 of 38 hypersensitive patients (18.42%) and in none of the control patients. Using the Structural Database of Allergenic Proteins, we confirmed that the examined peptides share some structural similarities with common environmental allergens. Therefore, the cross-reactivity between potentially present anti-allergen IgE with examined peptides cannot be excluded. Our study proved that BAT, together with other biological tests and specific databases of allergenic compounds, may serve as an initial selection of new active peptides and proteins.

The Impact of Intramedullary Nailing of Tibia Fractures on the Innate Immune System.

INTRODUCTION: Inflammation after trauma is thought to be aggravated by intramedullary nailing (IMN) and predisposes to acute respiratory distress syndrome. Polymorphonuclear granulocytes (PMNs) are the main effector cells in this process. However, in patients with a femur fracture, the injury severity was the decisive factor for the PMN phenotype. A tibia fracture is often caused by a more moderate injury and might allow for a window to assess the innate immune response caused by IMN. METHODS: A consecutive series of patients with a tibia fracture were included. The innate immune response was measured before and after IMN by plasma interleukin 6, PMN Mac1, and active FcγRII (FcγRII*) expression both before and after fMLF (N-formylmethionyl-leucyl-phenylalanine) stimulation. Furthermore, HLA-DR on monocytes was analyzed. RESULTS: Twenty-five consecutive patients were included. Polymorphonuclear granulocyte fMLF-induced Mac1 and FcγRII* were decreased. In concordance, HLA-DR expression on monocytes was decreased in patients compared with control subjects. Intramedullary nailing was associated with a further decrease of HLA-DR-positive monocytes, whereas no changes in PMN phenotype or plasma interleukin 6 levels were observed. CONCLUSION: Intramedullary nailing of a tibial fracture did not affect the PMN phenotype. The impact from injury determined the PMN phenotype. In contrast, the monocyte phenotype changed after the additional insult by IMN in patients with an isolated tibial fracture.

Promotion of experimental autoimmune encephalomyelitis upon neutrophil granulocytes' stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) peptide.

It has been acknowledged that neutrophil granulocytes, the common mediators of immune responses against extracellular bacteria, can also intercede autoimmune reactions such as experimental autoimmune encephalomyelitis (EAE). Formyl-methionyl-leucyl-phenylalanine (fMLP) is a microbial peptide that can be well-tolerated when intravenously administered and can directly lead to activation and accumulation of neutrophils into the blood circulation. Here, this antigenic peptide was injected to the mice at the induction of EAE, and the immunological and pathological outcomes were assessed. As a peripheral immune organ, spleen of the animals that received fMLP contained considerably high percentages of Gr-1(hi)Ly7/4(hi) mature neutrophils. In the sera samples, only a slight difference was determined in Th1/Th2/Th17-related cytokine levels. Expression of CXCR1 or CXCR2 chemokine receptors was not significantly modulated in EAE with or without fMLP which might indicate a direct role for this antigenic peptide on neutrophil accumulation. Even though fMLP administration did not propone the clinical (symptomatic) onset of the disease, the animals showed severe body conditions with higher EAE scores. Accordingly, the expression of TNF-α and CXCL1 inflammatory mediators in the brain was increased in fMLP-EAE group. In conclusion, with its potent capacity to mobilize neutrophils and to stimulate innate immune cells, fMLP peptide can be used to aggravate immune reactions in EAE. This observation may indicate that the strength of innate immune responses particularly at the induction phase of EAE might influence the clinical course of the disease.

Impact of priming on the response of neutrophils to human neutrophil alloantigen-3a antibodies.

BACKGROUND: Human neutrophil alloantigen-3a (HNA-3a) antibodies can induce transfusion-related acute lung injury (TRALI). The severity of TRALI varies largely among the affected patients. Severe comorbidity seems to increase the susceptibility for TRALI, potentially by priming of neutrophils. Thus, the impact of neutrophil priming on HNA-3a antibody-mediated neutrophil aggregation and CD11b surface expression was investigated. STUDY DESIGN AND METHODS: Neutrophils were primed using formyl-methionyl-leucyl-phenylalanine (fMLP) or bacterial lipopolysaccharide (LPS). Granulocyte aggregation and CD11b surface expression were evaluated by the granulocyte agglutination test and by flow cytometry (FC), respectively. Priming-induced changes in the surface expression of choline transporter-like protein 2 (CTL2) and the CTL2 mRNA expression were assessed by FC and quantitative real-time polymerase chain reaction, respectively. RESULTS: Priming of neutrophils lowered the amount of HNA-3a antibodies required for inducing granulocyte aggregation in a dose-dependent manner by 50% to 75%. The priming agent concentration necessary for this response differed between donors. Priming slightly enhanced binding of HNA-3a antibodies to neutrophils. However, CTL2 de novo synthesis was not induced after priming with LPS, indicating that increased HNA-3a antibody binding was likely caused by translocation of intracellular CTL2 to the surface or by increased affinity of HNA-3a antibodies to CTL2. HNA-3a antibodies influenced CD11b surface expression on neutrophils only marginally, which was also not potentiated by priming with fMLP or LPS. CONCLUSION: This study provides experimental evidence supporting the "threshold model" of TRALI. Priming of neutrophils with fMLP or LPS increases their aggregation response to HNA-3a antibodies by lowering the required antibody amount.

Isolation of healthy individuals' and rheumatoid arthritis patients' peripheral blood neutrophils by the gelatin and Ficoll-Hypaque methods: comparative efficiency and impact on the neutrophil oxidative metabolism and Fcγ receptor expression.

In vitro assessment of the functional responses of leukocytes sometimes requires their isolation from blood, joint and tissues. In this study, we compared the efficiency of two procedures - the gelatin method and Ficoll-Hypaque density centrifugation gradient - to isolate peripheral blood neutrophils of healthy individuals and patients with active rheumatoid arthritis (RA). We also assessed whether these procedures affect the neutrophil activation status. Both purification procedures were concluded in 90min, and yielded cell populations with similar degrees of purity (80-90%), number of neutrophils (1-2×10(6) cells per mL of blood), and viability (97-100%). In vitro neutrophil priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly increased the reactive oxygen species producing ability of the cells stimulated with n-formyl-methionyl-leucyl-phenylalanine (n-fMLP), soluble immune complexes (s-ICs), and insoluble immune complexes (i-ICs). Isolated neutrophils not treated with GM-CSF responded to n-fMLP and i-IC, but not to s-IC. Almost all of the neutrophils (98-100%) purified by both methods expressed FcγRII/CD32 and FcγRIII/CD16, but they did not express significant levels of FcγRI/CD64. Similar results were obtained for healthy individuals' and RA patients' neutrophils. In summary, the gelatin method was comparable to Ficoll-Hypaque gradient in terms of purity, yield, and viability of the neutrophil preparations. Both methods neither primed or activated the neutrophils, nor affected their functional responsiveness. Therefore, both methods are suitable to isolate peripheral blood neutrophils of healthy individuals and RA patients.

Neutrophil oxidative burst capacity for peri-operative immune monitoring in trauma patients.

BACKGROUND: Post injury immune dysfunction can result in serious complications. Measurement of biomarkers may guide the optimal timing of surgery in clinically borderline patients and therefore prevent complications. AIM: peri-operative measurement of neutrophil oxidative burst capacity as an indicator of the immune response to major orthopaedic surgical procedures. METHODS: Prospective cohort study of trauma patients aged ≥16 yrs with pelvic, acetabular, femoral shaft or tibial shaft fractures requiring surgical intervention. Blood samples were taken immediately pre-op and at 30 min, 7, 24 and 72-9 6 h post-operatively. Neutrophil oxidative burst capacity was measured both with and without stimulation by formyl-methionyl-leucyl-phenylalanine (fMLP, a chemotactic factor). Clinical outcomes measured were mortality, length of stay, MOF, pneumonia, acute respiratory distress syndrome (ARDS) and sepsis. RESULTS: 100 consecutive orthopaedic trauma patients were enrolled over a 16 month period. 78% were male, with a mean age of 42 ± 18 years and an average ISS of 19 ± 13. Neutrophil oxidative burst capacity was significantly elevated at 7 h (p = 0.006) and 24 h (p = 0.022) post operatively. Patients who developed infective complications (pneumonia and sepsis) had higher levels of oxidative burst capacity pre-operatively (pneumonia: 1.52 ± 0.93 v 0.99 ± 0.66 p = 0.032, sepsis: 1.39 ± 0.86 v 0.97 ± 0.56 p = 0.024) and at 24 h post op (pneumonia: 2.72 ± 2.38 v 1.12 ± 0.63 p = < 0.001, sepsis: 2.16 ± 2.09 v 1.10 ± 0.54 p = < 0.001). When analysed by operation type, no statistical difference was seen between major and minor operations. No correlation was found between length of stay, length of ICU stay, ISS or age and neutrophil oxidative burst capacity at any time point. CONCLUSIONS: Neutrophil oxidative burst capacity response to orthopaedic trauma surgery is associated with the infective post injury complications. There was no correlation between magnitude of injury or operation and oxidative burst capacity. These results are promising for the development of tools for prediction of post-operative complications and guidance for optimal timing for surgical intervention.

Garcimultiflorone G, a novel benzoylphloroglucinol derivative from Garcinia multiflora with inhibitory activity on neutrophil pro-inflammatory responses.

A novel benzoylphloroglucinol derivative, garcimultiflorone G (1), was isolated from the fruits of Garcinia multiflora. The structure of 1 was determined through extensive 1D- and 2D-NMR, and MS analyses. Garcimultiflorone G (1) showed inhibitory effects against superoxide anion (O·2(-) generation and elastase release by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB), with IC50 values of 6.97 ± 1.56 and 11.70 ± 1.58 µM, respectively.

Role of MHC class Ib molecule, H2-M3 in host immunity against tuberculosis.

The MHC class I family comprises both classical (class Ia) and non-classical (class Ib) members. While the prime function of classical MHC class I molecules (MHC class Ia) is to present peptide antigens to pathogen-specific cytotoxic T cells, non-classical MHC-I (MHC class Ib) antigens perform diverse array of functions in both innate and adaptive immunity. Vaccines against intracellular pathogens such as Mycobacterium tuberculosis need to induce strong cellular immune responses. Recent studies have shown that MHC class I molecules play an important role in the protective immune response to M. tuberculosis infection. Both MHC Ia-restricted and MHC class Ib-restricted M. tuberculosis -reactive CD8(+) T cells have been identified in humans and mice, but their relative contributions to immunity is still uncertain. Unlike MHC class Ia-restricted CD8(+) T cells, MHC class Ib-restricted CD8(+) T cells are constitutively activated in naive animals and respond rapidly to infection challenge, hence filling the temporal gap between innate and adaptive immunity. The present review article summarizes the general host immunity against M. tuberculosis infection highlighting the possible role of MHC class Ib molecule, H2-M3 and their ligands (N-formylated peptides) in protection against tuberculosis.

P2Y11 purinoceptor mediates the ATP-enhanced chemotactic response of rat neutrophils.

ATP and hydrolysis products of ATP like adenosine regulate the chemotaxis of neutrophils by activating purinoceptors and adenosine receptors. The present study was designed to examine exogenous ATP, activation of purinoceptors, and activation of A(3) adenosine receptor as key steps in the signal cascades that control cell orientation and migration of rat neutrophils. One or more of those steps might be potential therapeutic targets for treatment of inflammatory diseases. The chemotaxis of rat neutrophils was stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and measured with an EZ-TAXIScan apparatus. The effects of apyrase, exogenous ATP, suramin (P2X and P2Y blocker), PPADS (a P2X blocker), TNP-ATP (P2X(1) and P2X(3) antagonist), and Reactive Blue 2 (a P2Y blocker) on the chemotactic response were also investigated. Rat neutrophil chemotaxis was significantly suppressed by apyrase. fMLP induced rat neutrophil chemotaxis was potentiated by ATP, blocked by suramin, not affected by PPADS or TNP-ATP, and significantly inhibited by RB-2. Western blotting showed that A(3), P2Y(2), and P2Y(11) were expressed in rat neutrophils. The chemotactic response of rat neutrophils to fMLP stimulation is potentiated by ATP via P2Y(11) purinoceptors but not P2X purinoceptors or A(3) adenosine receptor, and that the response plays a critical role in host defense and pathogenicity.

The effect of HMGB1, a damage-associated molecular pattern molecule, on polymorphonuclear neutrophil migration depends on its concentration.

Polymorphonuclear neutrophils (PMN) play a key role in host defenses against invading microorganisms but also potentiate inflammatory reactions in case of excessive or misdirected responses. Release of the alarmin high-mobility group box 1 (HMGB1) by cells that die at an inflammatory site may act as an alert signal for the immune system. We studied the effect of HMGB1 on human PMN migration, using whole-blood samples to avoid cell activation associated with isolation procedures. HMGB1 50-100 ng/ml reduced baseline PMN migration as well as formyl-methionyl-leucyl-phenylalanine- and IL-8-induced PMN chemotaxis. This inhibitory effect was mediated by the RAGE receptor. In contrast, a higher HMGB1 concentration (5,000 ng/ml) had a chemoattractant effect on PMN through IL-8 production. This effect required the engagement of Toll-like receptors 2 and 4 in addition to the RAGE receptor. The A box component of HMGB1, which antagonizes the endogenous protein, reduced chemotaxis and also strongly inhibited the enhancement of PMN migration observed with the highest HMGB1 concentration. In contrast, the B box, reported to be the active form of HMGB1, exerted a chemoattractant effect. These results strongly point to a key regulatory role of HMGB1 in PMN recruitment to inflammatory tissues. The A box component could potentially serve to inhibit inappropriate PMN recruitment during chronic inflammatory disorders associated with excessive HMGB1 release.

Calcium-dependent potentiation of the pro-inflammatory functions of human neutrophils by tigecycline in vitro.

OBJECTIVES: Tigecycline is the prototype of the recently introduced, intravenously administered glycylcycline class of antibiotics, developed in response to the increasing problem of antibiotic resistance in Gram-positive bacteria, especially Staphylococcus aureus, as well as Gram-negative bacteria and anaerobes. However, relatively little is known about the immunomodulatory potential of tigecycline, specifically its interactions with human neutrophils. In the current study we investigated the effects of tigecycline at therapeutically relevant concentrations and greater (0.625-10 mg/L) on alterations in cytosolic Ca(2+) concentrations, generation of antimicrobial reactive oxygen species (ROS) and release of granule proteases [elastase, matrix metalloproteinase-8 (MMP-8) and matrix metalloproteinase-9 (MMP-9)] by human blood neutrophils activated with the chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 µM). METHODS: Cytosolic Ca(2+) concentrations were measured using fura-2/AM-based spectrofluorimetry and radiometric procedures, generation of ROS by oxygen consumption and myeloperoxidase-mediated auto-iodination, and protease release by ELISA procedures. RESULTS: Exposure of the cells to fMLP resulted in activation of the generation of ROS, as well as release of the granule proteases, all of which were significantly increased by pre-incubation of the cells with tigecycline in a dose-dependent manner. Tigecycline-mediated enhancement of these neutrophil functions was associated with elevations in the concentrations of cytosolic Ca(2+), which appeared to result from the Ca(2+) ionophore activity of tigecycline. CONCLUSIONS: Tigecycline, by functioning as a Ca(2+) ionophore, and independent of antimicrobial activity, potentiates the pro-inflammatory functions of human neutrophils in vitro.

Formylated peptides are important virulence factors in Staphylococcus aureus arthritis in mice.

BACKGROUND: Staphylococcus aureus is the most common pathogen causing septic arthritis in humans. The affected joints are often rapidly and permanently damaged despite antibiotic treatment, indicating that the elicited host immune response contributes substantially to joint destruction. Bacterial formylated peptides are important chemotactic molecules mediating neutrophil recruitment into infected tissues as an important first step of host defense against invading bacteria. The role of formylated peptides in S. aureus infections has been unknown. METHODS: Mice were intravenously inoculated with wild-type S. aureus strain RN4220 or its isogenic mutant strain (Δfmt) lacking the ability to produce formylated peptides. The development of arthritis was followed clinically and histopathologically. RESULTS: Mice inoculated with the formyl peptide-producing wild-type strain showed a significantly increased frequency and severity of arthritis and subsequent joint destruction as compared with Δfmt mutant strain-inoculated mice. The wild-type S. aureus strain also induced significantly more weight loss than the Δfmt mutant strain. The recruitment of neutrophils into infected kidneys and synovial tissue was significantly higher in mice inoculated with the wild-type strain. CONCLUSIONS: Our data show that formylated peptides function as important virulence factors in S. aureus arthritis, partly by mediating neutrophil recruitment, which contributes substantially to the joint damage.

Quercetin reduces neutrophil recruitment induced by CXCL8, LTB4, and fMLP: inhibition of actin polymerization.

Recent in vitro data have suggested that the flavonoid quercetin (1) does not affect the functioning of neutrophils. Therefore, we evaluated in vivo and in vitro whether or not 1 affects neutrophil function, focusing on recruitment. The in vivo treatment with 1 inhibited in a dose-dependent manner the recruitment of neutrophils to the peritoneal cavity of mice induced by known chemotatic factors such as CXCL1, CXCL5, LTB(4), and fMLP. Furthermore, 1 also inhibited in a concentration-dependent manner the chemoattraction of human neutrophils induced by CXCL8, LTB(4), and fMLP in a Boyden chamber. In vitro treatment with 1 did not affect human neutrophil surface expression of CXCR1, CXCR2, BLT1, or FLPR1, but rather reduced actin polymerization. These results suggest that 1 inhibits actin polymerization, hence, explaining the inhibition of neutrophil recruitment in vivo and in vitro and highlighting its possible usefulness to diminish excessive neutrophil migration during inflammation.