Azurophilic Research Articles

The N125S polymorphism in the cathepsin G gene (rs45567233) is associated with susceptibility to osteomyelitis in a Spanish population.

Osteomyelitis is a bone infection, most often caused by Staphylococcus aureus, in which neutrophils play a key role. Cathepsin G (CTSG) is a bactericidal serine protease stored in the neutrophil azurophilic granules. CTSG regulates inflammation, activating matrix metalloproteinases (MMPs), and coagulation. Lactoferrin (LF), a neutrophil glycoprotein, increases CTSG catalytic activity and induces inflammation. The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis. CTSG N125S polymorphism was genotyped in 329 osteomyelitis patients and 415 controls), Blood coagulation parameters, serum CTSG activity, LF, MMP-1, MMP-13, and soluble receptor activator for nuclear factor κ B ligand (sRANKL) levels were assessed in carriers of the different CTSG genotypes. CTSG N125S (AG) genotype was significantly more frequent among osteomyelitis patients than controls (15.5% vs. 9.4%, p = 0.014). CTSG N125S variant G allele (AG +GG) was also more frequent among osteomyelitis patients (8.1% vs. 4.7%, p = 0.01). Serum CTSG activity and LF levels were significantly higher in osteomyelitis patients carrying the G allele compared to those with the AA genotype, (p<0.04). Serum MMP-1 was lower in the G allele carriers (p = 0.01). There was no association between these genotypes and clinical characteristics of osteomyelitis, or coagulation parameters, MMP-13, and sRANKL serum levels. Differences in the CTSG gene might enhance osteomyelitis susceptibility by increasing CTSG activity and LF levels.

Pro-oxidant versus anti-oxidant effects of seeds aglycone extracts of Lepidium sativum and Eruca vesicaria Linn., in vitro, and on neutrophil nitro-oxidative functions.

This study evaluated the anti-inflammatory and antioxidant properties of seeds aglycone extracts from Lepidium sativum (LS) and Eruca vesicaria (EV) Linn., on oxidative damages in vitro and on neutrophil nitro-oxidative functions. The results showed that LS and EV aglycone extracts attenuated liver microsomal lipids and proteins oxidation through a potent antioxidant effect as attested by the dose dependent quenching of DPPH radical scavenging activity. LS and EV aglycone extracts inhibited dose dependently the production of superoxide anion by BALB/c mice-derived peritoneal neutrophils, whereas they slightly enhanced exocytosis of myeloperoxidase (MPO), a marker of azurophilic granules. Interestingly, only LS replenished glutathione (GSH) and nitric oxide levels, indicating a fine differential effect. This study highlighted the subtle oxidative and antioxidant capacity of LS and EV seeds aglycone extracts. These health promoting compounds could be used to finely modulate critical events involved in microbial infection, inflammation and nitro-oxidative stress.

Prolonged exposure to hypoxia induces an autophagy-like cell survival program in human neutrophils.

Neutrophils contribute to low oxygen availability at inflammatory sites through the generation of reactive oxidants. They are also functionally affected by hypoxia, which delays neutrophil apoptosis. However, the eventual fate of neutrophils in hypoxic conditions is unknown and this is important for their effective clearance and the resolution of inflammation. We have monitored the survival and function of normal human neutrophils exposed to hypoxia over a 48 h period. Apoptosis was delayed, and the cells remained intact even at 48 h. However, hypoxia promoted significant changes in neutrophil morphology with the appearance of many new cytoplasmic vesicles, often containing cell material, within 5hours of exposure to low O2 . This coincided with an increase in LC3B-II expression, indicative of autophagosome formation and an autophagy-like process. In hypoxic conditions, neutrophils preferentially lost myeloperoxidase, a marker of azurophil granules. Short-term (2 h) hypoxic exposure resulted in sustained potential to generate superoxide when O2 was restored, but the capacity for oxidant production was lost with longer periods of hypoxia. Phagocytic ability was unchanged by hypoxia, and bacterial killing by neutrophils in both normoxic and hypoxic conditions was substantially diminished after 24hours. However, pre-exposure to hypoxia resulted in an enhanced ability to kill bacteria by oxidant-independent mechanisms. Our data provide the first evidence for hypoxia as a driver of neutrophil autophagy that can influence the function and ultimate fate of these cells, including their eventual clearance and the resolution of inflammation.

Iterative Optimization of the Cyclic Peptide SFTI-1 Yields Potent Inhibitors of Neutrophil Proteinase 3.

Neutrophils produce at least four serine proteases that are packaged within azurophilic granules. These enzymes contribute to antimicrobial defense and inflammation but can be destructive if their activities are not properly regulated. Accordingly, they represent therapeutic targets for several diseases, including chronic obstructive pulmonary disease, cystic fibrosis, and rheumatoid arthritis. In this study, we focused on proteinase 3 (PR3), a neutrophil protease with elastase-like specificity, and engineered potent PR3 inhibitors based on the cyclic peptide sunflower trypsin inhibitor-1 (SFTI-1). We used an iterative optimization approach to screen targeted substitutions at the P1, P2, P2', and P4 positions of SFTI-1, and generated several new inhibitors with K i values in the low nanomolar range. These SFTI-variants show high stability in human serum and are attractive leads for further optimization.

PB1906 FROM ACADEMY TO REAL WORLD: THE GRAY AREA OF THE LARGE GRANULAR LYMPHOCYTES LEUKEMIA

Background:Large granular lymphocytes (LGLs) are defined as large white blood cells (WBC) that are morphologically characterized by an abundant cytoplasm with azurophilic granulation. LGLs belong to CD3+CD56− cytotoxic T cells (CT) or natural killer (NK) cells. The NK cells are roughly defined by a lack of the T cell marker CD3 and a presence of CD56 (or NCAM). LGL leukaemia represents a chronic lymphoproliferative disorder of CTLs, a malignancy that involves lymphocyte infiltration of multiple organs, including the BM, liver, and spleen.Aims:The aim of this retrospective study was to achieve information about LGL and the association between LGL and clinical characteristics.Methods:A retrospective study analyzing the global diagnosis of LGL leukaemia and factors influencing the disease evolution at the district hospital and included cases with the previous or recent diagnosis of LGL leukaemia between January 2015 and December 2018. Special emphasis was taken on the results of bone marrow examination, flow cytometry, immunophenotype, and results of molecular studies to assess for T‐cell receptor (TCR) gene rearrangements. A total of 26 patients were confirmed as having LGL leukaemia, and they fulfilled the WHO classification criteria. May‐Grünwald‐Giemsa–stained peripheral blood smears were examined for the presence of large granular lymphocytes. Bone marrow trephine biopsy and aspirate specimens (when was applicable) were obtained. Bone marrow cellularity and the pattern of lymphocyte infiltration were determined on core biopsy or aspirate clot specimens stained with H&amp;E. Statistic The Fisher exact test was used to analyze differences between the T‐LGL and T‐LGL/other LPD groups. The median time from completion of last treatment with rituximab to the diagnosis of LGL disorder was 30 months.Results:LGL leukaemia or T‐LGL expansion were identified in the global group. Near total of the patient 21 (80.7%) had a direct history of a clonal B‐cell lymphocytosis, monoclonal B‐cell lymphocytosis (MBL), or clonal B‐cell disorder concomitantly with or shortly after the LGL diagnosis. The other predominant diagnosis was rheumatoid conditions in 9 patients (34.6%) (including Felty syndrome) and monoclonal gammopathy of unknown significance (MGUS) 7 patients (26.9%). The median age at the time of diagnosis was 68.5 years (range, 20‐86 years). B‐symptoms were reported by 4 (15.38%) patients, as well as Lymphadenopathy/splenomegaly was identified in 5 (19.23%). 4 individuals received treatment prior to the diagnosis of LGL, and the predominant treatment was methotrexate/rituximab. The favourite regimens in our daily practice were cyclosporin, rituximab or intermittent G‐CSF. The overall response rate at 3 months and complete response rate for the various treatments were as follows: steroids (11% and 3%), methotrexate (55% and 21%), rituximab (64% and 47%), cyclosporine (19% and 3%), respectively. All patients responded to rituximab after methotrexate treatment had failed. The mean number of treatments was 3.4 (range, 1‐5).Summary/Conclusion:Whether or not the T‐LGL and T‐LGL/other LPD groups are correlated with specific clinical‐biological features remains uncertain and is the subject of ongoing research. We don’t perform routinely in our clinical practice an investigation for the Constitutive activation of STAT3. In our series, we have not reported direct LGL‐related deaths.

Multiple Myeloma with Intracytoplasmic Azurophilic Granules

Bone marrow examination plays an important role in the diagnosis of multiple myeloma. In some cases with multiple myeloma, marrow plasma cells with cytoplasmic inclusions are seen. In this study, a 46-year- old man was evaluated for multiple myeloma. In bone marrow aspiration, large intracytoplasmic azurophilic granules, resembling intracellular microorganisms were seen. IHC study demonstrated that these cells are CD138 positive. This is a rare histologic finding that usually results from the deposition of excess immunoglobulin.

Evaluation of anticancer activity of α-defensins purified from neutrophils trapped in leukoreduction filters

AimsThe α-defensins or human neutrophil peptides (HNP 1–3) that exist in azurophilic granules are found to have anticancer activity. The pattern of disulfide bonds in α-defensins is crucial for the functional properties. Therefore, synthesis using the chemical and recombinant approaches is a challenging. A safe source for the production of α-defensins can be the use of leukoreduction filters in blood banks that contain large quantities of neutrophils and are discarded after use. The aim of this study was to purify α-defensins from neutrophils trapped in leukofilters and to investigate its anticancer activity. Materials and methodsImmunoprecipitation was performed to purify α-defensins and the presence of protein was confirmed by Western Blot. The Jurkat T-cell line was incubated with different concentrations (5, 10 and 15 μg/ml) of purified HNP1–3 for 16 h. Cell viability was measured using a WST-1 assay and apoptosis was analyzed for Annexin V/PI markers. Caspase-3/7 activity was determined using fluorescence assay. The effects of purified α-defensins were compared to commercial HNP 1–3. Key findingsPurified HNP 1–3 decreased the viability at 10 and 15 μg/ml and commercial HNP 1–3 at 15 μg/ml concentrations. Following to the purified HNP1–3 treatment, the percentage of Annexin V positive population and caspase-3 activity were significantly increased compared to control (p = 0.000 and p = 0.001, respectively) and commercial HNP1–3 (p = 0.034 and p = 0.018, respectively). SignificanceResults indicated the anticancer activity of HNP1–3 which can be used as future chemotherapeutic drugs. Furthermore, leukofilters can be considered as economic source for purifying these peptides.

A rare morphological and immunophenotypic presentation of adult B-cell acute lymphoblastic leukemia with blasts in the monocytic zone on CD45 side scatter

&lt;p class="abstract"&gt;We present a case of B-acute lymphoblastic leukemia in an elderly patient who presented with severe weakness and pancytopenia. The patient was a 75 year old Female whose blasts had an unusual morphology in form of coarse azurophilic granules and cytoplasmic blebs and on flow cytometry the blasts were present in the bright CD45 zone with a high side scatter. Bone marrow aspirate sample was subjected to multicolour flow cytometry using Beckman Coulter Navios® which is an 8 colour flow cytometer. Flow cytometric analysis of the bone marrow aspirate showed blasts in the monocytic zone with a precursor B cell immunophenotype. Complete blood counts showed pancytopenia with peripheral blood film not showing any blasts. Bone marrow aspirate smears showed 20% blasts with coarse azurophilic granules and cytoplasmic blebs. The position of the blasts in this case which were in monocytic zone giving them a bright expression of CD45 and a high side scatter on the CD45 side scatter. This is not the usual position for blasts in B- acute lymphoblastic leukemia as these blasts are less complex. A bright expression of CD45 by blasts in B- acute lymphoblastic leukemia is known to be associated with a poor prognosis but the clinical significance of blasts being bright CD45 with a high side scatter is a very rare occurrence and more number of cases with a similar presentation are required to determine a prognostic significance.&lt;/p&gt;

The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration.

Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired azurophilic granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1-/- neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.

Correlation between human neutrophil peptide 1–3 secretion and azurophilic granule (CD63) expression in early childhood caries

In saliva, neutrophil constitutes the most prominent first-line defense of immune cells against pathogenic microbes. The importance of neutrophils to the host immune systems of neutropenic or patients disabled with regard to their neutrophil function results in a tendency toward serious infections, such as early childhood caries (ECC). The cytoplasmic granules present in neutrophils play a major role in neutrophil-mediated inflammation. Azurophilic granules contain antimicrobial proteins, such as defensin, a human antimicrobial peptide (HNP 1-3). The aim of this study is to analyze the correlation of HNP 1-3 secretion with CD63 expression on the surface of salivary neutrophils. This study constituted a cross-sectional, analytical observational study. Saliva taken from preschoolchildren between the ages of 4-6 years who had been divided into two groups, i.e., early childhood caries group with decayed, extracted, filled teeth (def-t) index >6 and caries free with def-t = 0, was subjected to a HNP 1-3 secretion test using ELISA assay and an expression test for CD63 by means of a flow cytometry test. The results obtained were analyzed using independent t-test and Pearson correlation (P < 0.05). The secretion of HNP 1-3 in the saliva of ECC was higher (172.6 ± 41.64) compared to that of caries-free cases (140.39 ± 31.91), whereas the level of CD63 salivary expression in ECC was lower (2.32 ± 0.57) than in the presence of caries (2.67 ± 0.46). In ECC cases, saliva increases HNP 1-3 secretion but decreases CD63 expression on the surface of salivary neutrophils.

NK Cells from Human Pluripotent Stem Cells for Immunotherapy

Natural Killer (NK) cells are a one of innate lymphocytes and show cytotoxicity against tumour cells without prior antigen specific stimulation. . NK cells can demonstrate stronger cytotoxicity than T cells in the absence of MHC Class I, and survive short lifespan from several weeks to one month. It suggested that NK cells show low risk of cytokine long-term secretion inside patient's body. Previous studies have developed peripheral blood mononuclear cells (PBMC) derived NK cells expansions or NK cells differentiation from cord blood (CB) cells for immunotherapy. Expansion trial using K562 tumor cell line, or with IL-15, or an anti-tumor antibody dasatinib is not sufficient to obtain NK cells with high cytotoxicity.More recently, NK cells induction from human pluripotent stem cells (hPSCs), taking the advantage of their unlimited growth potential, has been reported. Although previous studies regarding hPSC-derived NK cells seems impressive and successful, most systems used a bovine and human serum, which might result in the unstable yield and efficiency in the production of Hematopoietic progenitor cells (HPCs) and NK cells for immunotherapy. To resolve those problems, we tried to induce functional NK cells from hPSCs in xeno and serum free condition.This study used three hPSC cell lines; human ES cell (cell line: KhES1) and iPS cells (cell line: 409B2 and CB-A11) to check reproducibility. To differentiate hPSCs into hematopoietic cells, changed cytokine combinations and chemically defined medium in step-wise manner. We first induced HPC from hPSCs over 90% purity by 12 days culture. At this point, we selected two media to induce NK cells. We compared serum-containing medium that previous report used (Medium A) and chemically-defined medium (Medium B) by evaluating the differentiation efficiency and function of NK cells. NK cell marker CD56 (NCAM) was gradually expressed after additional 16 days culture (28 days of differentiation). Until hPSC-derived NK cells were maturated, we traced the expression of NK specific markers and transcriptional factors. On day48, the frequency of CD56 positive cells showed no significant differences between medium A (79.15 ± 5.30%) and medium B (80.90 ± 1.27%). In both conditions, NK cells expressed specific receptors such as CD161, NKG2D, killer immunoglobulin-like receptors (KIRs), NKG2a (CD94/CD159a heterodimeric inhibitory receptor), NKp44 and NKp46. hPSC-derived NK cells showed the compatible size and morphology to NK cells isolated from peripheral blood NK (PB-NK) cells: their nucleus was kidney-like shape and cytoplasm contained azurophilic granules. For functional assay, leukemia cell line K562 was incubated with 51 chromium (51Cr) for 1 hour at 37 degrees. After that, K562 was co-cultured with purified CD56 positive hPSC-derived NK cells for 4 hours at 37 degrees. The cytotoxic activity of NK cells was confirmed by 51Cr release from K562. PBMC-NK cells showed 49.65 ± 3.46% of killing activity against K562 target cells, while the killing potential of PSC-derived NK cell showed killing potential against K562 cells (Medium A: 25.4 ± 5.52%, Medium B: 23.25 ± 9.26%) which was slightly lower than that of PB-NK cells. Next trial, we are going to transplant hPSC-derived NK cells into immune deficiency mice. In detail, this mice was infected luciferase expressed K562. Using IVIS imaging system to detect intensity of luciferase, we characterized hPSC-derived NK cells potential in vivo.Here we have developed a novel and robust method to facilitate efficient NK cells differentiation in serum and xeno-free condition in all clones. They showed similar phenotypes compare to PBMC derived NK cells in terms of morphology, surface markers, translational factors and cytotoxicity against leukemia cell line K562 in vitro. This technology expected to be applicable not only to immunotherapy but also to model studies of the NK cells associating diseases. DisclosuresNo relevant conflicts of interest to declare.

Association of monocyte myeloperoxidase with incident cardiovascular disease: The Atherosclerosis Risk in Communities Study.

Myeloperoxidase (MPO) is a heme-containing peroxidase found in azurophilic granules of neutrophils and monocytes. Epidemiological studies have reported greater plasma MPO concentration to be associated with increased incidence of several cardiovascular diseases (CVD), but the association of intracellular monocyte MPO (mMPO) with CVD is unclear. The prospective population-based Atherosclerosis Risk in Communities (ARIC) cohort study measured mMPO using flow cytometry in 1,465 participants. The association of mMPO with incident cardiovascular disease (CVD, comprising incident coronary heart disease (CHD), heart failure, stroke, peripheral artery disease, and cardiovascular mortality) was examined over a median 9.6 years of follow-up (n = 290 CVD events). There was no statistically significant association between mMPO and all incident CVD events in either age, sex, and race-adjusted proportional hazards models (HR (95% CI) across tertiles of mMPO: 1, 1.09 (0.76, 1.57), and 0.78 (0.52, 1.15), P-trend = 0.21) or adjusted for other major CVD risk factors (HR (95% CI): 1, 1.17 (0.81, 1.69), and 0.87 (0.58, 1.29), P-trend = 0.50). There also was no association between mMPO tertiles and incident CHD, heart failure, or all-cause mortality, examined separately. In conclusion, intracellular monocyte myeloperoxidase was not associated with incident cardiovascular disease in this prospective population-based study.

Membrane-Associated Proteinase 3 on Granulocytes and Acute Myeloid Leukemia Inhibits T Cell Proliferation.

Proteinase 3 (P3), a serine protease expressed by myeloid cells, localized within azurophil granules, and also expressed on the cellular membrane of polymorphonuclear neutrophils (PMN), is the target of autoimmunity in granulomatosis with polyangiitis. PR1, an HLA-A2 restricted nonameric peptide derived from P3, has been targeted effectively in myeloid leukemia. We previously showed (Molldrem et al. 2003. JClinInvest 111: 639-647) that overexpression of P3 in chronic myeloid leukemia induces apoptosis of high-affinity PR1-specific T cells, leading to deletional tolerance and leukemia outgrowth. In this study, we investigated the effect of membrane P3 (mP3)-expressing PMN and acute myeloid leukemia (AML) blasts on the proliferation of CD4 and CD8 T cells in vitro. We demonstrate that mP3-expressing PMN significantly inhibits autologous healthy donor T cell proliferation but does not affect cytokine production in activated T cells and that this effect requires cell proximity and was abrogated by P3 blockade. This inhibition required P3 enzyme activity. However, suppression was not reversed by either the addition of catalase or the inhibition of arginase I. In addition to P3 blockade, anti-low density lipoprotein receptor-related protein 1 (LRP1) Ab also restored T cells' capacity to proliferate. Last, we show dose-dependent inhibition of T cell proliferation by mP3-expressing AML blasts. Together, our findings demonstrate a novel mechanism whereby PMN- and AML-associated mP3 inhibits T cell proliferation via direct LRP1 and mP3 interaction, and we identify P3 as a novel target to modulate immunity in myeloid leukemia and autoimmune disease.

Neutrophil-released enzymes can influence composition of circulating immune complexes in multiple sclerosis

During NET formation, the content of neutrophils granules is released into the intercellular milieu. Consisting of many proteases and ROS species, formed NETs were shown to degrade cytokines (Schauer, Nat Med, 2014); while the content of neutrophil’s azurophilic granules proved to contain glycosidases, secreted upon activation (Thaysen-Andersen, JBC, 2015), and formation of autoantibodies to neutrophil beta-glucoronidase was connected with the level of anti-MPO antibodies (Ab) (Martensson, Autoimmunity, 1992). Taking into account these facts, we aimed to investigate the possibility of NET-related changes in glycan composition on circulating IgG molecules and IgG–IgM immune complexes in multiple sclerosis (MS). This autoimmune disorder still has no reliable detection markers or established ways of treatment, besides widely accepted interferon therapy, making it a particularly interesting clinical condition. By applying capture lectin-ELISA, we analysed binding of α2,6 sialyl-specific lectins SNA, PSqL, and core α1,6-fucose specific lectin AAL to circulating IgG and related complexes in five groups of MS patients: untreated (17 persons); undergoing therapy with interferon (IFN) β-1 b (15 persons), corticosteroids (methylprednisolone) (12 persons) and anti-B-cell monoclonal Ab (12 persons: Ocrelizumab, 6 persons and alemtuzumab, 6 persons). A group of 23 healthy donors served as control. Significant increase in neutrophil elastase activity, observed in the group of patients under corticosteroid treatment was also accompanied by sialyl-specific PSqL and SNA lectin binding to captured IgG molecules. Subsequent analysis demonstrated that sialic acid residues were exposed on free IgG and on circulating IgG–IgM immune complexes. Increased lectin binding was not observed for anti-myelin basic protein (one of the major autoAb in MS) Ab compared to total serum Ab. IFN therapy was accompanied by low neutrophil elastase activity and low amount of circulating immune complexes. Incubation of in vitro generated NETs with human serum revealed the digestion of high-molecular weight immune complexes with subsequent exposure of hidden glycoepitops. Obtained data indicate the potential of neutrophil-derived proteases to modify (partially degrade) circulating immune complexes leading to exposure of internal glycoepitops.

Neuropeptides SP and CGRP Diminish the Moraxella catarrhalis Outer Membrane Vesicle- (OMV-) Triggered Inflammatory Response of Human A549 Epithelial Cells and Neutrophils.

Neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP) play both pro- and anti-inflammatory activities and are produced during infection and inflammation. Moraxella catarrhalis is one of the leading infectious agents responsible for inflammatory exacerbation in chronic obstructive pulmonary disease (COPD). Since the airway inflammation in COPD is connected with activation of both epithelial cells and accumulated neutrophils, in this study we determined the in vitro effects of neuropeptides on the inflammatory potential of these cells in response to M. catarrhalis outer membrane vesicle (OMV) stimulant. The various OMV-mediated proinflammatory effects were demonstrated. Next, using hBD-2-pGL4[luc2] plasmid with luciferase reporter gene, SP and CGRP were shown to inhibit the IL-1β-dependent expression of potent neutrophil chemoattractant, hBD-2 defensin, in transfected A549 epithelial cells (type II alveolar cells) upon OMV stimulation. Both neuropeptides exerted antiapoptotic activity through rescuing a significant fraction of A549 cells from OMV-induced cell death and apoptosis. Finally, CGRP caused an impairment of specific but not azurophilic granule exocytosis from neutrophils as shown by evaluation of gelatinase-associated lipocalin (NGAL) or CD66b expression and elastase release, respectively. Concluding, these findings suggest that SP and CGRP mediate the dampening of proinflammatory action triggered by M. catarrhalis OMVs towards cells engaged in lung inflammation in vitro.

Oxidation of cysteine by ceruloplasmin leads to formation of hydrogen peroxide, which can be utilized by myeloperoxidase

Myeloperoxidase (MPO) is the enzyme of azurophilic granules of neutrophils, which catalyzes two electron oxidation of either chloride or bromide in the so-called “halogenating cycle”. Interaction of hydrogen peroxide with MPO in the presence of chloride ions leads to formation of hypochlorous acid (HOCl). Ceruloplasmin (CP) is known to be an effective physiological inhibitor of the MPO activity. However, despite the large excess of CP in blood plasma, MPO-dependently modified biomolecules were found in variety of inflammation loci, including vessel walls. This study shows that CP, which is supposed to inhibit MPO, can provide its action in physiological conditions due to hydrogen peroxide formation during oxidation of free cysteine. The key role of labile copper ions in said process is also demonstrated.

Evaluation of the Functional Condition of Human Blood Neutrophills Based on the Registrationof Active Halogen Forms Production

The development of adequate methods for monitoring the functional state of neutrophils is an urgent task due to their important role in the development of the inflammatory response. In this study, the functional activity of neutrophils has been assessed using a complex approach, including the registration of generation of ROS by neutrophils as a result of the functioning of the NADPH oxidase complex; degranulation of azurophilic granules; as well as production of AFG, formed in reactions catalyzed by the enzyme azurophilic granules of neutrophils – MPO in the presence of substrates (Н2О2 and halide ions).

Morphine, a potential inhibitor of myeloperoxidase activity

Morphine is an opioid alkaloid commonly used in clinical practice for its analgesic properties. The phenolic hydroxyl group of that phenanthrene derivative is pivotal for binding to opioid receptors but it may also be responsible for the antioxidant behavior of morphine reported in several in vitro experiments. In this study, we assessed the effect of morphine on myeloperoxidase (MPO), a hemic enzyme from azurophilic granules of polymorphonuclear neutrophils involved in the production of cytotoxic and microbicidal reactive oxidants during inflammatory response. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling (docking) were performed to study the potential anti-catalytic action of morphine on MPO in comparison with the inhibitory effects of reference antioxidant molecules quercetin, gallic acid and ascorbic acid. The reducing action of morphine on the MPO peroxidase cycle has been investigated using electron paramagnetic resonance (EPR) and UV-visible absorption spectroscopy. Morphine acted as a reducing substrate in the peroxidase cycle of MPO and therefore protected the enzyme against the suicide action of its natural substrate, hydrogen peroxide. The SIEFED experiments associated with the docking study, further demonstrated a lack of strong and sustained anti-catalytic activity of morphine. In summary, from the results of this study, it appears that morphine acts as a weak and reversible inhibitor of MPO that may nonetheless contribute to immunomodulatory and antioxidant effects of this opioid analgesic in vivo.

New Insights into the Role of Zinc Acquisition and Zinc Tolerance in Group A Streptococcal Infection.

Zinc plays an important role in host innate immune function. However, the innate immune system also utilizes zinc starvation ("nutritional immunity") to combat infections. Here, we investigate the role of zinc import and export in the protection of Streptococcus pyogenes (group A Streptococcus; GAS), a Gram-positive bacterial pathogen responsible for a wide spectrum of human diseases, against challenge from host innate immune defense. In order to determine the role of GAS zinc import and export during infection, we utilized zinc import (ΔadcA ΔadcAII) and export (ΔczcD) deletion mutants in competition with the wild type in both in vitro and in vivo virulence models. We demonstrate that nutritional immunity is deployed extracellularly, while zinc toxicity is utilized upon phagocytosis of GAS by neutrophils. We also show that lysosomes and azurophilic granules in neutrophils contain zinc stores for use against intracellular pathogens.

Neutrophils and M1 macrophages contribute to vascular injury and lung Pathogenesis during Orientia tsutsugamushi infection

Abstract Acute lung injury (ALI) leading to acute respiratory distress syndrome is a prevailing pathologic manifestation during O. tsutsugamushi-induced severe scrub typhus in humans. In this study, we tested a hypothesis that lung pathology in scrub typhus is due in part to dysregulated activation of inflammatory responses, leading to vascular malfunction and ALI. Following infection with a lethal dose of O. tsutsugamushi in mice, lung tissues had a significant increase in ICAM1, VEGFR2, and angiopoietin-2 (Ang2), as measured by flow cytometry and immunofluorescence. Several unique features were also observed. First, a progressive decrease or loss of endothelial functional markers (Ang1 and Tie2) and CD41+ platelets, even after the peak of bacterial replication (around day 6), implying continued endothelial cell stress and tissue damage. Second, a sustained neutrophil influx and activation as disease progressed, and lung-recruited neutrophils became highly activated in releasing azurophilic granules or myeloperoxidase around day 10 (prior to host death). Finally, lung-derived macrophages were highly polarized to an M1 phenotype (CD80+CD64+CD11b+Ly6G−) at day 6-day 10, with no signs of M2 activation (CD206+CD64+CD11b+Ly6G−). This study reveals specific biomarkers for vascular stress/dysfunction and uncovers a type 1-skewed, but type 2-suppressed, immune responses in the lungs of lethally-infected mice. More importantly, it furthers findings from human patients and cells, implying an immunopathogenic role in ALI development during severe scrub typhus. Understanding of leukocyte effector molecules and endothelial stress pathways triggered by bacterial vs. host factors will help control this neglected tropical disease.