ResearchPad - neutrophils https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Differential expression of interferon-lambda receptor 1 splice variants determines the magnitude of the antiviral response induced by interferon-lambda 3 in human immune cells]]> https://www.researchpad.co/article/elastic_article_13835 Type III IFNs (IFN-λs) are antiviral cytokines that are thought to act on specific subsets of cells, especially to protect mucosal barriers. Here, we demonstrate that IFN-λ3 differentially binds multiple human immune cell subsets, indicating the specific receptor subunit, IFN-λR1, is more broadly expressed in the human immune system, compared to published mouse models. IFN-λR1 expression increased after cellular activation, and antiviral responses were inhibited by a soluble version of the receptor. The direct interaction of IFN-λs with human immune cells, and specific regulation of IFN-λR1 expression, has broad mechanistic implications in the modulation of inflammatory or anti-cancer immune responses, and future antiviral therapies.

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<![CDATA[Post-stroke infections associated with spleen volume reduction: A pilot study]]> https://www.researchpad.co/article/elastic_article_7682 Spleen volume reduction followed by re-expansion has been described in acute ischemic stroke in both animal and human studies. Splenic contraction might be partially due to sympathetic hyperactivity and might be accompanied by release of splenocytes in the peripheral circulation, leading to immunodepression.AimsTo investigate whether spleen volume changes in the first week after stroke are associated with post-stroke infections, changes in lymphocytes count and autonomic dysfunction.MethodsIn patients with acute ischemic stroke, spleen sizes were calculated from abdominal CT images on day one and day seven. Spleen size reduction was defined as > 10% spleen size reduction between day one and day seven. Post stroke infections were diagnosed during the first seven days after stroke onset using the modified criteria of the US Center of Disease Control and Prevention. We assessed the time course of leukocyte subsets and analysed pulse rate variability (PRV) indices.ResultsPost-stroke infections occurred in six out of 11 patients (55%) with spleen size reduction versus in five out of 27 patients (19%) without spleen size reduction (p = 0,047). Spleen size reduction was associated with a drop in lymphocytes and several lymphocyte subsets from admission to day one, and a higher NIHSS at admission and at day three (p = 0,028 and p = 0,006 respectively). No correlations could be found between spleen volume change and PRV parameters.ConclusionPost-stroke infections and a drop in lymphocytes and several lymphocyte subsets are associated with spleen volume reduction in acute ischemic stroke. ]]> <![CDATA[Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis]]> https://www.researchpad.co/article/Ndf7081dd-c312-4392-aa9c-ddf6cf67dfa0

Atherosclerosis, the predominant cause of death in well-resourced countries, may develop in the presence of plasma lipid levels within the normal range. Inflammation may contribute to lesion development in these individuals, but the underlying mechanisms are not well understood. Transgenic mice expressing α-def-1 released from activated neutrophils develop larger lipid and macrophage-rich lesions in the proximal aortae notwithstanding hypocholesterolemia caused by accelerated clearance of α-def-1/low-density lipoprotein (LDL) complexes from the plasma. The phenotype does not develop when the release of α-def-1 is prevented with colchicine. However, ApoE-/- mice crossed with α-def-1 mice or given exogenous α-def-1 develop smaller aortic lesions associated with reduced plasma cholesterol, suggesting a protective effect of accelerated LDL clearance. Experiments were performed to address this seeming paradox and to determine if α-def-1 might provide a means to lower cholesterol and thereby attenuate atherogenesis. We confirmed that exposing ApoE-/- mice to α-def-1 lowers total plasma cholesterol and decreases lesion size. However, lesion size was larger than in mice with total plasma cholesterol lowered to the same extent by inhibiting its adsorption or by ingesting a low-fat diet. Furthermore, α-def-1 levels correlated independently with lesion size in ApoE-/- mice. These studies show that α-def-1 has competing effects on atherogenesis. Although α-def-1 accelerates LDL clearance from plasma, it also stimulates deposition and retention of LDL in the vasculature, which may contribute to development of atherosclerosis in individuals with normal or even low plasma levels of cholesterol. Inhibiting α-def-1 may attenuate the impact of chronic inflammation on atherosclerotic vascular disease.

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<![CDATA[Pretreatment neutrophil-to-lymphocyte ratio predicts clinical relapse of ulcerative colitis after tacrolimus induction]]> https://www.researchpad.co/article/5c8acc85d5eed0c48498f939

Objectives

Although tacrolimus is useful as an induction therapy in patients with ulcerative colitis (UC), information regarding the long-term outcome after tacrolimus therapy is insufficient. The aim of this study was to evaluate the clinical significance of the pretreatment neutrophil-to-lymphocyte ratio (NLR) as a prognostic factor in patients with UC receiving tacrolimus, to aid treatment selection.

Materials and methods

Patients with moderate-to-severe active UC who received oral tacrolimus induction therapy and subsequent immunomodulatory maintenance therapy at our hospital between 2009 and 2017 and who showed clinical response at week 12, were retrospectively enrolled. Cox regression analysis was conducted to study the prognostic role of the pretreatment NLR. The combined impact of the NLR and other known prognostic factors was investigated with multivariate regression.

Results

Among 45 patients included in this study, 21 patients experienced relapse during a median follow-up period of 16.6 months. Multivariate Cox regression analysis identified the pretreatment NLR (hazard ratio [HR]: 0.82, 95% confidence interval [CI]: 0.72–0.94, P < 0.01) and the use of immunomodulators at the start of tacrolimus treatment (HR: 0.18, 95% CI: 0.05–0.66, P = 0.01) as independent predictors of clinical relapse.

Conclusions

The pretreatment NLR is an independent prognostic factor in patients with UC treated with tacrolimus.

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<![CDATA[The role of TLR9 on Leishmania amazonensis infection and its influence on intranasal LaAg vaccine efficacy]]> https://www.researchpad.co/article/5c7d95ebd5eed0c484734fa1

Leishmania (L.) amazonensis is one of the etiological agents of cutaneous leishmaniasis (CL) in Brazil. Currently, there is no vaccine approved for human use against leishmaniasis, although several vaccine preparations are in experimental stages. One of them is Leishvacin, or LaAg, a first-generation vaccine composed of total L. amazonensis antigens that has consistently shown an increase of mouse resistance against CL when administered intranasally (i.n.). Since Toll-like receptor 9 (TLR9) is highly expressed in the nasal mucosa and LaAg is composed of TLR9-binding DNA CpG motifs, in this study we proposed to investigate the role of TLR9 in both L. amazonensis infection and in LaAg vaccine efficacy in C57BL/6 (WT) mice and TLR9-/- mice. First, we evaluated, the infection of macrophages by L. amazonensis in vitro, showing no significant difference between macrophages from WT and TLR9-/- mice in terms of both infection percentage and total number of intracellular amastigotes, as well as NO production. In addition, neutrophils from WT and TLR9-/- mice had similar capacity to produce neutrophil extracellular traps (NETs) in response to L. amazonensis. L. amazonensis did not activate dendritic cells from WT and TLR9-/- mice, analysed by MHCII and CD86 expression. However, in vivo, TLR9-/- mice were slightly more susceptible to L. amazonensis infection than WT mice, presenting a larger lesion and an increased parasite load at the peak of infection and in the chronic phase. The increased TLR9-/- mice susceptibility was accompanied by an increased IgG and IgG1 production; a decrease of IFN-γ in infected tissue, but not IL-4 and IL-10; and a decreased number of IFN-γ producing CD8+ T cells, but not CD4+ T cells in the lesion-draining lymph nodes. Also, TLR9-/- mice could not control parasite growth following i.n. LaAg vaccination unlike the WT mice. This protection failure was associated with a reduction of the hypersensitivity response induced by immunization. The TLR9-/- vaccinated mice failed to respond to antigen stimulation and to produce IFN-γ by lymph node cells. Together, these results suggest that TLR9 contributes to C57BL/6 mouse resistance against L. amazonensis, and that the TLR9-binding LaAg comprising CpG motifs may be important for intranasal vaccine efficacy against CL.

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<![CDATA[Aspects of intradermal immunization with different adjuvants: The role of dendritic cells and Th1/Th2 response]]> https://www.researchpad.co/article/5c6b26a6d5eed0c484289df6

Intradermal (i.d.) application of vaccine is promising way how to induce specific immune response against particular pathogens. Adjuvants, substances added into vaccination dose with the aim to increase immunogenicity, play important role in activation of dendritic cells with subsequent activation of lymphocytes. They can, however, induce unwanted local reactions. The aim of the study was to determine the effect of i.d. administration of model antigen keyhole limped hemocyanine alone or with different adjuvants–aluminium hydroxide and oil-based adjuvants—on local histopathological reaction as well as dendritic cell activation at the site of administration and local cytokine and chemokine response. This was assessed at 4 and 24 hours after application. Selection of the adjuvants was based on the fact, that they differently enhance antibody or cell-mediated immunity. The results showed activation of dendritic cells and both Th1 and Th2 response stimulated by oil-based adjuvants. It was associated with higher expression of set of genes, incl. chemokine receptor CCR7 or Th1-associated chemokine CXCL10 and cytokine IFNγ. Application of the antigen with aluminium hydroxide induced higher expression of Th2-associated IL4 or IL13. On the other hand, both complete and incomplete Freund´s adjuvants provoked strong local reaction associated with influx of neutrophils. This was accompanied with high expression of proinflammatory IL1 or neutrophil chemoattractant CXCL8. Surprisingly, similarly strong local reaction was detected also after application of aluminium hydroxide-based adjuvant. The best balanced local reaction with sufficient activation of immune cells was detected after application of oil-based adjuvants Montanide and Emulsigen.

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<![CDATA[PepN is a non-essential, cell wall-localized protein that contributes to neutrophil elastase-mediated killing of Streptococcus pneumoniae]]> https://www.researchpad.co/article/5c5df336d5eed0c484580f0d

Streptococcus pneumoniae (Spn) is an asymptomatic colonizer of the human nasopharynx but can also cause disease in the inner ear, meninges, lung and blood. Although various mechanisms contribute to the effective clearance of Spn, opsonophagocytosis by neutrophils is perhaps most critical. Upon phagocytosis, Spn is exposed to various degradative molecules, including a family of neutrophil serine proteases (NSPs) that are stored within intracellular granules. Despite the critical importance of NSPs in killing Spn, the bacterial proteins that are degraded by NSPs leading to Spn death are still unknown. In this report, we identify a 90kDa protein in a purified cell wall (CW) preparation, aminopeptidase N (PepN) that is degraded by the NSP neutrophil elastase (NE). Since PepN lacked a canonical signal sequence or LPxTG motif, we created a mutant expressing a FLAG tagged version of the protein and confirmed its localization to the CW compartment. We determined that not only is PepN a CW-localized protein, but also is a substrate of NE in the context of intact Spn cells. Furthermore, in comparison to wild-type TIGR4 Spn, a mutant strain lacking PepN demonstrated a significant hyper-resistance phenotype in vitro in the presence of purified NE as well as in opsonophagocytic assays with purified human neutrophils ex vivo. Taken together, this is the first study to demonstrate that PepN is a CW-localized protein and a substrate of NE that contributes to the effective killing of Spn by NSPs and human neutrophils.

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<![CDATA[Role of tumor-associated neutrophils in regulation of tumor growth in lung cancer development: A mathematical model]]> https://www.researchpad.co/article/5c58d634d5eed0c4840318a9

Neutrophils display rapid and potent innate immune responses in various diseases. Tumor-associated neutrophils (TANs) however either induce or overcome immunosuppressive functions of the tumor microenvironment through complex tumor-stroma crosstalk. We developed a mathematical model to address the question of how phenotypic alterations between tumor suppressive N1 TANS, and tumor promoting N2 TANs affect nonlinear tumor growth in a complex tumor microenvironment. The model provides a visual display of the complex behavior of populations of TANs and tumors in response to various TGF-β and IFN-β stimuli. In addition, the effect of anti-tumor drug administration is incorporated in the model in an effort to achieve optimal anti-tumor efficacy. The simulation results from the mathematical model were in good agreement with experimental data. We found that the N2-to-N1 ratio (N21R) index is positively correlated with aggressive tumor growth, suggesting that this may be a good prognostic factor. We also found that the antitumor efficacy increases when the relative ratio (Dap) of delayed apoptotic cell death of N1 and N2 TANs is either very small or relatively large, providing a basis for therapeutically targeting prometastatic N2 TANs.

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<![CDATA[A single alcohol binge impacts on neutrophil function without changes in gut barrier function and gut microbiome composition in healthy volunteers]]> https://www.researchpad.co/article/5c5df322d5eed0c484580d6e

Alcohol binge drinking is a dangerous drinking habit, associated with neurological problems and inflammation. The impact of a single alcohol binge on innate immunity, gut barrier and gut microbiome was studied. In this cohort study 15 healthy volunteers received 2 ml vodka 40% v/v ethanol/kg body weight. Neutrophil function was studied by flow cytometry; markers of gut permeability and inflammation (lactulose/mannitol/sucrose test, zonulin, calprotectin, diamino-oxidase) were studied with NMR spectroscopy and enzyme-linked immunosorbent assay in urine, stool and serum respectively. Bacterial products in serum were quantified using different reporter cell lines. Gut microbiome composition was studied by 16S rDNA sequencing and bioinformatics analysis. After a single alcohol binge, neutrophils were transiently primed and the response to E.coli stimulation with reactive oxygen species (ROS) production was transiently increased, on the other hand the percentage of neutrophils that did not perform phagocytosis increased. No changes in gut permeability, inflammatory biomarker, bacterial translocation and microbiome composition could be detected up to 4 hours after a single alcohol binge or on the next day. A single alcohol binge in young, healthy volunteers transiently impacts on neutrophil function. Although the exact biological consequence of this finding is not clear yet, we believe that this strengthens the importance to avoid any alcohol binge drinking, even in young, otherwise healthy persons.

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<![CDATA[The 5’-nucleotidase S5nA is dispensable for evasion of phagocytosis and biofilm formation in Streptococcus pyogenes]]> https://www.researchpad.co/article/5c5ca309d5eed0c48441f036

5’-nucleotidases are widespread among all domains of life. The enzymes hydrolyze phosphate residues from nucleotides and nucleotide derivatives. In some pathobiontic bacteria, 5’-nucleotidases contribute to immune evasion by dephosphorylating adenosine mono-, di-, or tri-phosphates, thereby either decreasing the concentration of pro-inflammatory ATP or increasing the concentration of anti-inflammatory adenosine, both acting on purinergic receptors of phagocytic cells. The strict human pathogen Streptococcus pyogenes expresses a surface-associated 5’-nucleotidase (S5nA) under infection conditions that has previously been discussed as a potential virulence factor. Here we show that deletion of the S5nA gene does not significantly affect growth in human blood, evasion of phagocytosis by neutrophils, formation of biofilms and virulence in an infection model with larvae of the greater wax moth Galleria mellonella in S. pyogenes serotypes M6, M18 and M49. Hence, the surface-associated 5’-nucleotidase S5nA seems dispensable for evasion of phagocytosis and biofilm formation in S. pyogenes.

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<![CDATA[An optimized low-pressure tourniquet murine hind limb ischemia reperfusion model: Inducing acute ischemia reperfusion injury in C57BL/6 wild type mice]]> https://www.researchpad.co/article/5c536bb5d5eed0c484a49072

Acute ischemia reperfusion injury in skeletal muscle remains an important issue in several fields of regenerative medicine. Thus, a valid model is essential to gain deeper insights into pathophysiological relations and evaluate possible treatment options. While the vascular anatomy of mice regularly prevents sufficient vessel occlusion by invasive methods, there is a multitude of existing models to induce ischemia reperfusion injury without surgical procedures. Since there is no consensus on which model to prefer, this study aims to develop and evaluate a novel, optimized low-pressure tourniquet model. C57BL/6 mice underwent an ischemic procedure by either tourniquet or invasive artery clamping. A sham group served as control. With exception of the sham group, mice underwent 2 hours of ischemia followed by 4 hours of reperfusion. Groups were compared using microcirculatory and spectroscopic measurements, distinctions in tissue edema, histological and immunohistochemical analyses. Both procedures led to a significant decrease in tissue blood flow (- 97% vs. - 86%) and oxygenation (- 87% vs. - 75%) with a superiority of the low-pressure tourniquet. Tissue edema in the tourniquet cohort was significantly increased (+ 59%), while the increase in the clamping cohort was non-significant (+ 7%). Haematoxylin Eosin staining showed significantly more impaired muscle fibers in the tourniquet group (+ 77 p.p. vs. + 11 p.p.) and increased neutrophil infiltration/ROI (+ 51 vs. + 8). Immunofluorescence demonstrated an equal increase of p38 in both groups (7-fold vs. 8-fold), while the increase in apoptotic markers (Caspase-3, 3-Nitrotyrosine, 4-Hydroxynonenal) was significantly higher in the tourniquet group. The low-pressure tourniquet has been proven to produce reproducible and thus reliable ischemia reperfusion injury. In addition, significantly less force was needed than previously stated. It is therefore an important instrument for studying the pathophysiology of ischemia reperfusion injury and for the development of prophylactic as well as therapeutic interventions.

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<![CDATA[In vitro anti-inflammatory effects of AZD8999, a novel bifunctional muscarinic acetylcholine receptor antagonist /β2-adrenoceptor agonist (MABA) compound in neutrophils from COPD patients]]> https://www.researchpad.co/article/5c390bc1d5eed0c48491e2d8

Recent evidence indicates that AZD8999 (LAS190792), a novel muscarinic acetylcholine receptor antagonist and β2-adrenoceptor agonist (MABA) in development for chronic respiratory diseases, induces potent and sustained relaxant effects in human bronchi by adressing both muscarinic acetylcholine receptors and β2-adrenoceptor. However, the anti-inflammatory effects of the AZD8999 monotherapy or in combination with corticosteroids are unknown. This study investigates the anti-inflammatory effects of AZD8999 in monotherapy and combined with fluticasone propionate in neutrophils from healthy and chronic obstructive pulmonary disease (COPD) patients. Peripheral blood neutrophils from healthy and COPD patients were incubated with AZD8999 and fluticasone propionate, individually or in combination, for 1h followed by lipopolysaccharide (LPS) stimulation for 6h. The IL-8, MMP9, IL-1β, and GM-CSF release was measured in cell culture supernatants. AZD8999 shows ~ 50% maximum inhibitory effect and similar potency inhibiting the released cytokines in neutrophils from healthy and COPD patients. However, while fluticasone propionate suppresses mediator release in neutrophils from healthy patients, COPD neutrophils are less sensitive. The combination of non-effective concentrations of AZD8999 (0.01nM) with non-effective concentrations of fluticasone propionate (0.1nM) shows synergistic anti-inflammatory effects. The studied mechanisms that may be involved in the synergistic anti-inflammatory effects of this combination include the increase of glucocorticoid receptor (GR)α and MKP1 expression, the induction of glucocorticoid response element (GRE) activation and the decrease of ERK1/2, P38 and GR-Ser226 phosphorylations compared with monotherapies. In summary, AZD8999 shows anti-inflammatory effects in neutrophils from COPD patients and induces synergistic anti-inflammatory effects when combined with fluticasone propionate, supporting the use of MABA/ICS combination therapy in COPD.

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<![CDATA[Guanylate-binding protein 5 licenses caspase-11 for Gasdermin-D mediated host resistance to Brucella abortus infection]]> https://www.researchpad.co/article/5c2e7fe3d5eed0c48451bfc7

Innate immune response against Brucella abortus involves activation of Toll-like receptors (TLRs) and NOD-like receptors (NLRs). Among the NLRs involved in the recognition of B. abortus are NLRP3 and AIM2. Here, we demonstrate that B. abortus triggers non-canonical inflammasome activation dependent on caspase-11 and gasdermin-D (GSDMD). Additionally, we identify that Brucella-LPS is the ligand for caspase-11 activation. Interestingly, we determine that B. abortus is able to trigger pyroptosis leading to pore formation and cell death, and this process is dependent on caspase-11 and GSDMD but independently of caspase-1 protease activity and NLRP3. Mice lacking either caspase-11 or GSDMD were significantly more susceptible to infection with B. abortus than caspase-1 knockout or wild-type animals. Additionally, guanylate-binding proteins (GBPs) present in mouse chromosome 3 participate in the recognition of LPS by caspase-11 contributing to non-canonical inflammasome activation as observed by the response of Gbpchr3-/- BMDMs to bacterial stimulation. We further determined by siRNA knockdown that among the GBPs contained in mouse chromosome 3, GBP5 is the most important for Brucella LPS to be recognized by caspase-11 triggering IL-1β secretion and LDH release. Additionally, we observed a reduction in neutrophil, dendritic cell and macrophage influx in spleens of Casp11-/- and Gsdmd-/- compared to wild-type mice, indicating that caspase-11 and GSDMD are implicated in the recruitment and activation of immune cells during Brucella infection. Finally, depletion of neutrophils renders wild-type mice more susceptible to Brucella infection. Taken together, these data suggest that caspase-11/GSDMD-dependent pyroptosis triggered by B. abortus is important to infection restriction in vivo and contributes to immune cell recruitment and activation.

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<![CDATA[In vitro and in vivo properties of the bovine antimicrobial peptide, Bactenecin 5]]> https://www.researchpad.co/article/5c3fa5efd5eed0c484caa407

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum.

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<![CDATA[Macrophage expression of E3 ubiquitin ligase Grail protects mice from lipopolysaccharide-induced hyperinflammation and organ injury]]> https://www.researchpad.co/article/5c33d20cd5eed0c4845dd4de

Multiple organ dysfunction caused by hyperinflammation remains the major cause of mortality during sepsis. Excessive M1-macrophage activation leads to systemic inflammatory responses. Gene related to anergy in lymphocytes (Grail) is regarded as an important regulator of T cells that functions by diminishing cytokine production. However, its role in regulating macrophage activation and organ injury during sepsis remains unclear. Our aim was to examine the effects of Grail on macrophage reactivity and organ injury in endotoxemic animals. Wild-type and Grail knockout mice were injected with vehicle or Escherichia coli lipopolysaccharide and observed for 24 h. Changes in blood pressure, heart rate, blood glucose, and biochemical variables were then examined. Moreover, levels of neutrophil infiltration, MMP-9, and caspase 3 were analyzed in the lungs of animals. The expression of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages stimulated with LPS were also assessed in the presence or absence of Grail. Results indicated that loss of Grail expression enhances the induction of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages treated with LPS. Furthermore, LPS-induced macrophage hyperactivation was alleviated by ectopic Grail overexpression. In vivo studies showed that Grail deficiency exacerbates organ damage in endotoxemic animals. Levels of neutrophil infiltration, MMP-9, and caspase 3 were significantly increased in the lungs of Grail-deficient endotoxemic mice. Thus, these results suggest that Grail contributes to the attenuation of hyperinflammation caused by activated macrophages and prevents organ damage in endotoxemic mice. We suggest that Grail signaling could be a therapeutic target for endotoxemia.

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<![CDATA[Microbiota control acute arterial inflammation and neointimal hyperplasia development after arterial injury]]> https://www.researchpad.co/article/5c12cf15d5eed0c484913eab

Background

The microbiome has a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia development has been linked to inflammation, a direct role of the microbiota in neointimal hyperplasia has not yet been established. Germ-free (GF) mice are an invaluable model for studying causative links between commensal organisms and the host. We hypothesized that GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally raised (CONV-R) mice.

Methods

Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers at five days. Systemic inflammation was assessed by multiplex immunoassays of serum. CONV-R mice treated in the same manner served as the control cohort. GF and CONV-R mice were compared using standard statistical methods.

Results

All GF mice remained sterile during the entire study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia development compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media) area. The collagen content of the neointimal lesions appeared qualitatively similar on Masson’s trichrome staining. There was significantly reduced Ki67 immunoreactivity in the media and adventitia of GF carotid arteries 5 days after ligation. GF mice also had increased arterial infiltration of anti-inflammatory M2 macrophages compared to CONV-R mouse arteries and a reduced proportion of mature neutrophils. GF mice had significantly reduced serum IFN-γ-inducible protein (IP)-10 and MIP-2 5 days after carotid ligation, suggesting a reduced systemic inflammatory response.

Conclusions

GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which is likely related to altered kinetics of wound healing and acute inflammation. Recognizing the role of commensals in the regulation of arterial remodeling will provide a deeper understanding of the pathophysiology of restenosis and support strategies to treat or reduce restenosis risk by manipulating microbiota.

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<![CDATA[Associations between CT-determined visceral fat burden, hepatic steatosis, circulating white blood cell counts and neutrophil-to-lymphocyte ratio]]> https://www.researchpad.co/article/5bfdb383d5eed0c4845ca124

Visceral adiposity is associated with cardiovascular disease, an association that may be mediated in part by inflammation. We hypothesized that regional measures of visceral adiposity would associate with commonly obtained clinical measures of immune status. We consecutively studied 3,291 subjects (mean age, 49.8±9.8 years) who underwent an annual cardiovascular risk survey. Peri-cardial (PCF) and thoracic peri-aortic adipose tissue (TAT) volumes were determined by dedicated computed tomography (CT) software (Aquarius 3D Workstation, TeraRecon, San Mateo, CA, USA). Hepatic steatosis was assessed by abdominal ultrasonography. We explored cross-sectional associations between visceral fat measures and high-sensitivity C-reactive protein (hs-CRP), leukocyte counts, and the neutrophil-to-lymphocyte ration (NLR). Among 3,291 study participants, we observed positive linear associations between PCF and TAT, higher degree of hepatic steatosis and hs-CRP, various leukocyte counts, either total and its differential counts, and NLR (all trend p<0.001). Multi-variate linear and logistic regression models showed independent associations between PCF/TAT (ß-Coef: 0.14/0.16, both p<0.05) and total WBC counts, with only TAT further demonstrated significant relations with neutrophil counts and NLR (both p<0.05) and independently identified abnormally high WBC and NLR (Odds ratio: 1.18 & 1.21, both p<0.05). C-statistics showed significant incremental model prediction for abnormally high WBC and NLR (both ΔAUROC<0.05) when TAT was superimposed on traditional cardiovascular risks and biochemical information. Greater visceral adiposity burden and hepatic steatosis may be associated with higher circulating leukocyte counts and markers for atherosclerosis, with more pronounced influences for peri-aortic adiposity. Our data suggested the differential biological impacts for region-specific visceral adiposity.

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<![CDATA[Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo]]> https://www.researchpad.co/article/5b28b928463d7e146ff345cd

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei’s opportunity for macrophage parasitism and thereby enhancing this pathogen’s exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

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<![CDATA[Pimaradienoic Acid Inhibits Carrageenan-Induced Inflammatory Leukocyte Recruitment and Edema in Mice: Inhibition of Oxidative Stress, Nitric Oxide and Cytokine Production]]> https://www.researchpad.co/article/5989da1fab0ee8fa60b7e548

Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1β. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation.

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<![CDATA[Major Neutrophilia Observed in Acute Phase of Human Leptospirosis Is Not Associated with Increased Expression of Granulocyte Cell Activation Markers]]> https://www.researchpad.co/article/5989db10ab0ee8fa60bcbdf5

It has long been known that pathogenic Leptospira can mobilize the immune system but the specific contribution of neutrophils to control the infectious challenge remains to be clarified. We herein analyzed the phenotype of circulating neutrophils of patients with leptospirosis and healthy controls for the expression of toll-like receptor (TLR) type 2 (TLR2, to sense the leptospiral LPS) and several activation markers: interleukin 8 chemokine receptor CD182 (CXCR2), CD11b of the integrin/opsonin complement receptor type 3 (CR3) and CD15 (ligand of the selectin). The plasmatic level of the main CD182 ligand, interleukin 8 (CXCL8), was measured by ELISA. Hospitalized leptospirosis cases showed marked neutrophilia, particularly in the most severe cases. Interestingly, TLR2 was significantly increased in leptospirosis but identical levels of CD182 and CD11b were detected when compared to controls. CD15 was significantly decreased on neutrophils in leptospirosis but returned to normal within 1 month. Basal levels of IL-8 were measured in control subjects and were not increased in leptospirosis cases at the initial stage of the disease. In conclusion, we observed that neutrophils failed to regulate the expression of several of the receptors involved in cell activation and recruitment. This study further emphasizes the paradigm that neutrophils may be impaired in their overall capacity to thwart bacterial infection in leptospirosis patients.

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