Neutrophil-mediated Inflammation Research Articles

Pulmonary inflammation and Tie2/Angiopoietin mediated vascular dysregulation during lethal Orientia tsutsugamushi infection

Abstract Orientia tsutsugamushi infection can cause acute lung injury and high mortality in humans; however, the underlying mechanisms are unclear. Here, we tested a hypothesis that dysregulated pulmonary inflammation and Tie2-mediated endothelial malfunction contribute to lung damage. Using a murine model of lethal O. tsutsugamushi infection, we demonstrated pathological characteristics of lung damage: 1) a significant increase of ICAM-1, VEGFR2, and angiopoietin-2 (Ang2) proteins in inflamed tissues and lung-derived endothelial cells (EC), 2) a progressive loss of endothelial quiescent and junction proteins (Ang1, VE-cadherin), and 3) a profound impairment of Tie2 receptor at the transcriptional and functional levels. In vitro infection of primary human EC cultures and serum Ang2 proteins in scrub typhus patients support our animal studies, implying endothelial dysfunction in severe scrub typhus. Evidence for neutrophil-mediated inflammation and tissue damage included in vitro infection and activation of neutrophils, lung accumulation of activated (CD63+)neutrophils, co-localization of lung bacteria with neutrophils, and co-localization of MPO+ neutrophils with Ang2+ ECs. At the onset of disease, pulmonary macrophages (MF) were polarized toward an M1-like phenotype, with limited detection of M2-like cells. Comparative studies of LPS- versus IL-4-primed MFs revealed extensive bacterial replication in M2-type MFs that preferentially expressed the IL-10 and SOCS1 genes. This is the first detailed investigation of lung cellular immune responses during acute O. tsutsugamushi infection and highlights inflammatory biomarkers future therapeutic research for the control of this neglected tropical disease.

The MUB<sub>40</sub> Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

The MUB<sub>40</sub> Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

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.

Cerebrospinal Fluid Transcriptional Analyses Reveals Upregulation of IL-17, Type 1 Interferon Transcriptional Pathways and Neutrophil Persistence Genes Associated with Increased Mortality from Pneumococcal Meningitis in Adults

Background: Improving outcomes from pneumococcal meningitis (PM), particularly in populations with high HIV prevalence, requires better understanding of host inflammatory responses to infection. Methods: We compared the transcriptome in pre-antibiotic cerebrospinal fluid (CSF) and blood from Malawian adults with PM using RNA sequencing. We used network analyses and cellular/process deconvolution of the transcriptome to identify important patho-physiological associations with outcome. Findings: Blood transcriptional profiles were obtained in 28 patients (21 HIV co-infected; median age 33 years [26-66]; median CSF WCC 28 cells/mm3 [0-3660]; median bacterial load 4.7x106 copies/ml CSF [671-2x109]; in-hospital mortality 64%), paired CSF profiles were obtained in 13. Marked differences in gene expression by outcome were confined to the CSF. In non-survivors, differentially expressed genes in the CSF were co-correlated in a network of pro-inflammatory gene-clusters enriched for collagen degradation and platelet degranulation. In contrast, CSF gene expression networks from surviving patients were dominated by DNA repair, transcriptional regulation and immunological signalling. CSF expression of gene response-modules for IL-17, Type 1 interferons and IL-10 were enriched in non-survivors, expression of cell-specific response-modules did not differ by outcome. However, genes for neutrophil chemotaxis and persistence were highly over-expressed in non-survivors. Interpretation: These data suggest poor outcome in PM is associated with over-expression of IL-17 and T1-IFN associated pro-inflammatory responses in the CSF and suggest a role for neutrophil-mediated inflammation. These responses are unlikely to be effected by current adjunctive treatments. Improving poor outcomes from PM will require better-targeted interventions. Registration Number: (Current Controlled Trials registration ISRCTN96218197). Funding Statement: This study was funded by a Clinical Lecturer Starter Grant from the Academy of Medical Sciences (UK), by a PhD Fellowship in Global Health to EW from the Wellcome Trust (089671/B/09/Z) and by a Postdoctoral Clinical Research Fellowship to GP from the Wellcome Trust (WT101766). The Malawi-Liverpool-Wellcome Trust Clinical Research Programme is supported by a core grant from the Wellcome Trust (101113/Z/13/Z). This work was undertaken at UCLH/UCL who received a proportion of funding from the National Institute for Health Research University College London Hospitals Department of Health’s NIHR Biomedical Research Centre. JSB, MN and CV are supported by the Centre’s funding scheme. The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data and the final responsibility to submit for publication. Declaration of Interests: All authors have no conflicts of interest to declare. Ethics Approval Statement: All participants or nominated guardians gave written informed consent for inclusion. Ethical approval for the transcriptomics study was granted by both the College of Medicine Research and Ethics Committee (COMREC), University of Malawi, (P.01/10/980, January 2011), and the Liverpool School of Tropical Medicine Research Ethics Committee, UK (P10.70, November 2010) Committee, Liverpool, UK

Soluble Fas ligand blocks destructive corneal inflammation in mouse models of corneal epithelial debridement and LPS induced keratitis

Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using intra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation.

Inflammatory Mechanisms Contributing to Aortic Expansion and Rupture after Acute Aortic Dissection

In-hospital outcomes are generally acceptable with the conservative treatment of uncomplicated type B aortic dissection, but some patients present with undesirable complications, such as aortic expansion and rupture. Beyond mechanical and shear forces of blood flow affecting the weakened aortic wall, excessive inflammatory response has been shown to be associated with aortic expansion and adverse clinical outcomes. We have previously demonstrated the underlying mechanisms of catastrophic complications after acute aortic dissection (AAD) in mice. We propose that aortic dissection induces expression of the neutrophil chemoattractants CXCL1 and granulocyte-colony stimulating factor in the aortic tunica adventitia. These local environmental changes recruit neutrophils in combination with alteration of bone marrow milieu where reduced CXCL12 expression enhances neutrophil egress. Interleukin (IL)-6 production in the inflammatory adventitial neutrophils causes vascular inflammation, leading to vascular wall fragility. Targeting CXCR2 or IL-6 mitigates aortic expansion and prevents mice from aortic rupture. Collectively, adventitial neutrophil-mediated inflammation may be a potential therapeutic target to limit lethal complications after AAD.

Potential Role of Neutrophil-Mediated Vascular Inflammation and NET Formation in Atherosclerosis

Potential Role of Neutrophil-Mediated Vascular Inflammation and NET Formation in Atherosclerosis

Fcγ receptor III expression and morphological maturity on neutrophil are associated with higher iron level of major beta-thalassemia

Lifetime blood transfusion experienced by major β-thalassemia patients complicated with iron overload, therefore, may lead to their tissue injury. Ultimately, free toxic iron may alter immune response via dysregulation of immune cell activity producing prolonged effector reaction. Neutrophil as one of the vital innate immune cell despite serves as the first line of defense resulting acute inflammation has a pivotal role in chronic inflammation while releasing the toxic substance that interferes biological processes. This process is initiated by one of them by activation of Fcγ Receptor III (CD16), a neutrophil membrane-bound protein. A cross-sectional laboratory study involving lysed-erythrocyte heparinized whole blood of fifty pediatric major β-thalassemia patients treated with monoclonal antibodies i.e. CD16, CD14, and HLA-DR, dissected into CD16+ and CD16++ population using flow cytometry. Expression of Fcγ Receptor III was measured as Median Fluorescent Intensity (MFI). Hematology and iron status were measured. A correlation analysis was done. MFI of CD16 neutrophil [509.5 (371 - 796.5)] and ferritin level [(3209 µg/L, 1862 - 4564)] was positively correlated (r = 0.4, P = 0.007). Respectively, ferritin and serum iron were found negatively correlated with segmented neutrophils (r = -0.3, P = 0.02; r = -0.3, P = 0.02). Change in CD16 expression may implicate preliminarily neutrophil activation as a response of iron-overloaded tissue and result in chronic inflammation in β-thalassemia patients. However, the maturity of this cell may be altered. Future study in the understanding of neutrophil-mediated inflammation, particularly related to immune complexes and functionality, is imperative to be explored.

IL-6 Drives Neutrophil-Mediated Pulmonary Inflammation Associated with Bacteremia in Murine Models of Colitis

Inflammatory bowel disease (IBD) is associated with several immune-mediated extraintestinal manifestations. More than half of all IBD patients have some form of respiratory pathology, most commonly neutrophil-mediated diseases, such as bronchiectasis and chronic bronchitis. Using murine models of colitis, we aimed to identify the immune mechanisms driving pulmonary manifestations of IBD. We found increased neutrophil numbers in lung tissue associated with the pulmonary vasculature in both trinitrobenzenesulfonic acid- and dextran sulfate sodium-induced models of colitis. Analysis of systemic inflammation identified that neutrophilia was associated with bacteremia and pyrexia in animal models of colitis. We further identified IL-6 as a systemic mediator of neutrophil recruitment from the bone marrow of dextran sulfate sodium animals. Functional inhibition of IL-6 led to reduced systemic and pulmonary neutrophilia, but it did not attenuate established colitis pathology. These data suggest that systemic bacteremia and pyrexia drive IL-6 secretion, which is a critical driver for pulmonary manifestation of IBD. Targeting IL-6 may reduce neutrophil-associated extraintestinal manifestations in IBD patients.

Inflammation–coagulation response and thrombotic effects induced by silica nanoparticles in zebrafish embryos

Nowadays, nanotechnology environmental health and safety (nanoEHS) is gaining attention. We previously found that silica nanoparticles (SiNPs) could induce vascular endothelial damage. However, the subsequent toxicologic response to SiNPs-induced endothelial damage was still largely unknown. In this study, we explored the inflammation–coagulation response and thrombotic effects of SiNPs in endothelial cells and zebrafish embryos. For in vitro study, swollen mitochondria and autophagosome were observed in ultrastructural analysis. The cytoskeleton organization was disrupted by SiNPs in vascular endothelial cells. The release of proinflammatory and procoagulant cytokines including IL-6, IL-8, MCP-1, PECAM-1, TF and vWF, were markedly elevated in a dose-dependent manner. For in vivo study, based on the NOAEL for dosimetry selection, and using two transgenic zebrafish, Tg(mpo:GFP) and Tg(fli-1:EGFP), SiNPs-induced neutrophil-mediated inflammation and impaired vascular endothelial cells. With the dosage higher than NOAEL, SiNPs significantly decreased blood flow and velocity, exhibiting a blood hypercoagulable state in zebrafish embryos. The thrombotic effect was assessed by o-dianisidine staining, showed that an increasing of erythrocyte aggregation occurred in SiNPs-treated zebrafish. Microarray analysis was used to screen the possible genes for inflammation–coagulation response to SiNPs in zebrafish, and the JAK1/TF signaling pathway was further verified by qRT-PCR and Western blot assays. For in-deepth study, il6st was knocked down with specific morpholinos. The whole-mount in situ hybridization and qRT-PCR analysis showed that the expression jak1 and f3b were attenuated in il6st knockdown groups. In summary, our data demonstrated that SiNPs could induce inflammation–coagulation response and thrombotic effects via JAK1/TF signaling pathway.

CXCL8 hyper-signaling in the aortic abdominal aneurysm

There are indications for elevated CXCL8 levels in abdominal aortic aneurysm disease (AAA).CXCL8 is concurrently involved in neutrophil-mediated inflammation and angiogenesis, two prominent and distinctive characteristics of AAA. As such we considered an evaluation of a role for CXCL8 in AAA progression relevant.ELISA’s, real time PCR and array analysis were used to explore CXCL8 signaling in AAA wall samples. A role for CXCL8 in AAA disease was tested through the oral CXCR1/2 antagonist DF2156A in the elastase model of AAA disease.There is an extreme disparity in aortic wall CXCL8 content between AAA and aortic atherosclerotic disease (median [IQR] aortic wall CXCL8 content: 425 [141–1261] (AAA) vs. 23 [2.8–89] (atherosclerotic aorta) µg/g protein (P < 1 · 10−14)), and abundant expression of the CXCR1 and 2 receptors in AAA. Array analysis followed by pathway analysis showed that CXCL8 hyper-expression in AAA is followed increased by IL-8 signaling (Z-score for AAA vs. atherosclerotic control: 2.97, p < 0.0001).Interference with CXCL8 signaling through DF2156A fully abrogated AAA formation and prevented matrix degradation in the murine elastase model of AAA disease (p < 0.001).CXCL8-signaling is a prominent and distinctive feature of AAA, interference with the pathway constitutes a promising target for medical stabilization of AAA.

Systemic Loss of C-terminal Src Kinase Expression Elicits Spontaneous Suppurative Inflammation in Conditional Knockout Mice.

C-terminal Src kinase (Csk) is one of the critical negative regulators of the Src family of kinases. The Src family of kinases are nonreceptor tyrosine kinases that regulate inflammation, cell proliferation, motility, and adhesion. To investigate potential histologic lesions associated with systemic loss of Csk gene activity in adult mice, conditional Csk-knockout mice were examined. Cre-mediated systemic excision of Csk induced by tamoxifen treatment resulted in multiorgan inflammation. Specifically, induction of Csk gene excision with three days of tamoxifen treatment resulted in greater than 90% gene excision. Strikingly, these mice developed enteritis that ranged from minimal and suppurative to severe, fibrinonecrosuppurative and hemorrhagic. Other inflammatory lesions included suppurative pneumonia, gastritis, and myocarditis, and increased numbers of inflammatory cells within the hepatic parenchyma. When tamoxifen treatment was reduced from three days to one day in an effort to lower the level of Csk gene excision and limit lesion development, the mice developed severe suppurative to pyogranulomatous pneumonia and minimal to mild suppurative enteritis. Lesions observed secondary to Csk gene excision suggest important roles for Csk in downregulating the proinflammatory activity of the Src family of kinases and limiting neutrophil-mediated inflammation.

Infection Is Not Required for Mucoinflammatory Lung Disease in CFTR-Knockout Ferrets.

Classical interpretation of cystic fibrosis (CF) lung disease pathogenesis suggests that infection initiates disease progression, leading to an exuberant inflammatory response, excessive mucus, and ultimately bronchiectasis. Although symptomatic antibiotic treatment controls lung infections early in disease, lifelong bacterial residence typically ensues. Processes that control the establishment of persistent bacteria in the CF lung, and the contribution of noninfectious components to disease pathogenesis, are poorly understood. To evaluate whether continuous antibiotic therapy protects the CF lung from disease using a ferret model that rapidly acquires lethal bacterial lung infections in the absence of antibiotics. CFTR (cystic fibrosis transmembrane conductance regulator)-knockout ferrets were treated with three antibiotics from birth to several years of age and lung disease was followed by quantitative computed tomography, BAL, and histopathology. Lung disease was compared with CFTR-knockout ferrets treated symptomatically with antibiotics. Bronchiectasis was quantified from computed tomography images. BAL was evaluated for cellular differential and features of inflammatory cellular activation, bacteria, fungi, and quantitative proteomics. Semiquantitative histopathology was compared across experimental groups. We demonstrate that lifelong antibiotics can protect the CF ferret lung from infections for several years. Surprisingly, CF animals still developed hallmarks of structural bronchiectasis, neutrophil-mediated inflammation, and mucus accumulation, despite the lack of infection. Quantitative proteomics of BAL from CF and non-CF pairs demonstrated a mucoinflammatory signature in the CF lung dominated by Muc5B and neutrophil chemoattractants and products. These findings implicate mucoinflammatory processes in the CF lung as pathogenic in the absence of clinically apparent bacterial and fungal infections.

CXCR2 Inhibition \u2013 a novel approach to treating CoronAry heart DiseAse (CICADA): study protocol for a randomised controlled trial

BackgroundThere is emerging evidence of the central role of neutrophils in both atherosclerotic plaque formation and rupture. Patients with lower neutrophil counts following acute coronary syndromes tend to have a greater coronary flow reserve, which is a strong predictor of long-term cardiovascular health. But so far, no data are available regarding the impact of neutrophil inhibition on cardiovascular clinical or surrogate endpoints. Therefore, the aim of this study is to investigate the effects of AZD5069, a cysteine-X-cysteine chemokine receptor 2 (CXCR2) inhibitor, on coronary flow reserve and coronary structure and function in patients with coronary artery disease.Methods/DesignNinety subjects with coronary artery disease undergoing percutaneous coronary intervention will be included in this investigator-driven, randomised, placebo-controlled, double-blind, phase IIa, single-centre study. Participants will be randomised to receive either AZD5069 (40 mg) administered orally twice daily or placebo for 24 weeks. Change in coronary flow reserve as determined by 13N-ammonia positron emission tomography-computed tomography will be the primary outcome. Change in the inflammatory component of coronary plaque structure and the backward expansion wave, an invasive coronary physiological measure of diastolic function, will be assessed as secondary outcomes.DiscussionCardiovascular surrogate parameters, such as coronary flow reserve, may provide insights into the potential mechanisms of the cardiovascular effects of CXCR2 inhibitors. Currently, ongoing trials do not specifically focus on neutrophil function as a target of intervention, and we therefore believe that our study will contribute to a better understanding of the role of neutrophil-mediated inflammation in coronary artery disease.Trial registrationEudraCT, 2016-000775-24. Registered on 22 July 2016.International Standard Randomised Controlled Trial Number, ISRCTN48328178. Registered on 25 February 2016.

IFN-λ 'guts' neutrophil-mediated inflammation.

Interferon-λ (IFN-λ) curbs neutrophil-mediated intestinal inflammation by diminishing the production of reactive oxygen species and subsequent oxidative stress. This regulatory process is unique to IFN-λ and is independent of interferon-induced transcription and translation programs.

Abstract 243: Neutrophil-Mediated Inflammation Drives Sickle Cell Vasculopathy

Objectives: Many of the clinical complications associated with sickle cell disease (SCD), such as stroke, pain crises, proliferative retinopathy, renal and heart failure, can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of vascular repair following injury are largely unknown in SCD. Given our previous work showing the importance of reactive oxygen species in neovascularization, we aimed to delineate the immune mechanisms of oxidative stress during vascular repair in a humanized sickle cell mouse model (SS) in comparison to wildtype (AA). Methods: We performed limb ischemia (HLI) in mice by ligation of the femoral artery to evaluate vascular dysfunction in sickle cell mice. Vascular recovery was ascertained using weekly LASER Doppler perfusion imaging (LDPI) for 28 days. Voluntary running wheel test was used to determine spontaneous motor function recovery. Results: There was significant diminution in functional collateral vessel formation in SS mice following HLI as evaluated by LDPI (76 ±13 % recovery in AA vs 34±10 % recovery in SS by day 28, p &lt; 0.001 n=8 per group). This was characterized by dysfunctional Moyamoya-like sprouting vessels with impaired spontaneous motor function recovery in SS. Specifically, AA mice recovered 98% motor function by day 28 following HLI, vs 36% in SS mice, p &lt; 0.001. The phenotype was associated with persistent neutrophils in the hind limb muscle of SS mice up to 28 days, a time point by which all neutrophils were cleared in AA mice. Consequently, there was a 2.45 fold increased production of hydrogen peroxide in SS mice ischemic hind limbs at day 28, compared to AA mice (p&lt; 0.05). Importantly, in vivo depletion of neutrophils improved functional collateral vessel formation in the SS mice. Conclusions: Our data suggest that neutrophil-mediated excessive inflammation and oxidative stress drive dysfunctional collateral vessel formation in SS mice following ischemic injury. Targeting neutrophils may improve vascular dysfunction in SS disease.

Involvement of IL-17A-producing TCR γδ T cells in late protective immunity against pulmonary Mycobacterium tuberculosis infection

Abstract Interleukin (IL)-17A is a cytokine originally reported to induce neutrophil-mediated inflammation and anti-microbial activity. The CD4+ T cells which produce IL-17A have been well characterized as Th17 cells. On the other hand, IL-17A-producing TCR γδ+ T cells have been reported to participate in the immune response at an early stage of infection with Listeria monocytogenes and Mycobacterium bovis in mice. However, the involvement of IL-17A in protective immunity was not clearly demonstrated in the chronic stage of M. tuberculosis-infected mice. In this study, we found that TCR γδ+ T cells are a primary source of IL-17A, but that mycobacterial antigen-specific Th17 cells were hardly detected even at the chronic stage of M. tuberculosis infection. IL-17A-deficient mice showed a decreased survival rate, and increased bacterial burden in the lungs after the infection when compared to the wild-type mice. Furthermore, a histological analysis showed an impaired granuloma formation in the infected lungs of IL-17A-deficient mice, which was considered to be due to a decrease of IFN-γ and TNF-α at the chronic stage. These data suggest that the IL-17A-producing TCR γδ+ T cells, rather than the Th17 cells, in the infected lungs are an indispensable source of protective immunity against M. tuberculosis infection.

Alamandine abrogates neutrophil degranulation in atherosclerotic mice

Neutrophil-mediated inflammation was recently identified as an active contributor to athero-progression. Therapeutic strategies inhibiting neutrophil degranulation or recruitment were hypothesized to positively impact on plaque vulnerability. In this study, we investigated whether treatment with the recently discovered agonist of the Mas-related G-coupled receptor type D (MrgD) alamandine would impact on neutrophil degranulation in vivo and in vitro. Fifteen-week-old ApoE-/- mice were fed with a Western-type diet for an additional 11 weeks. After the first 2 weeks of diet, mice were surgically implanted with a carotid 'cast' device that alters the blood shear stress and induces different carotid plaque phenotypes. During the last 4 weeks before euthanasia, mice were randomly assigned to subcutaneously receive vehicle (NaCl 0·15 M) or alamandine (24 μg/kg/h) by micropump. For in vitro experiments, neutrophils were obtained after thioglycollate intraperitoneal injection in ApoE-/- mice. Treatment with alamandine was well-tolerated, but failed to affect lipid, macrophage, neutrophil or collagen content within carotid and aortic root plaques. Also, treatment with alamandine did not affect Th-cell polarization in lymphoid organs. Conversely, alamandine administration was associated with a reduction in serum levels of neutrophil granule enzymes, such as MMP-9 and MPO as well as MMP-9 content within aortic root plaques. In vitro, preincubation with alamandine dose-dependently abrogated PMA-induced neutrophil degranulation of MMP-9 and MPO. These results suggest that treatment with the MrgD agonist alamandine led to a reduced release of neutrophil granule products, potentially interfering with pro-atherosclerotic neutrophil activation.

Celastrol alleviates arthritis by modulating the inflammatory activities of neutrophils

ObjectiveNeutrophils play an important role in the pathogenesis of rheumatoid arthritis (RA). In this study, we used the adjuvant-induced arthritis murine model to evaluate the efficacy of celastrol on neutrophil-mediated inflammation in RA. MethodsFreund's complete adjuvant-induced arthritis was used as the murine model of RA. Celastrol was intraperitoneally administrated daily after onset of the disease. The joint diameter and inflammatory score were evaluated daily during the treatment period. Myeloperoxidase (MPO) and neutrophil elastase (NE) activities were evaluated by immunohistochemical analyses. Quantitative PCR and enzyme-linked immunoabsorbent assay were used to quantify the expression of cytokines. The expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 were evaluated by western blot. ResultsCelastrol suppressed inflammation in joints of arthritic mice and diminished the expression of MPO and NE in the joint tissue. Celastrol significantly inhibited the expression of TNFα and IL-6 induced by LPS in neutrophils in a dose-dependent manner in vitro. Moreover, celastrol induced apoptosis of LPS-stimulated neutrophils by increasing the expression of Bax and cleaved caspase-3 while decreasing Bcl-2 expression. ConclusionOur findings show that celastrol significantly alleviates murine arthritis by modulating the inflammatory activities of neutrophils. These results indicate that celastrol could serve as an alternative or adjunct modality for the treatment of RA.