Degranulation Responses Research Articles

Longitudinal proteomic investigation of COVID-19 vaccination.

Although the development of COVID-19 vaccines has been a remarkable success, the heterogeneous individual antibody generation and decline over time are unknown and still hard to predict. In this study, blood samples were collected from 163 participants who next received two doses of an inactivated COVID-19 vaccine (CoronaVac®) at a 28-day interval. Using TMT-based proteomics, we identified 1,715 serum and 7,342 peripheral blood mononuclear cells (PBMCs) proteins. We proposed two sets of potential biomarkers (seven from serum, five from PBMCs) at baseline using machine learning, and predicted the individual seropositivity 57 days after vaccination (AUC = 0.87). Based on the four PBMC's potential biomarkers, we predicted the antibody persistence until 180 days after vaccination (AUC = 0.79). Our data highlighted characteristic hematological host responses, including altered lymphocyte migration regulation, neutrophil degranulation, and humoral immune response. This study proposed potential blood-derived protein biomarkers before vaccination for predicting heterogeneous antibody generation and decline after COVID-19 vaccination, shedding light on immunization mechanisms and individual booster shot planning.

Hyaluronic Acid in Synovial Fluid Prevents Neutrophil Activation in Spondyloarthritis

Spondyloarthritis (SpA) patients suffer from joint inflammation resulting in tissue damage, characterized by the presence of numerous neutrophils in the synovium and synovial fluid (SF). As it is yet unclear to what extent neutrophils contribute to the pathogenesis of SpA, we set out to study SF neutrophils in more detail. We analyzed the functionality of SF neutrophils of 20 SpA patients and 7 disease controls, determining ROS production and degranulation in response to various stimuli. In addition, the effect of SF on neutrophil function was determined. Surprisingly, our data show that SF neutrophils in SpA patients have an inactive phenotype, despite the presence of many neutrophil-activating stimuli such as GM-CSF and TNF in SF. This was not due to exhaustion as SF neutrophils readily responded to stimulation. Therefore, this finding suggests that one or more inhibitors of neutrophil activation may be present in SF. Indeed, when blood neutrophils from healthy donors were activated in the presence of increasing concentrations of SF from SpA patients, degranulation and ROS production were dose-dependently inhibited. This effect was independent of diagnosis, gender, age, and medication in the patients from which the SF was isolated. Treatment of SF with the enzyme hyaluronidase strongly reduced the inhibitory effect of SF on neutrophil activation, indicating that hyaluronic acid that is present in SF may be an important factor in preventing SF neutrophil activation. This finding provides novel insights into the role of soluble factors in SF regulating neutrophil function and may lead to the development of novel therapeutics targeting neutrophil activation via hyaluronic acid or associated pathways.

MRGPRX2 Antagonist EP262 Potently Inhibits Agonist-Induced Mast Cell Degranulation In Vitro and In Vivo

Mas-Related G-Protein Coupled Receptor X2 (MRGPRX2) is a promiscuous receptor on mast cells that mediates IgE-independent mast cell activation and degranulation in response to a wide variety of structurally diverse compounds including neuropeptides and host defense peptides and has been implicated in inflammatory conditions involving mast cell activation. Our objective was to develop and characterize a small molecule antagonist that potently inhibits MRGPRX2-mediated mast cell degranulation as a novel therapeutic approach for mast cell-mediated diseases.

Human EGFRvIII chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma.

Glioblastoma is a highly aggressive and fatal brain malignancy, and effective targeted therapies are required. The combination of standard treatments including surgery, chemotherapy and radiotherapy is not curative. Chimeric antigen receptor (CAR) T cells are known to cross the blood-brain barrier, mediating antitumor responses. A tumor-expressed deletion mutant of the epidermal growth factor receptor (EGFRvIII) is a robust CAR Tcell target in glioblastoma. Here, we show our de novo generated, high-affinity EGFRvIII-specific CAR; GCT02, demonstrating curative efficacy in human orthotopic glioblastoma models. The GCT02 binding epitope was predicted using Deep Mutational Scanning (DMS). GCT02 CAR T cell cytotoxicity was investigated in three glioblastoma models in vitro using the IncuCyte platform, and cytokine secretion with a cytometric bead array. GCT02 in vivo functionality was demonstrated in two NSG orthotopic glioblastoma models. The specificity profile was generated by measuring T cell degranulation in response to coculture with primary human healthy cells. The GCT02 binding location was predicted to be located at a shared region of EGFR and EGFRvIII; however, the in vitro functionality remained exquisitely EGFRvIII specific. A single CAR T cell infusion generated curative responses in two orthotopic models of human glioblastoma in NSG mice. The safety analysis further validated the specificity of GCT02 for mutant-expressing cells. This study demonstrates the preclinical functionality of a highly specific CAR targeting EGFRvIII on human cells. This CAR could be an effective treatment for glioblastoma and warrants future clinical investigation.

Esomeprazole induces structural changes and apoptosis and alters function of in vitro canine neoplastic mast cells

Histamine-2 receptor antagonists such as famotidine and proton pump inhibitors such as esomeprazole are commonly used in canine MCT disease, but direct effects on dog MCs have not been evaluated. Omeprazole is a proton pump inhibitor which has been demonstrated to cause structural and functional changes to in vitro murine mast cells (MCs). It has not yet been determined if esomeprazole, the commercially available and commonly prescribed S-isomer of omeprazole, has similar effects. Our primary study objective was to evaluate and compare the effects of acid suppressants (esomeprazole and famotidine) on MC ultrastructure, viability, and function in vitro using both healthy and neoplastic MCs. Murine bone marrow derived mast cells (BMMC), human LAD2, and canine C2 and BR cells, were used for these studies, representing a single healthy (i.e., BMMCs) MC model and multiple neoplastic MC models (i.e., LAD2, C2, BR), respectively. The rat basophilic leukemic (RBL-2H3) and canine B cell lymphoma 17–71 cell lines served as granulocytic and agranulocytic control lines for experiments, respectively. The treatment effect of acid suppressants on MC ultrastructure was assessed via both light and transmission electron microscopy. Differences in MC viability was assessed between groups via MTS-based, colorimetric assays and flow cytometry. Degranulation was assessed by quantification of β-hexosaminidase (i.e., LAD2 and RBL-2H3). Esomeprazole-treated MCs of all lines exhibited dramatic time and concentration-dependent alterations in ultrastructure (i.e., increased vacuolization, compromise of cell membrane), increased apoptosis, and altered degranulation responses in comparison to famotidine and vehicle-treated cells. The canine B cell lymphoma cells consistently exhibited either no significant (i.e., cytotoxicity assays) or greatly diminished treatment responses (i.e., apoptosis) compared to MCs. Esomeprazole, but not famotidine, induces significant cytotoxicity, as well as alterations to cell structure and function to multiple lines of in vitro neoplastic MCs. Continued in vitro work investigating the specific mechanisms by which proton pump inhibitors induce these effects, as well as prospective, in vivo work comparing the treatment effects of acid suppressants on canine MCTs, are warranted.

SARS-CoV-2 vaccine excipients polyethylene glycol and trometamol do not induce mast cell degranulation, in an in vitro model for non-IgE-mediated hypersensitivity.

The development of vaccines against SARS-CoV2 brought about several challenges, including the management of hypersensitivity reactions to these formulations. The search for underlying mechanisms involved in these adverse events initially focused on excipients which may trigger mast cell activation responses via non-IgE pathways: polyethylene glycol and trometamol. We sought to determine whether these components, in their pure form, were capable of stimulating mast cells directly. To test this hypothesis, we used an in vitro model for non-IgE-mediated activation that has previously shown degranulation responses induced via MRGPRX2 with known drug agonists of the receptor. Human LAD2 mast cells were incubated with different concentrations (1, 10, 50 mg/ml) of trometamol and of purified polyethylene glycol/Macrogol (molecular weights: 2,000, 3,350, 4,000, and 6,000). Mast cell degranulation was assessed using a beta-hexosaminidase read-out. Interestingly, degranulation responses for all reagents tested showed no significant differences from those obtained from the negative control (basal degranulation). Receptor-silencing assays were therefore not conducted. In summary, purified PEG and trometamol did not induce mast cell degranulation in this in vitro model for the study of non-IgE mechanisms of drug hypersensitivity, previously shown to be useful in the investigation of MRGPRX2 ligands. Studies using complete vaccine formulations, lipid conjugates, and receptor gene variants are needed to further clarify mechanisms of vaccine hypersensitivity.

Impaired Heat Shock Protein Expression in Activated T Cells in B-Cell Lymphoma.

Heat shock proteins (HSPs) are molecular chaperones that act in a variety of cellular processes, ensuring protein homeostasis and integrity. HSPs play critical roles in the modulation of various immune cells. However, the role of HSPs in T cell activation is largely unknown. We show that HSPs are upregulated following CD3/CD28 stimulation, suggesting that HSP expression might be regulated via TCR. We found that B-cell lymphoma (BCL) patients have dysregulated expression of intracellular and extracellular HSPs, immune checkpoints PD-1, CTLA-4, and STAT3 in CD3/CD28-activated T cells. Consistent with previous findings, we show that HSP90 inhibition downregulated CD4 and CD8 surface markers in healthy controls and BCL patients. HSP90 inhibition alone or in combination with PD-1 or CTLA-4 inhibitors differentially affected CD4+ and CD8+ T cell degranulation responses when stimulated with allogeneic DCs or CD3/CD28 in BCL patients. Additionally, we showed that HSP90 inhibition does not significantly affect intracellular PD-1 and CTLA-4 expression in CD3/CD28-activated T cells. These findings may provide the basis for the discovery of novel immunological targets for the treatment of cancer patients and improve our understanding of HSP functions in immune cells.

Blood plasma proteome alteration after local tissue damage induced by Bothrops erythromelas snake venom in mice

Snakes of the genus Bothrops are responsible the most snakebites in the Brazil, causing a diverse and complex pathophysiological condition. Bothrops erythromelas is the main specie of medical relevance found in the Caatinga from the Brazilian Northeast region. The pathophysiological effects involving B. erythromelas snakebite as well as the organism reaction in response to this envenomation are not so explored. Thus, edema was induced in mice paws using 2.5 μg or 5.0 μg of B. erythromelas venom, and the percentage of edema was measured. Plasma was collected 30 minutes after the envenomation-induced in mice and analyzed by mass spectrometry. It was identified a total of 112 common plasma proteins differentially abundant among experimental groups, which are involved with the complement system and coagulation cascades, oxidative stress, neutrophil degranulation, platelets degranulation and inflammatory response. Apolipoprotein A1 (Apoa), serum amyloid protein A-4 (Saa4), adiponectin (Adipoq) showed up-regulated in mice plasma after injection of venom, while fibulin (Fbln1), factor XII (F12) and vitamin K-dependent protein Z (Proz) showed down-regulated. The results indicate a protein pattern of thrombo-inflammation to the B. erythromelas snakebite, evidencing potential biomarkers for monitoring this snakebite, new therapeutic targets and its correlations with the degree of envenomation once showed modulations in the abundance among the different groups according to the amount of venom injected into the mice.

CD56-negative NK cells: Frequency in peripheral blood, expansion during HIV-1 infection, functional capacity, and KIR expression.

Human NK cells are usually defined as CD3-CD56+ lymphocytes. However, a CD56-CD16+ (CD56neg) lymphocyte population that displays NK-associated markers expands during chronic viral infections such as HIV-1 and HCV, and, to lesser extent, in herpesvirus infections. This CD56neg NK cell subset has been understudied because it requires the exclusion of other lymphocytes to accurately identify its presence. Many questions remain regarding the origin, development, phenotype, and function of the CD56neg NK cell population. Our objective was to determine the frequency of this NK subset in healthy controls and its alteration in viral infections by performing a meta-analysis. In addition to this, we analyzed deposited CyTOF and scRNAseq datasets to define the phenotype and subsets of the CD56neg NK cell population, as well as their functional variation. We found in 757 individuals, from a combined 28 studies and 6 datasets, that the CD56neg subset constitutes 5.67% of NK cells in healthy peripheral blood, while HIV-1 infection increases this population by a mean difference of 10.69%. Meta-analysis of surface marker expression between NK subsets showed no evidence of increased exhaustion or decreased proliferation within the CD56neg subset. CD56neg NK cells have a distinctive pattern of KIR expression, implying they have a unique potential for KIR-mediated education. A perforin-CD94-NKG2C-NKp30- CD56neg population exhibited different gene expression and degranulation responses against K562 cells compared to other CD56neg cells. This analysis distinguishes two functionally distinct subsets of CD56neg NK cells. They are phenotypically diverse and have differing capacity for education by HLA class-I interactions with KIRs.

Platelet SR-PSOX/CXCL16-CXCR6 Axis Influences Thrombotic Propensity and Prognosis in Coronary Artery Disease.

Platelets express the transmembrane chemokine SR-PSOX/CXCL16, proteolytic cleavage of which generates the sCXCL16 soluble-(s) chemokine. The sCXCL16 engages CXCR6 on platelets to synergistically propagate degranulation, aggregation and thrombotic response. Currently, we have investigated the pro-thrombotic and prognostic association of platelet CXCL16–CXCR6 axis in CAD-(n = 240; CCS n = 62; ACS n = 178) patients. Platelet surface-associated-CXCL16 and CXCR6 surface expression ascertained by flow cytometry correlated significantly with platelet activation markers (CD62P denoting degranulation and PAC-1 binding denoting α2bβ3-integrin activation). Higher platelet CXCL16 surface association (1st quartile vs. 2nd–4th quartiles) corresponded to significantly elevated collagen-induced platelet aggregation assessed by whole blood impedance aggregometry. Platelet-CXCL16 and CXCR6 expression did not alter with dyslipidemia, triglyceride, total cholesterol, or LDL levels, but higher (>median) plasma HDL levels corresponded with decreased platelet-CXCL16 and CXCR6. Although platelet-CXCL16 and CXCR6 expression did not change significantly with or correlate with troponin I levels, they corresponded with higher Creatine Kinase-(CK) activity and progressively deteriorating left ventricular ejection fraction (LVEF) at admission. Elevated-(4th quartile) platelet-CXCL16 (p = 0.023) and CXCR6 (p = 0.030) measured at admission were significantly associated with a worse prognosis. However, after Cox-PH regression analysis, only platelet-CXCL16 was ascertained as an independent predictor for all-cause of mortality. Therefore, the platelet CXCL16–CXCR6 axis may influence thrombotic propensity and prognosis in CAD patients.

Marek's disease virus-specific T cells proliferate, express antiviral cytokines but have impaired degranulation response.

The major histocompatibility complex (MHC) haplotype is one of the major determinants of genetic resistance and susceptibility of chickens to Marek’s disease (MD) which is caused by an oncogenic herpesvirus; Marek’s disease virus (MDV). To determine differential functional abilities of T cells associated with resistance and susceptibility to MD, we identified immunodominant CD4+TCRvβ1 T cell epitopes within the pp38 antigen of MDV in B19 and B21 MHC haplotype chickens using an ex vivo ELISPOT assay for chicken IFN-gamma. These novel pp38 peptides were used to characterize differential functional abilities of T cells as associated with resistance and susceptibility to MD. The results demonstrated an upregulation of cytokines (IL-2, IL-4, IL-10) and lymphocyte lysis-related genes (perforin and granzyme B) in an antigen specific manner using RT-PCR. In the MD-resistant chickens (B21 MHC haplotype), antigen-specific and non-specific response was highly skewed towards Th2 response as defined by higher levels of IL-4 expression as well as lymphocyte lysis-related genes compared to that in the MD-susceptible chicken line (B19 MHC haplotype). Using CD107a degranulation assay, the results showed that MDV infection impairs cytotoxic function of T cells regardless of their genetic background. Taken together, the data demonstrate an association between type of T cell response to pp38 and resistance to the disease and will shed light on our understanding of immune response to this oncogenic herpesvirus and failure to induce sterile immunity.

Multifunctional regulation of VAMP3 in exocytic and endocytic pathways of RBL-2H3 cells.

Mast cells (MCs) are inflammatory cells involved in allergic reactions. Crosslinking of the high-affinity receptor for IgE (FcϵRI) with multivalent antigens (Ags) induces secretory responses to release various inflammatory mediators. These responses are largely mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Vesicle-associated membrane protein 3 (VAMP3) is a vesicular-SNARE that interacts with targeted SNARE counterparts, driving the fusion of MC secretory granules with the membrane and affecting subsequent assembly of the plasma membrane. However, the role of VAMP3 in FcϵRI-mediated MC function remains unclear. In this study, we comprehensively examined the role of VAMP3 and the molecular mechanisms underlying VAMP3-mediated MC function upon FcϵRI activation. VAMP3 shRNA transduction considerably decreased VAMP3 expression compared with non-target shRNA-transduced (NT) cells. VAMP3 knockdown (KD) cells were sensitized with an anti-DNP IgE antibody and subsequently stimulated with Ag. The VAMP3 KD cells showed decreased degranulation response upon Ag stimulation. Next, we observed intracellular granule formation using CD63-GFP fluorescence. The VAMP3 KD cells were considerably impaired in their capacity to increase the size of granules when compared to NT cells, suggesting that VAMP3 mediates granule fusion and therefore promotes granule exocytosis in MCs. Analysis of FcϵRI-mediated activation of signaling events (FcϵRI, Lyn, Syk, and intracellular Ca2+ response) revealed that signaling molecule activation was enhanced in VAMP3 KD cells. We also found that FcϵRI expression on the cell surface decreased considerably in VAMP3 KD cells, although the amount of total protein did not vary. VAMP3 KD cells also showed dysregulation of plasma membrane homeostasis, such as endocytosis and lipid raft formation. The difference in the plasma membrane environment in VAMP3 KD cells might affect FcϵRI membrane dynamics and the subsequent signalosome formation. Furthermore, IgE/Ag-mediated secretion of TNF-α and IL-6 is oppositely regulated in the absence of VAMP3, which appears to be attributed to both the activation of FcϵRI and defects in VAMP3-mediated membrane fusion. Taken together, these results suggest that enhanced FcϵRI-mediated signal transduction in VAMP3 KD cells occurs due to the disruption of plasma membrane homeostasis. Hence, a multifunctional regulation of VAMP3 is involved in complex secretory responses in MCs.

NK Cell Subset Redistribution and Antibody Dependent Activation after Ebola Vaccination in Africans.

Natural killer cells play an important role in the control of viral infections both by regulating acquired immune responses and as potent innate or antibody-mediated cytotoxic effector cells. NK cells have been implicated in control of Ebola virus infections and our previous studies in European trial participants have demonstrated durable activation, proliferation and antibody-dependent NK cell activation after heterologous two-dose Ebola vaccination with adenovirus type 26.ZEBOV followed by modified vaccinia Ankara-BN-Filo. Regional variation in immunity and environmental exposure to pathogens, in particular human cytomegalovirus, have profound impacts on NK cell functional capacity. We therefore assessed the NK cell phenotype and function in African trial participants with universal exposure to HCMV. We demonstrate a significant redistribution of NK cell subsets after vaccine dose two, involving the enrichment of less differentiated CD56dimCD57− and CD56dimFcεR1γ+ (canonical) cells and the increased proliferation of these subsets. Sera taken after vaccine dose two support robust antibody-dependent NK cell activation in a standard NK cell readout; these responses correlate strongly with the concentration of anti-Ebola glycoprotein specific antibodies. These sera also promote comparable IFN-γ production in autologous NK cells taken at baseline and post-vaccine dose two. However, degranulation responses of post-vaccination NK cells were reduced compared to baseline NK cells and these effects could not be directly attributed to alterations in NK cell phenotype after vaccination. These studies demonstrate consistent changes in NK cell phenotypic composition and robust antibody-dependent NK cell function and reveal novel characteristics of these responses after heterologous two dose Ebola vaccination in African individuals.

Bispecific CD8 Treg modulators regulate a novel regulatory CD8 T cell network and eliminate pathogenic CD4 T cells in live cell co-culture system

Abstract INTRODUCTION Others have described a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation through canonical T cell receptors results in their oligoclonal expansion and perforin dependent elimination of pathogenic CD4 T cells. We have described the CD8 Treg network in Celiac patients and its potential to eliminate pathogenic CD4 T cells. Here we describe bispecific CD8 Treg modulators that activate CD8 Tregs, resulting in pathogenic CD4 T cell death. METHODS Novel bispecific CD8 Treg modulators were tested for target specificity by Octet and cell binding. The functional impact of CD8 Treg modulators was evaluated using flow and Luminex assays, and in a live cell co-culture system using Celiac patient derived CD8 Tregs and gliadin activated CD4 T cell targets. CD8 Treg modulators with specific molecular formats were then ranked for functional efficacy. RESULTS We tested a panel of bispecific CD8 Treg modulators with monovalent binding affinities in the low nanomolar range that selectively bind the CD8 Treg subset. Within 48 hours of CD8 Treg modulator addition to T cell co-cultures, CD8 Tregs had a rapid increase of cytolytic markers and degranulation in response to CD4 T cell targets. We observed a concomitant increase of activated CD4 T cell death and decrease of pro-inflammatory cytokines. CONCLUSIONS Our results indicate that a targeted approach to engage and activate CD8 Treg reduces pathogenic CD4 T cells. As this newly described CD8 Treg cell network is observed in many autoimmune diseases, we postulate that bispecific CD8 T cell modulators may represent a novel, selective and broadly applicable therapeutic approach for the treatment of human autoimmune diseases.

Aberrant HSP90 Expression in Lymphocytes and HSP90 Response to Anti-PD-1 Therapy in Lymphoma Patients.

HSP90 family of molecular chaperones has been shown to be implicated in various stages of tumor growth and development. Recent studies have highlighted the role of extracellular HSP90 in tumor immunology, however, the role that HSP90 plays in the regulation of immune responses and the impact of cancer immunotherapy, including immune checkpoint blockade, on HSP90 is still unclear. Here we assessed the surface and intracellular expression of constitutive cytosolic HSP90β isoform, mitochondrial HSP90 homolog TRAP1 and co-chaperone STIP1/HOP in T, NK, B and NKT cells derived from peripheral blood and bone marrow samples of patients with Hodgkin and B-cell Non-Hodgkin lymphomas. HSP90β and STIP1 were overexpressed in B lymphocytes, while TRAP1 expression was decreased in T, B, NK and NKT cells of lymphoma patients. HSP90 overexpression in B cells was not associated with malignant B cell clones, since no clonotypic B cells were detected by immunoglobulin heavy chain (IgH) gene rearrangements. PD-1 blockade was found to differently affect the intracellular and surface HSP90 in T, B, NK and NKT cells in patients with relapsed or refractory classical Hodgkin lymphoma. Modulating HSP90 was found to affect the NK cell degranulation response and IFNγ production in lymphoma patients. These findings provide the rationale to further explore HSP90 homologs for improving patient response to cancer immunotherapy.

Mast cell activation test: A new asset in the investigation of the chlorhexidine cross-sensitization profile.

Insights into the IgE cross-sensitization and possible cross-reactivity patterns of sera reactive to chlorhexidine (CHX) are still incomplete and are likely to benefit from a functional exploration using a passive mast cell activation test (pMAT). Therefore, we want to study whether the pMAT with CHX-specific IgE (sIgE) enables to depict effector cell degranulation in response to alexidine (ALX), octenidine (OCT) and/or polyhexamethylene biguanide (PHMB) indicative of cross-reactivity between these compounds and CHX. Serum of 10 CHX-allergic patients, nine individuals with an isolated sIgE CHX and five healthy controls were included. Human cultured mast cells (MCs) were, before and after sensitization, challenged with CHX, ALX, OCT or PHMB. Degranulation was measured via quantification of upregulation of CD63. Mast cell responsiveness to ALX and OCT was demonstrable with 4/10 and 3/10 of the sera of CHX-allergic patients respectively. Percentage of degranulation varied between 12 and 34% for ALX-reactive MCs and between 4 and 22% for OCT-reactive MCs. No reactivity to ALX or OCT was demonstrable when using sera obtained from individuals with an isolated sIgE CHX or from healthy controls. Unlike CHX, ALX and OCT, PHMB turned out to be a direct MC activator via occupation of MRGPRX2. PHMB-reactive sIgEs were demonstrable in some patients with an isolated sIgE CHX but were unable to trigger PHMB-induced degranulation in MRGPRX2 knockdown MCs. Mast cells constitute an attractive tool to explore cross-reactivity between structurally similar compounds. Along with the identification of safe alternatives for the individual patient, the pMAT can advance our insights into sIgE cross-reactivity patterns including assessment of molecules not yet approved for human use.

An experimental in silico study on COVID-19: Response of neutrophil-related genes to antibiotics.

Background and AimsAll components of the immune system are involved in alleviating severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection. Further research is required to provide detailed insights into COVID‐19‐related immune compartments and pathways. In addition, a significant percentage of hospitalized COVID‐19 patients suspect bacterial infections and antimicrobial resistance occurs following antibiotics treatment. The aim of this study was to evaluate the possible effects of antibiotics on the response of neutrophil‐related genes in SARS‐CoV‐2 patients by an experimental in silico study.MethodsThe two data sets GSE1739 and GSE21802 including 10 SARS positive patients and 35 influenza A (H1N1) patients were analyzed, respectively. Differentially expressed genes (DEGs) between these two data sets were determined by GEO2R analysis and the Venn diagram online tool. After determining the hub genes involved in immune responses, the expression of these genes in 30 COVID‐19 patients and 30 healthy individuals was analyzed by real‐time polymerase chain reaction (PCR). All patients received antibiotics, including levofloxacin, colistin, meropenem, and ceftazidime.ResultsGEO2R analysis detected 240 and 120 DEGs in GSE21802 and GSE1739, respectively. Twenty DEGs were considered as enriched hub genes involved in immune processes such as neutrophil degranulation, neutrophil activation, and antimicrobial humoral response. The central nodes were attributed to the genes of neutrophil elastase (ELANE), arginase 1 (ARG‐1), lipocalin 2 (LCN2), and defensin 4 (DEFA4). Compared to the healthy subjects, the expression of LCN2 and DEFA4 were significantly reduced in COVID‐19 patients. However, no significant differences were observed in the ELANE and AGR‐1 levels between COVID‐19 subjects and the control group.ConclusionsActivation and degranulation of neutrophils were observed mainly in SARS, and H1N1 infection processes and antibiotics administration could affect neutrophil activity during viral infection. It can be suggested that antibiotics can decrease inflammation by restoring the expression of neutrophil‐related genes in COVID‐19 patients.

Gene Differential Expression and Interaction Networks Illustrate the Biomarkers and Molecular Mechanisms of Atherosclerotic Cerebral Infarction.

Atherosclerotic cerebral infarction (ACI) seriously threatens the health of the senile patients, and the strategies are urgent for the diagnosis and treatment of ACI. This study investigated the mRNA profiling of the patients with ischemic stroke and atherosclerosis via excavating the datasets in the GEO database and attempted to reveal the biomarkers and molecular mechanism of ACI. In this study, GES16561 and GES100927 were obtained from Gene Expression Omnibus (GEO) database, and the related differentially expressed genes (DEGs) were analyzed with R language. Furthermore, the DEGs were analyzed with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Besides, the protein-protein interaction (PPI) network of DEGs was analyzed by STRING database and Cytoscape. The results showed that 133 downregulated DEGs and 234 upregulated DEGs were found in GES16561, 25 downregulated DEGs and 104 upregulated DEGs were found in GSE100927, and 6 common genes were found in GES16561 and GES100927. GO enrichment analysis showed that the functional models of the common genes were involved in neutrophil activation, neutrophil degranulation, neutrophil activation, and immune response. KEGG enrichment analysis showed that the DEGs in both GSE100927 and GSE16561 were connected with the pathways including Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Phagosome, Antigen processing and presentation, and Staphylococcus aureus infection. The PPI network analysis showed that 9 common DEGs were found in GSE100927 and GSE16561, and a cluster with 6 nodes and 12 edges was also identified by PPI network analysis. In conclusion, this study suggested that FCGR3A and MAPK pathways were connected with ACI.

Macrophages secrete murinoglobulin-1 and galectin-3 to regulate neutrophil degranulation after myocardial infarction.

Inflammation presides early after myocardial infarction (MI) as a key event in cardiac wound healing. Ischemic cardiomyocytes secrete inflammatory cues to stimulate infiltration of leukocytes, predominantly macrophages and neutrophils. Infiltrating neutrophils degranulate to release a series of proteases including matrix metalloproteinase (MMP)-9 to break down extracellular matrix and remove necrotic myocytes to create space for the infarct scar to form. While neutrophil to macrophage communication has been explored, the reverse has been understudied. We used a proteomics approach to catalogue the macrophage secretome at MI day 1. Murinoglobulin-1 (MUG1) was the highest-ranked secreted protein (4.1-fold upregulated at MI day 1 vs. day 0 pre-MI cardiac macrophages, p = 0.004). By transcriptomics evaluation, galectin-3 (Lgals3) was 2.2-fold upregulated (p = 0.008) in MI day 1 macrophages. We explored the direct roles of MUG1 and Lgals3 on neutrophil degranulation. MUG1 blunted while Lgals3 amplified neutrophil degranulation in response to phorbol 12-myristate 13-acetate or interleukin-1β, as measured by MMP-9 secretion. Lgals3 itself also stimulated MMP-9 secretion. To determine if MUG1 regulated Lgals3, we co-stimulated neutrophils with MUG1 and Lgals3. MUG1 limited degranulation stimulated by Lgals3 by 64% (p < 0.001). In vivo, MUG1 was elevated in the infarct region at MI days 1 and 3, while Lgals3 increased at MI day 7. The ratio of MUG1 to Lgals3 positively correlated with infarct wall thickness, revealing that MUG1 attenuated infarct wall thinning. In conclusion, macrophages at MI day 1 secrete MUG1 to limit and Lgals3 to accentuate neutrophil degranulation to regulate infarct wall thinning.

NAD+-boosting molecules suppress mast cell degranulation and anaphylactic responses in mice.

Nicotinamide adenine dinucleotide (NAD+) acts as a cofactor for multiple biological processes. While previous research has revealed that the NAD+ declines associated with aging contributes to an impairment of immune cells, its role in mast cell function, especially in response to an anaphylactic condition, has remained unexplored. We tested whether the restoration of cellular NAD+ concentration by the supplementation of NAD+ boosting molecules prevented mast cell degranulation and anaphylactic responses.Methods: Bone marrow derived mast cells (BMMCs) and human cord blood derived mast cells were treated with NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR), and FcεRI downstream signaling was assessed. Animal models of passive systemic anaphylaxis (PSA) and passive cutaneous anaphylaxis (PCA) were used to investigate the effects of NAD+ precursors in the anaphylactic responses of mice.Results: Treatment of murine BMMCs and human cord blood derived mast cells with NAD+ precursors repressed intracellular signaling downstream of FcεRI, as well as the release of inflammatory cytokines and lipid mediators. The intraperitoneal administration of NMN or NR also markedly attenuated IgE-mediated anaphylactic responses in mouse models of PSA and PCA. These beneficial effects of NAD+ precursors, however, were attenuated in mast cell-specific Sirt6 knockout mice, indicating a Sirt6 dependency for their action.Conclusion: NAD+ precursors may serve as an effective therapeutic strategy that limits mast cell-mediated anaphylactic responses.