Expression Of Surface Activation Markers Research Articles

Mononuclear cell composition and activation in blood and mucosal tissue of eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a chronic, inflammatory, antigen-driven disease of the esophagus. Tissue EoE pathology has previously been extensively characterized by novel transcriptomics and proteomic platforms, however the majority of surface marker determination and screening has been performed in blood due to mucosal tissue size limitations. While eosinophils, CD4+ T cells, mast cells and natural killer (NK) T cells were previously investigated in the context of EoE, an accurate picture of the composition of peripheral blood mononuclear cells (PBMC) and their activation is missing. In this study, we aimed to comprehensively analyze the composition of peripheral blood mononuclear cells and their activation using surface marker measurements with multicolor flow cytometry simultaneously in both blood and mucosal tissue of patients with active EoE, inactive EoE, patients with gastroesophageal reflux disease (GERD) and controls. Moreover, we set out to validate our data in co-cultures of PBMC with human primary esophageal epithelial cells and in a novel inducible mouse model of eosinophilic esophagitis, characterized by extensive IL-33 secretion in the esophagus. Our results indicate that specific PBMC populations are enriched, and that they alter their surface expression of activation markers in mucosal tissue of active EoE. In particular, we observed upregulation of the immunomodulatory molecule CD38 on CD4+ T cells and on myeloid cells in biopsies of active EoE. Moreover, we observed significant upregulation of PD-1 on CD4+ and myeloid cells, which was even more prominent after corticosteroid treatment. With co-culture experiments we could demonstrate that direct cell contact is needed for PD-1 upregulation on CD4+ T cells. Finally, we validated our findings of PD-1 and CD38 upregulation in an inducible mouse model of EoE. Herein we show significant alterations in the PBMC activation profile of patients with active EoE in comparison to inactive EoE, GERD and controls, which could have potential implications for treatment. To our knowledge, this study is the first of its kind expanding the multi-color flow cytometry approach in different patient groups using in vitro and in vivo translational models.

Age-dependent inflammatory response is altered in an ex vivo model of bacterial pneumonia

BackgroundAging is associated with an increased incidence and mortality of Pseudomonas aeruginosa-induced pneumonias. This might be partly due to age-dependent increases in inflammatory mediators, referred to as inflamm-aging and a decline in immune functions, known as immunosenescence. Still, the impact of dysregulated immune responses on lung infection during aging is poorly understood. Here, we aimed to mimic inflamm-aging using ex vivo precision-cut lung slices (PCLS) and neutrophils – as important effector cells of innate immunity – from young and old mice and investigated the influence of aging on inflammation upon infection with P. aeruginosa bacteria.MethodsMurine PCLS were infected with the P. aeruginosa standard lab strain PAO1 and a clinical P. aeruginosa isolate D61. After infection, whole-transcriptome analysis of the tissue as well as cytokine expression in supernatants and tissue lysates were performed. Responses of isolated neutrophils towards the bacteria were investigated by quantifying neutrophil extracellular trap (NET) formation, cytokine secretion, and analyzing expression of surface activation markers using flow cytometry.ResultsInflamm-aging was observed by transcriptome analysis, showing an enrichment of biological processes related to inflammation, innate immune response, and chemotaxis in uninfected PCLS of old compared with young mice. Upon P. aeruginosa infection, the age-dependent pro-inflammatory response was even further promoted as shown by increased production of cytokines and chemokines such as IL-1β, IL-6, CXCL1, TNF-α, and IL-17A. In neutrophil cultures, aging did not influence NET formation or cytokine secretion during P. aeruginosa infection. However, expression of receptors associated with inflammatory responses such as complement, adhesion, phagocytosis, and degranulation was lower in neutrophils stimulated with bacteria from old mice as compared to young animals.ConclusionsBy using PCLS and neutrophils from young and old mice as immunocompetent ex vivo test systems, we could mimic dysregulated immune responses upon aging on levels of gene expression, cytokine production, and receptor expression. The results furthermore reflect the exacerbation of inflammation upon P. aeruginosa lung infection as a result of inflamm-aging in old age.

Characterization of the effect of histone deacetylation inhibitors on CD8+ T cells in the context of aging

BackgroundPosttranslational protein modifications regulate essential cellular processes, including the immune cell activation. Despite known age-related alterations of the phenotype, composition and cytokine profiles of immune cells, the role of acetylation in the aging process of the immune system was not broadly investigated. Therefore, in the current study the effect of acetylation on the protein expression profiles and function of CD8+ T cells from donors of distinct age was analyzed using histone deacetylase inhibitors (HDACi).MethodsCD8+ T cells isolated from peripheral blood mononuclear cells of 30 young (< 30 years) and 30 old (> 60 years) healthy donors were activated with anti-CD3/anti-CD28 antibodies in the presence and absence of a cocktail of HDACi. The protein expression profiles of untreated and HDACi-treated CD8+ T cells were analyzed using two-dimensional gel electrophoresis. Proteins with a differential expression level (less than 0.66-fold decrease or more than 1.5-fold increase) between CD8+ T cells of young and old donors were identified by matrix-associated laser desorption ionization—time of flight mass spectrometry. Functional enrichment analysis of proteins identified was performed using the online tool STRING. The function of CD8+ T cells was assessed by analyses of cytokine secretion, surface expression of activation markers, proliferative capacity and apoptosis rate.ResultsThe HDACi treatment of CD8+ T cells increased in an age-independent manner the intracellular acetylation of proteins, in particular cytoskeleton components and chaperones. Despite a strong similarity between the protein expression profiles of both age groups, the functional activity of CD8+ T cells significantly differed with an age-dependent increase in cytokine secretion and expression of activation markers for CD8+ T cells from old donors, which was maintained after HDACi treatment. The proliferation and apoptosis rate of CD8+ T cells after HDACi treatment was equal between both age groups.ConclusionsDespite a comparable effect of HDACi treatment on the protein signature of CD8+ T cells from donors of different ages, an initial higher functionality of CD8+ T cells from old donors when compared to CD8+ T cells from young donors was detected, which might have clinical relevance.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products—An In-Depth Analysis

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

Preservation of antigen-specific responses in cryopreserved CD4+ and CD8+ T cells expanded with IL-2 and IL-7

ObjectivesWe sought to develop medium throughput standard operating procedures for screening cryopreserved human peripheral blood mononuclear cells (PBMCs) for CD4+ and CD8+ T cell responses to potential autoantigens. MethodsDendritic cells were loaded with a peptide cocktail from ubiquitous viruses or full-length viral protein antigens and cocultured with autologous T cells. We measured expression of surface activation markers on T cells by flow cytometry and cytometry by time of flight 24–72 h later. We tested responses among T cells freshly isolated from healthy control PBMCs, cryopreserved T cells, and T cells derived from a variety of T cell expansion protocols. We also compared the transcriptional profile of CD8+ T cells rested with interleukin (IL)7 for 48 h after 1) initial thawing, 2) expansion, and 3) secondary cryopreservation/thawing of expanded cells. To generate competent antigen presenting cells from PBMCs, we promoted differentiation of PBMCs into dendritic cells with granulocyte macrophage colony stimulating factor and IL-4. ResultsWe observed robust dendritic cell differentiation from human PBMCs treated with 50 ng/mL GM-CSF and 20 ng/mL IL-4 in as little as 3 days. Dendritic cell purity was substantially increased by magnetically enriching for CD14+ monocytes prior to differentiation. We also measured antigen-dependent T cell activation in DC-T cell cocultures. However, polyclonal expansion of T cells with anti-CD3/antiCD28 abolished antigen-dependent upregulation of CD69 in our assay despite minimal transcriptional differences between rested CD8+ T cells before and after expansion. Furthermore, resting these expanded T cells in IL-2, IL-7 or IL-15 did not restore the antigen dependent responses. In contrast, T cells that were initially expanded with IL-2 + IL-7 rather than plate bound anti-CD3 + anti-CD28 retained responsiveness to antigen stimulation and these responses strongly correlated with responses measured at initial thawing. SignificanceWhile screening techniques for potential pathological autoantibodies have come a long way, comparable full-length protein target assays for screening patient T cells at medium throughput are noticeably lacking due to technical hurdles. Here we advance techniques that should have broad applicability to translational studies investigating cell mediated immunity in infectious or autoimmune diseases. Future studies are aimed at investigating possible CD8+ T cell autoantigens in MS and other CNS autoimmune diseases.

Autoantibodies to red blood cell surface Glycophorin A impact the activation poise of circulating leukocytes.

Both M and N alleles encode antigens on Glycophorin A (GPA), a red blood cell (RBC) surface sialoglycoprotein. Interaction between RBC GPA and leukocyte surface lectins may downregulate their activation. The current study investigates if RBC autoantibodies against GPA, such as auto-anti-M/N, prime an activated phenotype in peripheral blood leukocytes. Leukocyte activation was assessed in whole blood from patients with auto-anti-GPA (anti-M/N) and compared to those with allo-anti-M/N and healthy subjects. Control samples from healthy subjects with no antibodies incubated in vitro with either anti-GPA or anti-Rh were analyzed for neutrophil and monocyte surface activation marker expression, reactive oxygen species (ROS) content, and formation of aggregates with RBCs. Samples incubated with an IgG1 isotype antibody served as controls. Ex vivo, neutrophil CD66b and monocyte CD63 surface expression was increased in patients with auto-anti-M/N compared to those with allo anti-M/N (p=.1757; p=.0698) and to healthy subjects (p=.0186; p=.013). In vitro, neutrophil CD66b and monocyte CD63 surface expression was increased following incubation with anti-GPA compared to anti-Rh (p=.0003; p=.0328) and isotype control (p=.000; p=.0062). Intracellular ROS content increased in both neutrophils and monocytes incubated with anti-GPA compared to anti-Rh (p=.0012; p=.0693) and isotype control (p=.001; p=.0021). Percentage of neutrophil-RBC aggregates was decreased when incubated with anti-GPA compared to isotype control (p < .01). Neutrophils and monocytes in peripheral blood exposed to an antibody directed against GPA on RBC surfaces, such as M or N antigens, may be primed towards an activated phenotype.

Intranasal Administration of Codium fragile Polysaccharide Elicits Anti-Cancer Immunity against Lewis Lung Carcinoma.

Natural polysaccharides have shown promising effects on the regulation of immunity in animals. In this study, we examined the immune stimulatory effect of intranasally administered Codium fragile polysaccharides (CFPs) in mice. Intranasal administration of CFPs in C57BL/6 mice induced the upregulation of surface activation marker expression in macrophages and dendritic cells (DCs) in the mediastinal lymph node (mLN) and the production of interleukin-6 (IL-6), IL-12p70, and tumor necrosis factor-α in bronchoalveolar lavage fluid. Moreover, the number of conventional DCs (cDCs) was increased in the mLNs by the upregulation of C-C motif chemokine receptor 7 expression, and subsets of cDCs were also activated following the intranasal administration of CFP. In addition, the intranasal administration of CFPs promoted the activation of natural killer (NK) and T cells in the mLNs, which produce pro-inflammatory cytokines and cytotoxic mediators. Finally, daily administration of CFPs inhibited the infiltration of Lewis lung carcinoma cells into the lungs, and the preventive effect of CFPs on tumor growth required NK and CD8 T cells. Furthermore, CFPs combined with anti-programmed cell death-ligand 1 (PD-L1) antibody (Ab) improved the therapeutic effect of anti-PD-L1 Ab against lung cancer. Therefore, these data demonstrated that the intranasal administration of CFP induced mucosal immunity against lung cancer.

Impact of Plasmodium falciparum small-sized extracellular vesicles on host peripheral blood mononuclear cells

Background: Exagerated immune activation has a key role in the pathogenesis of malaria. During blood-stage infection, Plasmodium falciparum can interact directly with host immune cells through infected red blood cells (PfiRBCs), or indirectly by the release of extracellular vesicles (EVs). Here, we compared the impact of PfiRBCs and P. falciparum small-sized EVs (PfsEVs, also known as exosomes) from a Kenyan clinical isolate (PfKE12) adapted to short-term laboratory culture conditions on host peripheral blood mononuclear cells (PBMC). Methods: PfsEVs were isolated from cell-free culture-conditioned media by ultracentrifugation while mature trophozoite PfiRBCs were purified by magnetic column separation. The PfsEVs and the PfiRBCs were co-cultured for 18 hours with PBMC. Cellular responses were quantified by cell surface expression of activation markers (CD25, CD69) and cytokine/chemokine levels in the supernatant. Results: Relative to negative control conditions, PfsEVs induced CD25 expression on CD4+, CD19+ and CD14+ cells, while PfiRBCs induced on CD19+ and CD14+ cells. Both PfsEVs and PfiRBCs induced CD69 on CD4+, CD8+ and CD19+ cells. In addition, PfiRBCs induced higher expression of CD69 on CD14+ cells. CD69 induced by PfiRBCs on CD4+ and CD19+ cells was significantly higher than that induced by PfsEVs. Secretion of MIP1α, MIP1β, GM-CSF, IL-6, IL-8, and TNFα were significantly induced by both PfsEVs and PfiRBCs whereas MCP-1, IL-10, IL-17α were preferentially induced by PfsEVs and IP-10 and IFN-γ by PfiRBCs. Prior exposure to malaria (judged by antibodies to schizont extract) was associated with lower monocyte responses to PfsEVs. Conclusions: PfsEVs and PfiRBCs showed differential abilities to induce secretion of IL-17α and IFN-γ, suggesting that the former are better at inducing Th17, whilst the latter induce Th1 immune responses respectively. Prior exposure to malaria significantly reduces the ability of PfsEVs to activate monocytes, suggesting immune tolerance to PfsEVs may play a role in naturally acquired anti-disease immunity.

Laquinimod dampens IL-1β signaling and Th17-polarizing capacity of monocytes in patients with MS.

ObjectiveTo assess the impact of laquinimod treatment on monocytes and to investigate the underlying immunomodulatory mechanisms in MS.MethodsIn this cross-sectional study, we performed in vivo and in vitro analyses of cluster of differentiation (CD14+) monocytes isolated from healthy donors (n = 15), untreated (n = 13), and laquinimod-treated patients with MS (n = 14). Their frequency and the expression of surface activation markers were assessed by flow cytometry and the viability by calcein staining. Cytokine concentrations in the supernatants of lipopolysaccharide (LPS)-stimulated monocytes were determined by flow cytometry. The messenger ribonucleic acid (mRNA) expression level of genes involved in cytokine expression was measured by quantitative PCR. The LPS-mediated nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation was determined by the quantification of the phosphorylation level of the p65 subunit. Laquinimod-treated monocytes were cocultured with CD4+ T cells, and the resulting cytokine production was analyzed by flow cytometry after intracellular cytokine staining. The interleukin (IL)-17A concentration of the supernatant was assessed by ELISA.ResultsLaquinimod did not alter the frequency or viability of circulating monocytes, but led to an upregulation of CD86 expression. LPS-stimulated monocytes of laquinimod-treated patients with MS secreted less IL-1β following a downregulation of IL-1β gene expression. Phosphorylation levels of the NF-κB p65 subunit were reduced after laquinimod treatment, indicating a laquinimod-associated inhibition of the NF-κB pathway. T cells primed with laquinimod-treated monocytes differentiated significantly less into IL-17A–producing T helper (Th)-17 cells.ConclusionsOur findings suggest that inhibited NF-κB signaling and downregulation of IL-1β expression in monocytes contributes to the immunomodulatory effects of laquinimod and that the impairment of Th17 polarization might mediate its disease-modifying activity in MS.

NADPH oxidase 1 is a novel pharmacological target for the development of an antiplatelet drug without bleeding side effects.

Growing evidence supports a central role of NADPH oxidases (NOXs) in the regulation of platelets, which are circulating cells involved in both hemostasis and thrombosis. Here, the use of Nox1-/- and Nox1+/+ mice as experimental models of human responses demonstrated a critical role of NOX1 in collagen-dependent platelet activation and pathological arterial thrombosis, as tested in vivo by carotid occlusion assays. In contrast, NOX1 does not affect platelet responses to thrombin and normal hemostasis, as assayed in tail bleeding experiments. Therefore, as NOX1 inhibitors are likely to have antiplatelet effects without associated bleeding risks, the NOX1-selective inhibitor 2-acetylphenothiazine (2APT) and a series of its derivatives generated to increase inhibitory potency and drug bioavailability were tested. Among the 2APT derivatives, 1-(10H-phenothiazin-2-yl)vinyl tert-butyl carbonate (2APT-D6) was selected for its high potency. Both 2APT and 2APT-D6 inhibited collagen-dependent platelet aggregation, adhesion, thrombus formation, superoxide anion generation, and surface activation marker expression, while responses to thrombin or adhesion to fibrinogen were not affected. In vivo administration of 2APT or 2APT-D6 led to the inhibition of mouse platelet aggregation, oxygen radical output, and thrombus formation, and carotid occlusion, while tail hemostasis was unaffected. Differently to in vitro experiments, 2APT-D6 and 2APT displayed similar potency in vivo. In summary, NOX1 inhibition with 2APT or its derivative 2APT-D6 is a viable strategy to control collagen-induced platelet activation and reduce thrombosis without deleterious effects on hemostasis. These compounds should, therefore, be considered for the development of novel antiplatelet drugs to fight cardiovascular diseases in humans.

Impact of Plasmodium falciparum small-sized extracellular vesicles on host peripheral blood mononuclear cells

Background: Exagerated immune activation has a key role in the pathogenesis of malaria. During blood-stage infection, Plasmodium falciparum can interact directly with host immune cells through infected red blood cells (PfiRBCs), or indirectly by the release of extracellular vesicles (EVs). Here, we compared the impact of PfiRBCs and P. falciparum small-sized EVs (PfsEVs, also known as exosomes) from a Kenyan clinical isolate (PfKE12) adapted to short-term laboratory culture conditions on host peripheral blood mononuclear cells (PBMC). Methods: PfsEVs were isolated from cell-free culture-conditioned media by ultracentrifugation while mature trophozoite PfiRBCs were purified by magnetic column separation. The PfsEVs and the PfiRBCs were co-cultured for 18 hours with PBMC. Cellular responses were quantified by cell surface expression of activation markers (CD25, CD69) and cytokine/chemokine levels in the supernatant. Results: Relative to negative control conditions, PfsEVs induced CD25 expression on CD4+, CD19+ and CD14+ cells, while PfiRBCs induced on CD19+ and CD14+ cells. Both PfsEVs and PfiRBCs induced CD69 on CD4+, CD8+ and CD19+ cells. In addition, PfiRBCs induced higher expression of CD69 on CD14+ cells. CD69 induced by PfiRBCs on CD4+ and CD19+ cells was significantly higher than that induced by PfsEVs. Secretion of MIP1α, MIP1β, GM-CSF, IL-6, IL-8, and TNFα were significantly induced by both PfsEVs and PfiRBCs whereas MCP-1, IL-10, IL-17α were preferentially induced by PfsEVs and IP-10 and IFN-γ by PfiRBCs. Prior exposure to malaria (judged by antibodies to schizont extract) was associated with lower monocyte responses to PfsEVs. Conclusions: PfsEVs and PfiRBCs showed differential abilities to induce secretion of IL-17α and IFN-γ, suggesting that the former are better at inducing Th17, whilst the latter induce Th1 immune responses respectively. Prior exposure to malaria significantly reduces the ability of PfsEVs to activate monocytes, suggesting immune tolerance to PfsEVs may play a role in naturally acquired anti-disease immunity.

Cytokine Pre-activated γδT cells in Cancer Adoptive Immunotherapy

Abstract Adoptive cell therapy harnesses the power of immune system by stimulating and expanding immune cells to combat cancer. The application of tumour-specific αβT cell-based therapy may be hindered by limited tumour antigens. γδT cells are promising candidates for cellular immunotherapy because of their abundant IFN-γ or IL-17 production and MHC-independent cytotoxicity against a broad spectrum of tumors. Numerous attempts to use γδT cells in cancer immunotherapy have been made and resulted in variable efficacy and an overall good safety profile. Better expansion strategies are needed to improve the efficacy of the clinical trials using γδT cell-based immunotherapies. In the current project, we aim to explore the efficacy of cytokine pre-activated γδT cells in cancer immunotherapy. Pre-activated γδT cells by a combination of cytokines were characterised for their surface activation marker expression, cytokine production, and cytotoxicity against tumour cells. We found that IL-12/18, IL-12/15/18, IL-12/18/21 and IL-12/15/18/21 are the most effective cytokine combinations for in vitro expansion of γδT cells in terms of activation and cytokine production. IL-21 upregulated their cytotoxicity against tumour cells in murine systems. All four combinations showed upregulated cytotoxicity of human γδT cells. These findings were extended to in vivo studies using murine hepatocellular carcinoma model.

Dual Pro- and Anti-Inflammatory Features of Monocyte-Derived Dendritic Cells.

The transcription factor β-catenin is able to induce tolerogenic/anti-inflammatory features in different types of dendritic cells (DCs). Monocyte-derived dendritic cells (moDCs) have been widely used in dendritic cell-based cancer therapy, but so far with limited clinical efficacy. We wanted to investigate the hypothesis that aberrant differentiation or induction of dual pro- and anti-inflammatory features may be β-catenin dependent in moDCs. β-catenin was detectable in both immature and lipopolysaccharide (LPS)-stimulated DCs. The β-catenin inhibitor ICG-001 dose-dependently increased the pro-inflammatory signature cytokine IL-12p70 and decreased the anti-inflammatory signature molecule IL-10. The β-catenin activator 6-bromoindirubin-3′-oxime (6-BIO) dose-dependently increased total and nuclear β-catenin, and this was associated with decreased IL-12p70, increased IL-10, and reduced surface expression of activation markers, such as CD80 and CD86, and increased expression of inhibitory markers, such as PD-L1. 6-BIO and ICG-001 competed dose-dependently regarding these features. Genome-wide mRNA expression analyses further underscored the dual development of pro- and anti-inflammatory features of LPS-matured moDCs and suggest a role for β-catenin inhibition in production of more potent therapeutic moDCs.

Factor VIII Fc Fusion Protein but not FVIII Drives Human Monocyte-Derived Dendritic Cell Activation via FcγRIIa.

This study compares the effect of recombinant Factor VIII Fc fusion protein (rFVIII-Fc) with recombinant FVIII (rFVIII) on monocyte-derived dendritic cells (moDC's). Cells treated with rFVIII-Fc showed morphological changes typical for cell activation, had a significant up-regulation of cell activation markers and produced higher levels of pro-inflammatory cytokines. Even after stimulation with Lipopolysaccharides, the addition of rFVIII-Fc led to increased expression of activation markers, indicating that rFVIII-Fc is capable of amplifying the maturation signal. On the contrary, cultivation of moDC's with rFVIII did not alter cell morphology or increase surface activation marker expression and pro-inflammatory cytokine production. The binding of the Fc domain to the activating Fcγ receptor IIa (FcγRIIa) can cause cell activation. Therefore, the effect of rFVIII-Fc on FcγRIIa was analyzed in detail. Cultivation of moDC's with rFVIII-Fc led to increased phosphorylation of FcγRIIa, which was not detected for rFVIII. Blocking FcγRIIa prior to the cultivation with rFVIII-Fc significantly reduced the activating effect of rFVIII-Fc, indicating that rFVIII-Fc-induced moDC activation was caused by FcγRIIa. Moreover, rFVIII-Fc bound to FCGR2A-transfected human embryonic kidney 293 cells. Taken together, our data present a new mechanism of moDC activation by rFVIII-Fc via FcγRIIa.

Platelet PI3K Modulates Innate Leukocyte Extravasation during Acid-Induced Acute Lung Inflammation.

Blood platelets are increasingly recognized as modulators of leukocyte effector functions in various pathologies including acute lung injury (ALI). ALI is a life-threatening disease, caused by damage to the alveolar epi- and endothelium. Excessive accumulation of leukocytes leads to severe lung inflammation, resulting in impaired lung function and hypoxemia. Since leukocyte migration is modulated by activated platelets and phosphatidylinositol 3-kinase (PI3K) signaling is involved in platelet function, we aimed to elucidate the effect of PI3K on platelet-mediated immune responses. We generated a mouse model with a platelet-specific deletion of p85α, the most important regulatory subunit of the class IA PI3K, and evaluated platelet function and platelet-leukocyte interactions. Moreover, we analyzed the impact of platelet-specific p85α gene deficiency during sterile peritonitis and acid-induced ALI. In vitro analyses of platelets revealed that lack of p85α led to decreased downstream signaling and diminished expression of surface activation markers, for example, CD62P and CD63, as well as reduced platelet aggregation. Moreover, platelet PI3K essentially mediated direct interactions of platelets with monocytes and neutrophils. In mice, platelet-specific p85α deficiency prevented leukocyte infiltration into the peritoneum and the bronchoalveolar compartment during sterile peritonitis and ALI, respectively. Additionally, the release of the inflammatory cytokine interleukin-12/23 was diminished in platelet p85α-deficient mice during ALI. In contrast to PI3K, neither overexpression nor depletion of platelet phosphatase and tensin homolog, the endogenous antagonist of PI3K, significantly modulated platelet function. Our data indicate a crucial role of platelet PI3K signaling for leukocyte extravasation upon inflammatory stimuli in various diseases models.

A Plate-based Cytotoxicity Assay for the Assessment of Rat Placental Natural Killer Cell Cytolytic Function.

It is well known that decidual natural killer (NK) cells play a critical role in establishment and maintenance of normal pregnancy. Recent studies have demonstrated an altered population of circulating and decidual NK cells in women who suffer from adverse pregnancy complications such as recurrent miscarriage and preeclampsia. Studies from our group have shown that hypertension in pregnancy is associated with an increased population of activated NK cells in the placenta based on the expression of surface activation markers. This manuscript provides a detailed protocol to assess the cytotoxic function of NK cells isolated from placentas in a preeclampsia-like animal model of surgically induced placental ischemia. The following steps are described in detail: generation of single cell suspension, NK cell isolation, ex vivo stimulation, effector:target cell co-culture, and the cytotoxicity assay.

Role of Escherichia coli endopeptidases and dd-carboxypeptidases in infection and regulation of innate immune response

The low-molecular-mass penicillin-binding proteins, involved in peptidoglycan recycling can also produce peptidoglycan fragments capable of activating an innate immune response in host. To investigate how these proteins in Enterobacteriaceae play a role to elicit/evade innate immune responses during infections, we deleted certain endopeptidases and dd-carboxypeptidases from Escherichia coli CS109 and studied the viability of these mutants in macrophages. The ability of infected macrophages to exert oxidative killing, express surface activation markers TLR2, MHC class II and release TNFα, were assessed. Immune responses were elevated in macrophages infected with dd-carboxypeptidase mutants but reduced for endopeptidase mutants. However, the NFκB, iNOS, and TLR2 transcripts remained elevated in macrophages infected with both mutant types. Overall, we have shown, under normal conditions endopeptidases have a tendency to elicit the immune response but their effect is suppressed by the presence of dd-carboxypeptidases. Conversely, DD-carboxypeptidases, normally, tend to reduce immune responses, as their deletions enhanced the same in macrophages. Therefore, we conclude that the roles of endopeptidases and dd-carboxypeptidases are possibly counter-active in wild-type cells where either class of enzymes suppresses each other's immunogenic properties rendering overall maintenance of low immunogenicity that helps E. coli in evading the host immune responses.

Assessing cross-reactivity to neuromuscular blocking agents by skin and basophil activation tests in patients with neuromuscular blocking agent anaphylaxis

BackgroundFollowing diagnosis of neuromuscular blocking agent (NMBA) anaphylaxis, identifying safe alternatives for subsequent anaesthesia is critical. A patient with anaphylaxis to one NMBA can also have an allergic reaction to other NMBAs (cross-reactivity). Whilst drug provocation testing is standard for identifying or excluding allergy, there is significant risk. In vitro, after an allergen activates basophils, basophils express surface activation markers that can be measured by basophil activation testing (BAT). We compared cross-reactivity between NMBAs assessed by BAT against that by skin testing. MethodsAll patients attending an anaesthetic allergy clinic in Sydney, Australia between May 2017 and July 2018 diagnosed with NMBA anaphylaxis qualified for this study comparing intradermal skin tests and BAT with a panel of NMBAs (rocuronium, vecuronium, pancuronium, suxamethonium, cisatracurium). ResultsOf the 61 patients participating, sensitisation on skin testing and on BAT completely matched in only nine patients (15%). Sensitisation was not in agreement for pancuronium, cisatracurium and rocuronium, but was in agreement for vecuronium and suxamethonium. Nine patients with negative skin tests subsequently tolerated cisatracurium, and one false positive on BAT to cisatracurium was detected. ConclusionsThe utility of BAT in identifying safe NMBAs for subsequent anaesthesia needs further evaluation. BAT detects a different cross-reactivity profile to skin tests. Negative skin testing and BAT might increase confidence in performing drug provocation testing, but this and follow-up of subsequent anaesthesia in our cohort is necessary to determine the clinical significance of BAT sensitisation.

GAS6/TAM Pathway Signaling in Hemostasis and Thrombosis.

The GAS6/TYRO3-AXL-MERTK (TAM) signaling pathway is essential for full and sustained platelet activation, as well as thrombus stabilization. Inhibition of this pathway decreases platelet aggregation, shape change, clot retraction, aggregate formation under flow conditions, and surface expression of activation markers. Transgenic mice deficient in GAS6, or any of the TAM family of receptors that engage this ligand, exhibit in vivo protection against arterial and venous thrombosis but do not demonstrate either spontaneous or prolonged bleeding compared to their wild-type counterparts. Comparable results are observed in wild-type mice treated with pharmacological inhibitors of the GAS6-TAM pathway. Thus, GAS6/TAM inhibition offers an attractive novel therapeutic option that may allow for a moderate reduction in platelet activation and decreased thrombosis while still permitting the primary hemostatic function of platelet plug formation.

Immunomodulatory properties of Brucella melitensis lipopolysaccharide determinants on mouse dendritic cells in vitro and in vivo

ABSTRACT The lipopolysaccharide (LPS) is a major virulence factor of Brucella, a facultative intracellular pathogenic Gram-negative bacterium. Brucella LPS exhibits a low toxicity and its atypical structure was postulated to delay the host immune response, favouring the establishment of chronic disease. Here we carried out an in-depth in vitro and in vivo characterisation of the immunomodulatory effects of Brucella LPS on different dendritic cell (DC) subpopulations. By using LPSs from bacteria that share some of Brucella LPS structural features, we demonstrated that the core component of B. melitensis wild-type (Bm-wt) LPS accounts for the low activation potential of Brucella LPS in mouse GM-CSF-derived (GM-) DCs. Contrary to the accepted dogma considering Brucella LPS a poor TLR4 agonist and DC activator, Bm-wt LPS selectively induced expression of surface activation markers and cytokine secretion from Flt3-Ligand-derived (FL-) DCs in a TLR4-dependent manner. It also primed in vitro T cell proliferation by FL-DCs. In contrast, modified LPS with a defective core purified from Brucella carrying a mutated wadC gene (Bm-wadC), efficiently potentiated mouse and human DC activation and T cell proliferation in vitro. In vivo, Bm-wt LPS promoted scant activation of splenic DC subsets and limited recruitment of monocyte- DC like cells in the spleen, conversely to Bm-wadC LPS. Bm-wadC live bacteria drove high cytokine secretion levels in sera of infected mice. Altogether, these results illustrate the immunomodulatory properties of Brucella LPS and the enhanced DC activation ability of the wadC mutation with potential for vaccine development targeting Brucella core LPS structure.