Abstract 17368: The Novel P2X7 Receptor Antagonist SMW139 Inhibits Inflammasome Activation in STEMI Monocytes

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Thomas Hansen ◽  
Kristen Bubb ◽  
Michael Kassiou ◽  
Gemma Figtree
Keyword(s):  
Mononuclear Cells ◽  
Ex Vivo ◽  
Inflammasome Activation ◽  
Healthy Controls ◽  
Novel Therapy ◽  
Peripheral Blood Mononuclear ◽  
Caspase 1 ◽  
Coronary Syndromes ◽  
Artery Disease

Introduction: The inflammasome and the downstream pro-inflammatory cytokine interleukin-1ß (IL-1ß) have emerged as critical modulators of atherosclerosis and acute coronary syndromes (ACS). It therefore represents an important therapeutic target to treat coronary artery disease. This study investigates the activation of the inflammasome in circulating monocytes of ACS patients, and the effectiveness of SMW139- a compound selected from a panel of over 70 novel P2X7 antagonists based upon its excellent pharmacokinetic properties- on modulating the downstream inflammatory signalling pathway. Hypothesis: The P2X7 antagonist SMW139 reduces inflammasome activation. Methods: In vitro: THP-1 monocytic cells were treated with interferon- γ and lipopolysaccharide for 3 hours, then pre-incubated with SMW139 (1-1000nM), prior to ATP stimulation to activate the inflammasome, and collection of supernatant for IL-1ß measurement. Ex vivo: Peripheral blood mononuclear cells (PBMCs) were collected from 19 STEMI patients at time of infarct, and 3 healthy controls. Monocytes were pre-incubated with SMW139 or DMSO control prior to ATP stimulation. IL-1ß and caspase-1 activity were measured by ELISA and fluorometric assay. Results: In vitro: Sigmoidal dose-response curve demonstrated a LogEC50 of -8.069±0.16. Ex vivo: High levels of detectable P2X7 expression in PBMCs was confirmed by flow cytometry. Baseline levels of secreted IL-1ß were significantly elevated in PBMCs from STEMI patients compared with healthy controls (STEMI: 8.2±0.6, Control: 4.2±0.1;p=0.02). There was a significant increase in IL-1ß secretion post ATP-induced inflammasome activation in STEMI PBMCs (53.8±9.4;p<0.0001) but not control (9.8±0.9). P2X7 inhibition significantly attenuated this ATP-induced IL-1ß secretion in STEMI cells (13.9±2.4;p<0.0001) but had no effect in controls (7.5±2.8). Similarly, P2X7 inhibition resulted in approximately 60% reduction of ATP-induced caspase-1 activity in STEMI cells (p=0.01). Conclusions: The P2X7 antagonist compound SMW139 effectively inhibits the inflammasome, causing a significant reduction in caspase-1 activity and IL-1ß cytokine levels, and holds promise as a novel therapy for patients with atherosclerosis and ACS.

scholarly journals Classical complement and inflammasome activation converge in CD14highCD16- monocytes in HIV associated TB-immune reconstitution inflammatory syndrome

2021 ◽  
Vol 17 (3) ◽  
pp. e1009435
Author(s):  
Silvia Lucena Lage ◽  
Chun-Shu Wong ◽  
Eduardo Pinheiro Amaral ◽  
Daniel Sturdevant ◽  
Denise C. Hsu ◽  
...  
Keyword(s):  
Immune Reconstitution Inflammatory Syndrome ◽  
Immune Reconstitution ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Caspase 1 ◽  
Inflammatory Syndrome ◽  
Classical Complement

Inflammasome-derived cytokines, IL-1β and IL-18, and complement cascade have been independently implicated in the pathogenesis of tuberculosis (TB)-immune reconstitution inflammatory syndrome (TB-IRIS), a complication affecting HIV+ individuals starting antiretroviral therapy (ART). Although sublytic deposition of the membrane attack complex (MAC) has been shown to promote NLRP3 inflammasome activation, it is unknown whether these pathways may cooperatively contribute to TB-IRIS. To evaluate the activation of inflammasome, peripheral blood mononuclear cells (PBMCs) from HIV-TB co-infected patients prior to ART and at the IRIS or equivalent timepoint were incubated with a probe used to assess active caspase-1/4/5 followed by screening of ASC (apoptosis-associated speck-like protein containing a CARD domain) specks as a readout of inflammasome activation by imaging flow cytometry. We found higher numbers of monocytes showing spontaneous caspase-1/4/5+ASC-speck formation in TB-IRIS compared to TB non-IRIS patients. Moreover, numbers of caspase-1/4/5+ASC-speck+ monocytes positively correlated with IL-1β/IL-18 plasma levels. Besides increased systemic levels of C1q and C5a, TB-IRIS patients also showed elevated C1q and C3 deposition on monocyte cell surface, suggesting aberrant classical complement activation. A clustering tSNE analysis revealed TB-IRIS patients are enriched in a CD14highCD16- monocyte population that undergoes MAC deposition and caspase-1/4/5 activation compared to TB non-IRIS patients, suggesting complement-associated inflammasome activation during IRIS events. Accordingly, PBMCs from patients were more sensitive to ex-vivo complement-mediated IL-1β secretion than healthy control cells in a NLRP3-dependent manner. Therefore, our data suggest complement-associated inflammasome activation may fuel the dysregulated TB-IRIS systemic inflammatory cascade and targeting this pathway may represent a novel therapeutic approach for IRIS or related inflammatory syndromes.

scholarly journals The Anthrax Vaccine Adsorbed Vaccine Generates Protective Antigen (PA)-Specific CD4+ T Cells with a Phenotype Distinct from That of Naïve PA T Cells

2008 ◽  
Vol 76 (10) ◽  
pp. 4538-4545 ◽  
Author(s):  
William W. Kwok ◽  
Junbao Yang ◽  
Eddie James ◽  
John Bui ◽  
Laurie Huston ◽  
...  
Keyword(s):  
T Cells ◽  
Mononuclear Cells ◽  
Protective Antigen ◽  
Ex Vivo ◽  
Cytokine Profile ◽  
T Cell Epitopes ◽  
Peripheral Blood Mononuclear ◽  
Anthrax Vaccine ◽  
Cellular Immune

ABSTRACT Cellular immune responses against protective antigen (PA) of Bacillus anthracis in subjects that received the anthrax vaccine adsorbed (AVA) vaccine were examined. Multiple CD4+ T-cell epitopes within PA were identified by using tetramer-guided epitope mapping. PA-reactive CD4+ T cells with a CD45RA− phenotype were also detected by direct ex vivo staining of peripheral blood mononuclear cells (PBMC) with PA-specific tetramers. Surprisingly, PA-specific T cells were also detected in PBMC of nonvaccinees after a single cycle of in vitro PA stimulation. However, PA-reactive CD4+ T cells in nonvaccinees occurred at lower frequencies than those in vaccinees. The majority of PA-reactive T cells from nonvaccinees were CD45RA+ and exhibited a Th0/Th1 cytokine profile. In contrast, phenotyping and cytokine profile analyses of PA-reactive CD4+ T cells from vaccinees indicated that vaccination leads to commitment of PA-reactive T cells to a Th2 lineage, including generation of PA-specific, pre-Th2 central memory T cells. These results demonstrate that the current AVA vaccine is effective in skewing the development of PA CD4+ T cells to the Th2 lineage. The data also demonstrated the feasibility of using class II tetramers to analyze CD4+ cell responses and lineage development after vaccination.

scholarly journals Reprogramming of bone marrow myeloid progenitor cells in patients with severe coronary artery disease

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marlies P Noz ◽  
Siroon Bekkering ◽  
Laszlo Groh ◽  
Tim MJ Nielen ◽  
Evert JP Lamfers ◽  
...  
Keyword(s):  
Coronary Artery Disease ◽  
Bone Marrow ◽  
Coronary Artery ◽  
Immune Cells ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Ex Vivo ◽  
Peripheral Blood Mononuclear ◽  
Artery Disease ◽  
Myeloid Progenitors

Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation of innate immune cells occurs at the level of myeloid progenitors, which adds exciting opportunities for novel treatment strategies.

Transduction of inflammation from peripheral immune cells to the hippocampus induces neuronal hyperexcitability mediated by Caspase-1 activation

2020 ◽  
Author(s):  
Tarek Shaker ◽  
Bidisha Chattopadhyaya ◽  
Bénédicte Amilhon ◽  
Graziella Di Cristo ◽  
Alexander G. Weil
Keyword(s):  
Mononuclear Cells ◽  
Culture Media ◽  
Ectopic Expression ◽  
Peripheral Inflammation ◽  
Potential Contribution ◽  
Inflammation Markers ◽  
Peripheral Blood Mononuclear ◽  
Caspase 1 ◽  
Therapeutic Benefits

Abstract Background Recent studies report infiltration of peripheral blood mononuclear cells (PBMCs) into the central nervous system (CNS) in epileptic disorders, suggestive of a potential contribution of PBMC extravasation to the generation of seizures. Nevertheless, the underlying mechanisms involved in PBMC infiltrates promoting neuronal predisposition to ictogenesis remain unclear. Therefore, we developed an in vitro model mimicking PBMC infiltration into the brain in order to investigate potential transduction of inflammatory signals from PBMCs to the CNS.MethodsTo establish our model, we first extracted PBMCs from rat spleen, then, we immunologically primed PBMCs with lipopolysaccharide (LPS), followed by immunological activation with Nigericin. Thereafter, we cultured PBMCs on top of organotypic cortico-hippocampal brain slice cultures (OCHSCs) derived from the same rat, and compared PBMC-OCHSC co-cultures to OCHSCs exposed to PBMCs in the culture media. Also, we targeted a potential molecular pathway underlying transduction of peripheral inflammation to OCHSCs by incubating OCHSCs with the Caspase-1 inhibitor VX-765 prior to co-culturing PBMCs with OCHSCs. After 24 hours, we immunohistochemically analyzed inflammation markers in the cortex and the hippocampus. In addition, we performed whole-cell patch-clamp recordings in cortical layer II/III and hippocampal CA1 pyramidal neurons.ResultsIn the cortex, co-culturing immunoreactive PBMCs treated with LPS + Nigericin on top of OCHSCs induced ectopic expression of inflammation markers and enhanced neuronal excitation. In contrast, no excitability changes were detected after adding primed PBMCs, i.e. treated with LPS only, to OCHSCs. Strikingly, in the hippocampus, both immunoreactive and primed PBMCs elicited similar pro-inflammatory and pro-excitatory effects. However, when immunoreactive and primed PBMCs were cultured in the media separately from OCHSCs, only immunoreactive PBMCs gave rise to neuroinflammation and hyperexcitability in the hippocampus, whereas primed PBMCs failed to produce any significant changes. Finally, VX-765 application to OCHSCs, co-cultured with either immunoreactive or primed PBMCs, protected them from neuroinflammation and hippocampal hyperexcitability.ConclusionsOur study shows a higher susceptibility of the hippocampus to peripheral inflammation as compared to the cortex, mediated via Caspase-1-dependent signaling pathways. Thus, our findings suggest that Caspase-1 inhibition may potentially provide therapeutic benefits during hippocampal neuroinflammation and hyperexcitability secondary to peripheral innate immunity.

scholarly journals Ex Vivo Expanded Human Vγ9Vδ2 T-Cells Can Suppress Epithelial Ovarian Cancer Cell Growth

2019 ◽  
Vol 20 (5) ◽  
pp. 1139 ◽  
Author(s):  
Tsui Mao ◽  
Carol Miao ◽  
Yi Liao ◽  
Ying Chen ◽  
Chia Yeh ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
T Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Γδ T Cells ◽  
Ovarian Cancer Cells ◽  
Cytotoxic Activities ◽  
Antitumor Effects ◽  
Peripheral Blood Mononuclear

γδ-T-cells have attracted attention because of their potent cytotoxicity towards tumors. Most γδ-T-cells become activated via a major histocompatibility complex (MHC)-independent pathway by the interaction of their receptor, Natural Killer Group 2 Member D (NKG2D) with the tumor-specific NKG2D ligands, including MHC class I-related chain A/B (MICA/B) and UL16-binding proteins (ULBPs), to kill tumor cells. However, despite their potent antitumor effects, the treatment protocols specifically targeting ovarian tumors require further improvements. Ovarian cancer is one of the most lethal and challenging female malignancies worldwide because of delayed diagnoses and resistance to traditional chemotherapy. In this study, we successfully enriched and expanded γδ-T-cells up to ~78% from peripheral blood mononuclear cells (PBMCs) with mostly the Vγ9Vδ2-T-cell subtype in the circulation. We showed that expanded γδ-T-cells alone exerted significant cytotoxic activities towards specific epithelial-type OVCAR3 and HTB75 cells, whereas the combination of γδ-T cells and pamidronate (PAM), a kind of aminobisphosphonates (NBPs), showed significantly enhanced cytotoxic activities towards all types of ovarian cancer cells in vitro. Furthermore, in tumor xenografts of immunodeficient NSG mice, γδ-T-cells not only suppressed tumor growth but also completely eradicated preexisting tumors with an initial size of ~5 mm. Thus, we concluded that γδ-T-cells alone possess dramatic cytotoxic activities towards epithelial ovarian cancers both in vitro and in vivo. These results strongly support the potential of clinical immunotherapeutic application of γδ-T-cells to treat this serious female malignancy.

Human Erythroblasts Generated in Vitro Remain Functional with a Normal Karyotype 8 Years after Cryopreservation: Implications for Ex Vivo Generated Erythroid Transfusion Products.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2303-2303 ◽  
Author(s):  
Massimo Sanchez ◽  
Amanda Leblanc ◽  
Annalisa Mancini ◽  
Francesca Masiello ◽  
Valentina Tirelli ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Blood Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Genomic Rearrangements ◽  
Multicolor Fish ◽  
Peripheral Blood Mononuclear ◽  
Differentiation Potential ◽  
Blood Mononuclear Cells

Abstract The safety and adequacy of the blood supply is threatened by natural disasters, social and political events, epidemics, and emerging infections. During shortages, frozen blood is used to supplement the blood supply. Current regulations allow red blood cells to be stored frozen up to ten years; however, the shelf-life of such products is limited once blood is thawed. Cultured human erythroid cells derived in vitro from either fresh or cryopreserved CD34+ cells or peripheral blood mononuclear cells potentially represent an alternative source of erythrocytes for transfusion. However, it is unknown if normal erythroid cells undergoing ex-vivo expansion with growth factors will remain functional or develop genetic rearrangements in culture making them unsuitable for transfusion. We have compared the proliferative and differentiation potential of human erythroblasts obtained in culture from the peripheral blood mononuclear cells (PBMC) of adult donors. This analysis included freshly expanded erythroblasts as well as erythroblasts cryopreserved and stored for short (1 month) and long (8 years) periods. PBMC from four volunteer blood donors were prepared using gradient-density centrifugation and cryopreserved in DMSO in June 2000. One months later, 2x107 PBMC from one of the donors were thawed and cultured under conditions that allow massive ex vivo generation of erythroblasts (HEMA culture, Migliaccio et al Blood Cells Mol Dis2002;28:169-80). These cultures were stimulated with recombinant hSCF (50ng/mL), hGM-CSF (1ng/ml), hIL3 (1U/mL), hEPO (1U/mL) and contained dexamethasone and estradiol (each 10−6 M). Twenty million PBMC from the three additional donors were thawed and cultured under HEMA conditions in 2008. In all the three cases, the day 9 cultures contained an average of 10x107 cells, 95% of which were erythroid by CD36 and CD235a staining. These day 9 cells were either cultured for 4 additional days or cryopreserved (&gt;10 individual vials per donor containing 5x106 each). Cells were subcultured and maintained either under HEMA conditions (to assess their proliferation ability) or stimulated with EPO alone (5U/ mL) (to assess maturation). In May 2008, aliquots of the erythroblasts obtained from all donors were thawed and cultured again and amplification and differentiation potential of the freshly expanded and thawed cells were compared. Cells thawed after few months or 8 years of cryopreservation gave similar results and the data were pooled. The viability of the erythroblasts after thawing was 60–70%. After 4 days under HEMA conditions, both freshly expanded and cryopreserved erythroblasts doubled in numbers and retained an immature erythroid phenotype (CD36highCD235alow). On the other hand, in cultures containing EPO alone, the erythroblasts remained constant in number but progressed to a mature CD36posCD235ahigh phenotype. The results are summarized in the following table: Proliferation and Maturation Profile of Fresh and Cryopreserved Human Erythroblasts Fold Increase Phenotype CD36highCD235alow CD36highCD235ahigh Fresh cells HEMA culture 2 53% 40% EPO alone 1 15% 80% Thawed Cells HEMA culture 2 46% 36% EPO alone 1 5% 90% The eight-years cryopreserved erythroblasts expanded in culture were also cytogenetically evaluated. Karyotype and multicolor FISH analyses demonstrated a normal 46,XY karyotype with no obvious genomic rearrangements. To determine whether cells carried any known in utero leukemic genomic rearrangements, interphase FISH studies were performed for TEL/ETV6-AML1, MLL, 5q31 (EGR1) and 7q31 loci. In 800 evaluated interphase nuclei, all loci were present in disomy. This data indicates that human erythroblasts obtained in culture can be efficiently cryopreserved, remain functional in culture and do not acquire chromosomal abnormalities detectable by multicolor FISH analysis. These observations suggest that cultured erythroblasts should be further evaluated to determine if they represent a more suitable long term storage product than cryopreserved mature red blood cells.

scholarly journals The Expression and Pathophysiological Role of Osteopontin in Graves' Disease

2011 ◽  
Vol 96 (11) ◽  
pp. E1866-E1870 ◽  
Author(s):  
Lingyan Xu ◽  
Xinran Ma ◽  
Yanyan Wang ◽  
Xiaoli Li ◽  
Yicheng Qi ◽  
...  
Keyword(s):  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Graves Disease ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Pathophysiological Role ◽  
Blood Mononuclear Cells

Abstract Context: Graves' disease (GD) is a common autoimmune disease that affects the thyroid gland. Its pathogenesis is tightly involved with aberrant proinflammatory cytokine production. Osteopontin (OPN), an extracellular matrix protein of pleiotropic properties, has recently been recognized as a potent inflammatory cytokine in several autoimmune diseases. Objective: This study sought to explore the pathophysiological role of OPN in GD by comparing OPN levels in initial GD patients and healthy controls. Methods: Seventy-six patients who met criteria for initial GD and sixty-five healthy controls were recruited. OPN and other clinical GD diagnosis parameters were measured. In addition, the coexpression of several OPN receptors as well as various nuclear factor-κB (NF-κB) downstream target genes were examined in peripheral blood mononuclear cells from human subjects. The effect of OPN on NF-κB activation was determined by in vitro assays. Results: We demonstrated for the first time that the OPN levels are enhanced in serum from GD patients. OPN levels are strongly associated with clinical serum parameters for GD diagnosis. The coexpression of selective OPN receptors and inflammatory response genes was enhanced in peripheral blood mononuclear cells from GD patients. Furthermore, serum from GD patients activated NF-κB activity in vitro, which was significantly suppressed by OPN monoclonal antibody abrogation. Conclusion: These data indicated a clinical correlation between serum OPN levels and GD. OPN could affect GD development through NF-κB activation and the subsequent changes in inflammatory milieu. OPN could serve as a novel biomarker for GD as well as a potential target for GD treatment.

scholarly journals The Influence of Methylsulfonylmethane on Inflammation-Associated Cytokine Release before and following Strenuous Exercise

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Mariè van der Merwe ◽  
Richard J. Bloomer
Keyword(s):  
Whole Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Knee Extension ◽  
Strenuous Exercise ◽  
Peripheral Blood Mononuclear ◽  
Physically Active ◽  
Inflammatory Molecules

Background. Inflammation is associated with strenuous exercise and methylsulfonylmethane (MSM) has been shown to have anti-inflammatory properties.Methods. Physically active men were supplemented with either placebo or MSM (3 grams per day) for 28 days before performing 100 repetitions of eccentric knee extension exercise.Ex vivoandin vitrotesting consisted of evaluating cytokine production in blood (whole blood and isolated peripheral blood mononuclear cells (PBMCs)) exposed to lipopolysaccharide (LPS), before and through 72 hours after exercise, whilein vivotesting included the evaluation of cytokines before and through 72 hours after exercise.Results. LPS stimulation of whole blood after MSM supplementation resulted in decreased induction of IL-1β, with no effect on IL-6, TNF-α, or IL-8. After exercise, there was a reduced response to LPS in the placebo, but MSM resulted in robust release of IL-6 and TNF-α. A small decrease in resting levels of proinflammatory cytokines was noted with MSM, while an acute postexercise increase in IL-10 was observed with MSM.Conclusion. Strenuous exercise causes a robust inflammatory reaction that precludes the cells from efficiently responding to additional stimuli. MSM appears to dampen the release of inflammatory molecules in response to exercise, resulting in a less incendiary environment, allowing cells to still have the capacity to mount an appropriate response to an additional stimulus after exercise.

scholarly journals Transduction of inflammation from peripheral immune cells to the hippocampus induces neuronal hyperexcitability mediated by Caspase-1 activation

2020 ◽  
Author(s):  
Tarek Shaker ◽  
Bidisha Chattopadhyaya ◽  
Bénédicte Amilhon ◽  
Graziella Di Cristo ◽  
Alexander G. Weil
Keyword(s):  
Pyramidal Neurons ◽  
Mononuclear Cells ◽  
Culture Media ◽  
Cortical Layer ◽  
Peripheral Inflammation ◽  
Potential Contribution ◽  
Peripheral Blood Mononuclear ◽  
Caspase 1 ◽  
Therapeutic Benefits

Abstract 1.1. Background Recent studies report infiltration of peripheral blood mononuclear cells (PBMCs) into the central nervous system (CNS) in epileptic disorders, suggestive of a potential contribution of PBMC extravasation to the generation of seizures. Nevertheless, the underlying mechanisms involved in PBMC infiltrates promoting neuronal predisposition to ictogenesis remain unclear. Therefore, we developed an in vitro model mimicking infiltration of activated PBMCs into the brain in order to investigate potential transduction of inflammatory signals from PBMCs to the CNS.1.2. Methods To establish our model, we first extracted PBMCs from rat spleen, then, immunologically primed PBMCs with lipopolysaccharide (LPS), followed by further activation with nigericin. Thereafter, we co-cultured these activated PBMCs with organotypic cortico-hippocampal brain slice cultures (OCHSCs) derived from the same rat, and compared PBMC-OCHSC co-cultures to OCHSCs exposed to PBMCs in the culture media. We further targeted a potential molecular pathway underlying transduction of peripheral inflammation to OCHSCs by incubating OCHSCs with the Caspase-1 inhibitor VX-765 prior to co-culturing PBMCs with OCHSCs. After 24 hours, we analyzed inflammation markers in the cortex and the hippocampus using semiquantitative immunofluorescence. In addition, we analyzed neuronal activity by whole-cell patch-clamp recordings in cortical layer II/III and hippocampal CA1 pyramidal neurons.1.3. Results In the cortex, co-culturing immunoreactive PBMCs treated with LPS + nigericin on top of OCHSCs upregulated inflammatory markers and enhanced neuronal excitation. In contrast, no excitability changes were detected after adding primed PBMCs (i.e. treated with LPS only), to OCHSCs. Strikingly, in the hippocampus, both immunoreactive and primed PBMCs elicited similar pro-inflammatory and pro-excitatory effects. However, when immunoreactive and primed PBMCs were cultured in the media separately from OCHSCs, only immunoreactive PBMCs gave rise to neuroinflammation and hyperexcitability in the hippocampus, whereas primed PBMCs failed to produce any significant changes. Finally, VX-765 application to OCHSCs, co-cultured with either immunoreactive or primed PBMCs, protected them from neuroinflammation and hippocampal hyperexcitability.1.4. Conclusions Our study shows a higher susceptibility of the hippocampus to peripheral inflammation as compared to the cortex, mediated via Caspase-1-dependent signaling pathways. Thus, our findings suggest that Caspase-1 inhibition may potentially provide therapeutic benefits during hippocampal neuroinflammation and hyperexcitability secondary to peripheral innate immunity.

scholarly journals Bruton’s tyrosine kinase drives neuroinflammation and anxiogenic behavior in mouse models of stress

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Simantini Ghosh ◽  
Zaidan Mohammed ◽  
Itender Singh
Keyword(s):  
Mouse Models ◽  
Peripheral Blood Mononuclear Cells ◽  
Nlrp3 Inflammasome ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Specific Inhibitor ◽  
Inflammasome Activation ◽  
Peripheral Blood Mononuclear ◽  
Caspase 1 ◽  
Nlrp3 Inflammasome Activation

Abstract Background Current therapies targeting several neurotransmitter systems are only able to partially mitigate the symptoms of stress- and trauma-related disorder. Stress and trauma-related disorders lead to a prominent inflammatory response in humans, and in pre-clinical models. However, mechanisms underlying the induction of neuroinflammatory response in PTSD and anxiety disorders are not clearly understood. The present study investigated the mechanism underlying the activation of proinflammatory NLRP3 inflammasome and IL1β in mouse models of stress. Methods We used two mouse models of stress, i.e., mice subjected to physical restraint stress with brief underwater submersion, and predator odor stress. Mice were injected with MCC950, a small molecule specific inhibitor of NLRP3 activation. To pharmacologically inhibit BTK, a specific inhibitor ibrutinib was used. To validate the observation from ibrutinib studies, a separate group of mice was injected with another BTK-specific inhibitor LFM-A13. Seven days after the induction of stress, mice were examined for anxious behavior using open field test (OFT), light–dark test (LDT), and elevated plus maze test (EPM). Following the behavior tests, hippocampus and amygdale were extracted and analyzed for various components of NLRP3–caspase 1–IL1β pathway. Plasma and peripheral blood mononuclear cells were also used to assess the induction of NLRP3–Caspase 1–IL-1β pathway in stressed mice. Results Using two different pre-clinical models of stress, we demonstrate heightened anxious behavior in female mice as compared to their male counterparts. Stressed animals exhibited upregulation of proinflammatory IL1β, IL-6, Caspase 1 activity and NLRP3 inflammasome activation in brain, which were significantly higher in female mice. Pharmacological inhibition of NLRP3 inflammasome activation led to anxiolysis as well as attenuated neuroinflammatory response. Further, we observed induction of activated Bruton’s tyrosine kinase (BTK), an upstream positive-regulator of NLRP3 inflammasome activation, in hippocampus and amygdala of stressed mice. Next, we conducted proof-of-concept pharmacological BTK inhibitor studies with ibrutinib and LFM-A13. In both sets of experiments, we found BTK inhibition led to anxiolysis and attenuated neuroinflammation, as indicated by significant reduction of NLRP3 inflammasome and proinflammatory IL-1β in hippocampus and amygdala. Analysis of plasma and peripheral blood mononuclear cells indicated peripheral induction of NLRP3–caspase 1–IL1β pathway in stressed mice. Conclusion Our study identified BTK as a key upstream regulator of neuroinflammation, which drives anxiogenic behavior in mouse model of stress. Further, we demonstrated the sexually divergent activation of BTK, providing a clue to heightened neuroinflammation and anxiogenic response to stress in females as compared to their male counterparts. Our data from the pharmacological inhibition studies suggest BTK as a novel target for the development of potential clinical treatment of PTSD and anxiety disorders. Induction of pBTK and NLRP3 in peripheral blood mononuclear cells of stressed mice suggest the potential effect of stress on systemic inflammation.

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