Expression Of CD86 Research Articles

Immune profiling and functional analysis of NK and T cells in ataxia telangiectasia.

Ataxia telangiectasia (AT) is a rare autosomal-recessive disorder characterized by profound neurodegeneration, combined immunodeficiency, and an increased risk for malignant diseases. Treatment options for AT are limited, and the long-term survival prognosis for patients remains grim, primarily due to the emergence of chronic respiratory pathologies, malignancies, and neurological complications. Understanding the dysregulation of the immune system in AT is fundamental for the development of novel treatment strategies. In this context, we performed a retrospective longitudinal immunemonitoring of lymphocyte subset distribution in a cohort of AT patients (n = 65). Furthermore, we performed FACS analyses of peripheral blood mononuclear cells from a subgroup of 12 AT patients to examine NK and T cells for the expression of activating and functional markers. We observed reduced levels of peripheral blood CD3+CD8+ cytotoxic T cells, CD3+CD4+ T helper cells, and CD19+ B cells, whereas the amount of CD3--CD56+ NK cells and CD3+CD56+ NKT-like cells was similar compared with age-matched controls. Notably, there was no association between the age-dependent kinetic of T-, B-, or NK-cell counts and the occurrence of malignancy in AT patients. Additionally, our results indicate an altered NK- and T-cell response to cytokine stimulation in AT with increased levels of TRAIL, FasL, and CD16 expression in NK cells, as well as an elevated activation level of T cells in AT with notably higher expression levels of IFN-γ, CD107a, TRAIL, and FasL. Together, these findings imply function alterations in AT lymphocytes, specifically in T and NK cells, shedding light on potential pathways for innovative therapies.

Development and validation of a stromal-immune signature to predict prognosis in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is a highly desmoplastic tumor with poor prognosis even after curative resection. We investigated the associations between the composition of the ICC stroma and immune cell infiltration and aimed to develop a stromal-immune signature to predict prognosis in surgically treated ICC. We recruited 359 ICC patients and performed immunohistochemistry to detect α-smooth muscle actin (α-SMA), CD3, CD4, CD8, Foxp3, CD68, and CD66b. Aniline was used to stain collagen deposition. Survival analyses were performed to detect prognostic values of these markers. Recursive partitioning for a discrete-time survival tree was applied to define a stromal-immune signature with distinct prognostic value. We delineated an integrated stromal-immune signature based on immune cell subpopulations and stromal composition to distinguish subgroups with different recurrence-free survival (RFS) and overall survival (OS) time. We defined four major patterns of ICC stroma composition according to the distributions of α-SMA and collagen: dormant (α-SMAlow/collagenhigh), fibrogenic (α-SMAhigh/collagenhigh), inert (α-SMAlow/collagenlow), and fibrolytic (α-SMAhigh/collagenlow). The stroma types were characterized by distinct patterns of infiltration by immune cells. We divided patients into six classes. Class I, characterized by high CD8 expression and dormant stroma, displayed the longest RFS and OS, whereas Class VI, characterized by low CD8 expression and high CD66b expression, displayed the shortest RFS and OS. The integrated stromal-immune signature was consolidated in a validation cohort. We developed and validated a stromal-immune signature to predict prognosis in surgically treated ICC. These findings provide new insights into the stromal-immune response to ICC.

Monocyte Single-Cell Multimodal Profiling in Cardiovascular Disease Risk States.

Monocytes are a critical innate immune system cell type that serves homeostatic and immunoregulatory functions. They have been identified historically by the cell surface expression of CD14 and CD16. However, recent single-cell studies have revealed that they are much more heterogeneous than previously realized. We utilized cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and single-cell RNA sequencing to describe the comprehensive transcriptional and phenotypic landscape of 437 126 monocytes. This high-dimensional multimodal approach identified vast phenotypic diversity and functionally distinct subsets, including IFN-responsive, MHCIIhi (major histocompatibility complex class II), monocyte-platelet aggregates, as well as nonclassical, and several subpopulations of classical monocytes. Using flow cytometry, we validated the existence of MHCII+CD275+ MHCIIhi, CD42b+ monocyte-platelet aggregates, CD16+CD99- nonclassical monocytes, and CD99+ classical monocytes. Each subpopulation exhibited unique characteristics, developmental trajectories, transcriptional regulation, and tissue distribution. In addition, alterations associated with cardiovascular disease risk factors, including race, smoking, and hyperlipidemia were identified. Moreover, the effect of hyperlipidemia was recapitulated in mouse models of elevated cholesterol. This integrative and cross-species comparative analysis provides a new perspective on the comparison of alterations in monocytes in pathological conditions and offers insights into monocyte-driven mechanisms in cardiovascular disease and the potential for monocyte subpopulation targeted therapies.

Effect of physical activity in lymphocytes senescence burden in patients with COPD.

Chronic obstructive pulmonary disease (COPD) is regarded as an accelerated-age disease in which chronic inflammation, maladaptive immune responses, and senescence cell burden coexist. Accordingly, cellular senescence has emerged as a potential mechanism involved in COPD pathophysiology. In this study, 25 stable patients with COPD underwent a daily physical activity promotion program for 6 mo. We reported that increase of physical activity was related to a reduction of the senescent cell burden in circulating lymphocytes of patients with COPD. Senescent T-lymphocyte population, characterized by absence of surface expression of CD28, was reduced after physical activity intervention, and the reduction was associated to the increase of physical activity level. In addition, the mRNA expression of cyclin-dependent kinase inhibitors, a hallmark of cell senescence, was reduced and, in accordance, the proliferative capacity of lymphocytes was improved postintervention. Moreover, we observed an increase in functionality in T cells from patients after intervention, including improved markers of activation, enhanced cytotoxicity, and altered cytokine secretions in response to viral challenge. Lastly, physical activity intervention reduced the potential of lymphocytes' secretome to induce senescence in human primary fibroblasts. In conclusion, our study provides, for the first time, evidence of the potential of physical activity intervention in patients with COPD to reduce the senescent burden in circulating immune cells.NEW & NOTEWORTHY For the first time, we identified in patients with COPD a relation between physical activity intervention with respiratory function improvement and cellular senescence burden in lymphocytes that improved the T cell functionality and proliferative capacity of patients. In addition, our experiments highlight the possible impact of T-cell senescence in other cell types which could be related to some of the clinical lung complications observed in COPD.

Association of CD24, CD27, and co-stimulatory molecules CD80 immunological marker expression on B-cells of human peripheral blood with development of celiac disease

Abstract: BACKGROUND: B-cells express a variety of clusters of differentiation markers during development. These markers determine the basic function of the immune phenotype specific to B-cells. Changes in the expression of these markers are linked to the development of many diseases, including chronic inflammation, autoimmune diseases, and immunodeficiency. OBJECTIVE: The current study aimed to investigate a change in CD24, CD27, and co-stimulatory molecules CD80 expression on peripheral blood B-cells and the extent of their contribution to celiac disease. MATERIALS AND METHODS: A total of 60 male children, whose ages ranged between 8 and 14 years, participated in this study. Thirty-five were identified as having celiac disease, while the control group comprised 25 children with anti-tissue transglutaminase (TG) (immunoglobulin G [IgG]), anti-tissue TG (IgA), and deamidated gliadin peptide (DGP) IgG levels normal. The frequency CD24, CD27, and CD80 expression were measured by flow cytometry. RESULTS: Celiac disease patients showed a substantial decrease in the percentage of CD24, CD27, and CD80 expression on B-cells compared to control groups. CONCLUSION: These findings suggest that numerical deficiency of CD24, CD27, and CD80 expression on B-cells in the peripheral blood mononuclear cell population, that may involve the loss of auto-tolerance that plays an important role in the immune response associated with inflammation and tissue damage in celiac disease. These immunological markers may be used as diagnostic indicators for this disease.

Epidemic influenza virus nucleoprotein gene incorporated into vaccine influenza virus strain genome to optimize systemic and local T-cell immune response against live attenuated influenza vaccine

Introduction. Optimization of the vaccine-induced T-cell repertoire is one of the strategies to expand the spectrum of protective potential for live attenuated influenza vaccine (LAIV). LAIV cross-protective properties can be improved by introducing the nucleoprotein (NP) gene derived from epidemic parental virus into vaccine strain genome, i.e. by replacing the classical 6:2 genome formula with 5:3. The main objective of the present study was to detail evaluation for virus-specific systemic and tissue-resident memory T-cells subsets in mice immunized with seasonal H1N1 LAIV of the genome formula 6:2 and 5:3. Materials and methods. Two H1N1 LAIV strains with varying NP genes (LAIV 6:2 and LAIV 5:3) were generated using reverse genetics techniques. C57BL/6J mice were immunized intranasally with the vaccine candidates, twice, 3 weeks apart. Cells from the spleen and lung tissues were isolated 7 days after booster immunization to be stimulated with whole H1N1 influenza virus for assessing cytokine-producing memory CD44+CD62L– T-cells as well as expression of CD69 and CD103 surface markers using flow cytometry. Humoral murine serum immunity against H1N1 virus was assessed by ELISA. Results. The LAIV 5:3 vs classical 6:2 vaccine strain carrying the epidemic parental NP gene induced significantly more pronounced humoral immune response against recent influenza virus. The group of mice immunized with LAIV 5:3 demonstrated higher levels of virus-specific CD4+ and CD8+ effector memory T cells (TEM) in the spleen, including a subset of polyfunctional (IFNγ+TNFα+IL-2+) CD4+ TEM, compared to LAIV 6:2 group. Virus-specific memory T cell levels in lung tissues after immunization with LAIV 5:3 vs LAIV 6:2 also tended to increase, but no significant difference in stimulated tissue-resident CD69+CD103– and CD69+CD103+ T cells between the groups were found. Conclusion. Modification of the seasonal LAIV strain genome for updating its epitope composition allowed to enhance the virus-specific T-cell immune response both at systemic level and in lung tissues, thereby shoeing that the effectiveness of the vaccine against circulating influenza viruses can be potentially increased.

The immune landscape of the inflamed joint defined by spectral flow cytometry.

Cellular phenotype and function are altered in different microenvironments. For targeted therapies it is important to understand site-specific cellular adaptations. Juvenile Idiopathic Arthritis (JIA) is characterised by autoimmune joint inflammation, with frequent inadequate treatment responses. To comprehensively assess the inflammatory immune landscape, we designed a 37-parameter spectral flow cytometry panel delineating mononuclear cells from JIA synovial fluid (SF) of autoimmune inflamed joints, compared to JIA and healthy control blood. Synovial monocytes and NK cells (CD56bright) lack Fc-receptor CD16, suggesting antibody-mediated targeting may be ineffective. B cells and DCs, both in small frequencies in SF, undergo maturation with high 4-1BB, CD71, CD39 expression, supporting T cell activation. SF effector and regulatory T cells were highly active with newly described co-receptor combinations that may alter function, and suggestion of metabolic reprogramming via CD71, TNFR2 and PD-1. Most SF effector phenotypes, as well as an identified CD4-Foxp3+ T cell population, were restricted to the inflamed joint, yet specific SF-predominant CD4+Foxp3+ Treg subpopulations were increased in blood of active but not inactive JIA, suggesting possible recirculation and loss of immunoregulation at distal sites. This first comprehensive dataset of the site-specific inflammatory landscape at protein level will inform functional studies and the development of targeted therapeutics to restore immunoregulatory balance and achieve remission in JIA.

Immunization of recombinant NS3 protein (protease region) of dengue virus induces high levels of CTLA-4 and apoptosis in splenocytes of BALB/c mice.

DENV infection outcomes depend on the host's variable expression of immune receptors and mediators, leading to either resolution or exacerbation. While the NS3 protein is known to induce robust immune responses, the specific impact of its protease region epitopes remains unclear. This study investigated the effect of recombinant NS3 protease region proteins from all four DENV serotypes on splenocyte activation in BALB/c mice (n = 5/group). Mice were immunized with each protein, and their splenocytes were subsequently stimulated with homologous antigens. We measured the expression of costimulatory molecules (CD28, CD80, CD86, CD152) by flow cytometry, along with IL-2 production, CD25 expression, and examined the antigen-specific activation of CD4 + and CD8 + T cells. Additionally, the expression of IL-1, IL-10, and TGF-β1 in splenocytes from immunized animals was assessed. Apoptosis was evaluated using Annexin V/PI staining and DNA fragmentation analysis. Stimulation of splenocytes from immunized mice triggered apoptosis (phosphatidylserine exposure and caspase 3/7 activation) and increased costimulatory molecule expression, particularly CD152. Low IL-2 production and low CD25 expression, as well as sustained expression of the IL-10 gene. These results suggest that these molecules might be involved in mechanisms by which the NS3 protein contributes to viral persistence and disease pathogenesis.

Dendritic cells transfected with DNA constructs encoding CCR9, IL-10, and type II collagen demonstrate induction of immunological tolerance in an arthritis model.

Restoring immune tolerance is a promising area of therapy for autoimmune diseases. One method that helps restore immunological tolerance is the approach using tolerogenic dendritic cells (tolDCs). In our study, we analyzed the effectiveness of using dendritic cells transfected with DNA constructs encoding IL-10, type II collagen, and CCR9 to induce immune tolerance in an experimental model of arthritis. Dendritic cell cultures were obtained from bone marrow cells of Balb/c mice. Dendritic cells (DCs) cultures were transfected with pmaxCCR9, pmaxIL-10, and pmaxCollagen type II by electroporation. The phenotype and functions of DCs were studied using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Migration of electroporated DCs was assessed in vitro. Induction of antigen-collagen induced arthritis (ACIA) was carried out according to the protocol in Balb/c mice. DCs were then administered to ACIA mice. The development of arthritis was monitored by measuring paw swelling with a caliper at different time points. The immunological changes were assessed by analyzing the content of antibodies to type II collagen using enzyme immunoassay. Additionally, a histological examination of the joint tissue was conducted, followed by data analysis. DCs were obtained, characterized by reduced expression of CD80, CD86, and H-2Db (MHC class I), increased expression of CCR9, as well as producing IL-10 and having migratory activity to thymus cells. Transfected DCs induced T-regulatory cells (T-reg) and increased the intracellular content of IL-10 and TGF-β in CD4+T cells in their co-culture, and also suppressed their proliferative activity in response to antigen. The administration of tolDCs transfected with DNA constructs encoding type II collagen, IL-10, and CCR9 to mice with ACIA demonstrated a reduction in paw swelling, a reduction in the level of antibodies to type II collagen, and a regression of histological changes. The study presents an approach by which DCs transfected with DNA constructs encoding epitopes of type II collagen, IL-10 and CCR9 promote the development of antigen-specific tolerance, control inflammation and reduce the severity of experimental arthritis through the studied mechanisms: induction of T-reg, IL-10, TGF-β.

Coronavirus spike protein fragment-containing chimeric virus-like particles stimulate human dendritic cell maturation

Introduction. Viral capsid proteins can assemble into virus-like particles lacking infectivity and bearing parental virus antigens or artificially introduced antigens from other pathogens. At least some of such particles are highly immunogenic and could serve as a platform for promising vaccines. In this work, we assessed an effect of virus-like particles decorated with a SARS-CoV-2 spike protein fragment on human dendritic cell phenotype and functional properties. Materials and methods. The virus-like particles were assembled using chimeric molecules obtained by fusing genetic sequences encoding a norovirus major capsid protein VP1 fragment and a coronavirus spike protein fragment, including the receptor-binding domain. Dendritic cells were obtained from monocytes in vitro. Results. Incubation of immature dendritic cells with virus-like particles induced their phenotypic and functional maturation. The former was revealed by significantly increased expression of HLA-DR, CD80, CD86 and CD83. Dendritic cell phenotype after incubation with virus-like particles at the maximum concentration of 10 μg/ml did not differ significantly from that of mature dendritic cells in positive control. Along with phenotypic maturation, virus-like particles caused a manifold increase in the production of pro-inflammatory tumor necrosis factor-α, anti-inflammatory interleukin-10, as well as interleukin-6, which can stimulate both antibody synthesis and cellular pro-inflammatory reactions. The pronounced stimulation of dendritic cells by virus-like particles coated with coronavirus antigens evidence about successful particle recognition. Finally, we discuss plausible mechanisms for recognition of such virus-like particles by dendritic cell receptors. Conclusion. It has been shown that chimeric virus-like particles induced phenotypic and functional dendritic cell maturation, which is manifested by markedly elevated expression of functionally important membrane molecules, as well as a manifold rise in production of cytokines with a wide functional range. In our opinion, the data obtained indicate a promise of using virus-like particles based on norovirus proteins to display SARS-CoV-2 antigens.

Apolipoprotein E deficiency leads to the polarization of splenic macrophages towards M1 phenotype by increasing iron content.

Apolipoprotein E (ApoE) plays a crucial role in iron homeostasis in the body, while macrophages are the principal cells responsible for handling iron in mammals. However, it is unknown whether ApoE can affect the functional subtypes and the iron handling capacity of splenic macrophages (SM). Here, we investigated the effects of ApoE deficiency (ApoE-/-) on the polarization and iron content of SM and its potential mechanisms. ApoE-/- was found to induce a significant increase in the expressions of M1 marker genes CD86, IL-1β, IL-6, IL-12, TNF-α and iNOS and a reduction in M2 marker genes CD206, Arg-1, IL-10 and Ym-1 in SM of mice aged 28 weeks, Meanwhile, ApoE-/- caused a significant increase in iron content and expression of ferritin, transferrin receptor 1 (TfR1), iron regulatory protein 1 (IRP1) and heme oxygenase-1 (HO-1) and a reduction in ferroportin1 (Fpn1) in spleen and/or SM of mice aged 28 weeks. It was concluded that ApoE-/- can increase iron content through increased iron uptake mediated by TfR/ IRPs and decreased iron release mediated by Fpn1, leading to polarization of the SM to M1 phenotype.

Siraitia grosvenorii Extract Protects Lipopolysaccharide-Induced Intestinal Inflammation in Mice via Promoting M2 Macrophage Polarization.

Siraitia grosvenorii has anti-inflammatory, antioxidant, and immune-regulating effects, while macrophages play an important role in reducing inflammation. However, it is still unclear whether Siraitia grosvenorii extract (SGE) is effective in reducing inflammation by regulating macrophages. This study investigated the regulatory effect of SGE on macrophage polarization in a lipopolysaccharide (LPS)-induced intestinal inflammation model after establishing the model in vitro and in vivo. The results from the in vivo model showed that, compared with the LPS group, SGE significantly improved ileal morphology, restored the ileal mucosal barrier, and reduced intestinal and systemic inflammation by increasing CD206 and reducing iNOS proteins. In the in vitro model, compared with the LPS group, SGE significantly reduced the expression of iNOS protein and cytokines (TNF-α, IL-1β, and IFN-γ) while significantly increasing the protein expression of CD206 in RAW264.7 cells. In conclusion, SGE can alleviate intestinal inflammation, protect the mucus barrier, and block the systemic immunosuppressive response by increasing M2 macrophages.

Linderanine C regulates macrophage polarization by inhibiting the MAPK signaling pathway against ulcerative colitis

Ulcerative colitis (UC) is a chronic non-specific inflammatory disease involving the mucosa and submucosa of the rectum and colon. Lindera aggregate (Sims) Kosterm is a traditional Chinese herb used for thousands of years in the treatment of gastrointestinal diseases. Previously, we have demonstrated that the extracts of Lindera aggregate have good anti-UC effects, but their pharmacodynamic active components have not been fully clarified. Therefore, we explored the therapeutic effect of Linderanine C (LDC), a characteristic component of Lindera aggregata, on UC and its mechanism in this study. Firstly, we found that LDC could significantly reduce the disease activity index of UC and improve shortened colon and pathological changes in vivo. Colon tissue transcriptomics suggested that the anti-UC effect of LDC might be related to its anti-inflammatory activity. Cellular experiments revealed that LDC could inhibit the expression of the M1 cell marker CD86 in RAW264.7 cells, reduce the production of inflammatory mediators such as IL-6 and TNF-α, and have good anti-inflammatory activity in vitro. Cellular transcriptomics reveal the potential involvement of the MAPK signaling pathway in the anti-inflammatory effect of LDC. The co-culture assay confirmed that LDC could significantly reduce inflammation-mediated intestinal epithelial cell injury. In conclusion, LDC was able to inhibit macrophage M1 polarization and reduce inflammatory mediator production by inhibiting the MAPK signaling pathway, effectively improving UC.

Establishment of an in vitro evaluation method for immunomodulatory functions of yeast strains.

Saccharomyces cerevisiae, a widely studied yeast known for its industrial applications, is increasingly recognized for its potential in immunomodulation. This study aimed to systematically analyze and compare the immune-modulating properties of various S. cerevisiae strains under controlled experimental conditions. Three essential signals crucial for immune response activation were evaluated to elucidate the immunological responses elicited by these strains, i.e., dendritic cells (DC) cytokine secretion profiles, maturation status, and T cell polarization. Analysis of DC cytokine secretion profiles and maturation status revealed that all tested yeast strains induced DC activation, characterized by significant IL-6 secretion and modest IL-10 induction, as well as upregulation of MHC II molecules. Additionally, strain-specific effects were observed, particularly, strain AJM109 and Y1383 uniquely enhanced CD86 and PD-L1 expression, respectively, suggesting differential impacts on DC co-stimulatory signaling. Furthermore, strain Y1383 showed a unique capacity to support Treg-mediated immune suppression, demonstrating its potential in immune tolerance induction. These findings underscore the complexity of S. cerevisiae-based immune modulation and emphasize the importance of standardized evaluation methods to distinguish their specific immunological effects.

Knock down of transforming growth factor beta improves expressions of co-stimulatory molecules, type I interferon-regulated genes, and pro-inflammatory cytokine in PRRSV-inoculated monocyte-derived macrophages

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a poor innate immune response following infection. This study evaluates the effects of transforming growth factor beta 1 (TGFβ1) up-regulated by PRRSV on gene expressions of co-stimulatory molecules, type I interferon (IFN), type I IFN-regulated genes (IRGs), pattern recognition receptors, and pro-inflammatory cytokines in PRRSV-inoculated monocyte-derived macrophages (MDMs). Phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) specific to various regions of porcine TGFβ1 mRNA were synthesized, and those specific to the AUG region efficiently knockdown TGFβ1 mRNA expression and protein translation. Transfection of TGFβAS ODNs in MDMs inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) significantly reduced TGFβ1 mRNA expression and significantly increased mRNA expressions of CD80, CD86, IFNβ, IRGs (i.e. IFN regulatory factor 3 (IRF3), IRF7, myxovirus resistance 1, osteopontin, and stimulator of IFN genes), Toll-like receptor 3, and tumor necrosis factor-alpha. Transfection of TGFβAS ODNs in MDMs inoculated with HP-PRRSV-2 also significantly increased mRNA expressions of IFNα, IFNγ, and 2’-5’-oligoadenylate synthetase 1. The quantity of PRRSV-2 RNA copy numbers was significantly reduced in MDMs transfected with TGFβAS ODNs as compared to untransfected MDMs. Recombinant porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα) sustained and reduced the yields of PRRSV-2 RNA copy numbers in PRRSV-2 inoculated MDMs, respectively. These findings demonstrate a strategy of PRRSV for innate immune suppression via an induction of TGFβ expression. These findings also suggest TGFβ as a potential parameter that future PRRSV vaccine and vaccine adjuvant candidates should take into consideration.

Endoplasmic Reticulum Stress Promotes Telomerase Reverse Transcriptase Expression Contributes to Development of Allergic Rhinitis.

The Th2 cell polarization is a crucial factor in the pathogenesis of allergic diseases. The underlying mechanism requires further investigation. Telomerase has an immune-regulating ability. The aim of this study is to elucidate the association between telomerase and Th2 cell polarization in patients with allergic rhinitis (AR). CD4+ T cells were isolated from blood samples collected from AR patients and healthy control subjects. RNA sequencing was employed to analyze RNA samples extracted from CD4+ T cells. An AR mouse model was established using the ovalbumin-alum protocol. High telomerase gene activity and high endoplasmic reticulum (ER) stress status were observed in CD4+ T-cells in patients with AR. Positive correlation between the telomerase reverse transcriptase (TERT) gene expression in CD4+ T cells and AR response in patients with AR. TERT facilitated the degradation of Foxp3 proteins in CD4+ T cells, resulting in the polarization of Th2 cells. Sensitization with the ovalbumin-alum protocol enhanced the Tert expression in CD4+ T cells by exacerbating ER stress. Conditional inhibition of the Tert or eukaryotic translation initiation factor 2-α (Eif2a) expression in CD4+ T cells effectively attenuated experimental AR in mice. Elevated amounts of telomerase in CD4+ T cells were found in CD4+ T cells of subjects with AR. Telomerase promoted Th2 cell polarization by inducing Foxp3 protein degradation and promotes GATA3 activation. Inhibition of TERT or eIF2a alleviated experimental AR.

Abstract We075: Stimulator of Interferon Genes (STING) Regulates T Cell Fate in Cardiometabolic Heart Failure with Preserved Ejection Fraction (HFpEF)

Introduction: Combined obesity and hypertension alter the endoplasmic reticulum (ER) stress response by downregulating X-box binding protein 1 ( Xbp1 ) in T cells, which heightens T cell inflammatory potential and persistence in Heart Failure with Preserved Ejection Fraction (HFpEF), a prevalent syndrome with no cure. The ER protein Stimulator of Interferon Genes (STING), in concert with Xbp1 , coordinates T cell inflammation and death in other contexts, yet its role in the T cell inflammatory response in cardiometabolic HFpEF is unknown. Hypothesis: STING activation and dysregulated ER stress responses drive T cell inflammation in HFpEF by balancing T cell activation and persistence in response to combined comorbidities. Methods: C67BL/6J (wild type, WT) mice were fed high-fat diet/L-NAME (H/L) or standard chow (STD) for 5 weeks, and STING activation was measured in splenic CD4 + T cells by Western blotting. Splenic CD4 + T cells from WT or STING-deficient mice (STING -/- ) were activated with αCD3/αCD28 for 3 days in vitro , then treated with complete media (CM) or CM with 0.2 mM palmitate (abundant in dietary fat)/0.2 mM L-NAME (PALNA) for up to 48 hours. T cell gene expression, activation, and death were assessed using qPCR, flow cytometry, Western blotting, and Lionheart live-cell microscopy. Results: Mice fed H/L had significantly increased splenic T cell expression of phospho-STING, alongside decreased T cell Xbp1 expression and increased cardiac inflammation, compared to STD-fed mice. Ex vivo incubation of CD4 + T cells with PALNA significantly decreased Xbp1 gene expression and increased apoptosis, indicated by cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) expression, compared to cells in CM. However, the remaining live PALNA-treated cells showed higher expression of the activation protein CD69, and this was sustained over time, compared to cells in CM. Strikingly, live cell imaging revealed that STING -/- T cells had improved survival and decreased CD69 expression in response to PALNA compared to WT PALNA-treated cells. Conclusions: STING contributes to T cell inflammation in experimental cardiometabolic HFpEF by synergizing with dysregulated ER stress responses to promote the emergence of hyperactivated T cells.

Validation of reliable reference genes for qPCR of CD4+ Tcells exposed to compressive strain.

For accurate interpretation of quantitative real-time PCR (qPCR) data, stable reference genes are essential for normalization of target genes. To date, there is no information on reliable housekeeping genes in CD4+ Tcells in athree-dimensional (3D) matrix under pressure stimulation. This in vitro study describes for the first time amethod for pressure stimulation of CD4+ Tcells in a3D matrix in the context of orthodontic tooth movement (OTM) and identifies aset of reliable reference genes. CD4+ Tcells were isolated from murine spleen and activated with anti-CD3/-CD28 Dynabeads (Thermo Fisher, Langenselbold, Germany) on standard cell culture plates or in 3D scaffolds with or without compressive strain. Expression stability of nine potential reference genes was examined using four mathematical algorithms. Gene expression of Il2 was normalized to all potential reference genes to highlight the importance of correct normalization. Cell proliferation and the expression of the surface markers CD25 and CD69 were also determined. The 3D matrix did not inhibit proliferation after immunological activation of Tcells and embedded the cells sufficiently to expose them to pressure load. Expression of ubiquitin C (Ubc) and hypoxanthine phosphoribosyltransferase (Hprt) was the most stable under all conditions tested. Acombination of these two genes was suitable for normalization of qPCR data. Normalization of Il2 gene expression showed highly variable results depending on the reference gene used. Pressure reduced cell proliferation and the number of CD69-positive Tcells. This study provides abasis for performing valid and reliable qPCR experiments with CD4+ Tcells cultured in 3D scaffolds and exposed to compressive forces simulating OTM.

Abstract Tu099: GDF11 accelerates NF-kB-mediated inflammation prior to reduction in cardiac fibrosis in response to experimental pressure overload.

Background: Cardiac fibrosis is a common pathophysiology in a broad variety of heart diseases. GDF11, a member of the TGF-beta superfamily, can reduce cardiac hypertrophy and fibrosis in the pressure overload model of experimental Trans Aortic Constriction (TAC). However, the molecular mechanisms by which GDF11 can reduce cardiac fibrosis are incompletely described. Hypothesis: GDF11 is known to activate both NF-kB as well as SMAD2/3 signaling which are essential in cardiac fibrosis and hypertrophy processes. We hypothesized that exogenous GDF11 can change the dynamics of early inflammatory NF-kB target genes during the response to pressure overload. Methods: We used mice implanted with AngII-osmotic pumps to induce left ventricular fibrosis. The mice were harvested at different time points: 3, 7, 14, and 28 days post-pump implantation (ppi) to follow the evolution of cardiac fibrosis and cardiac hypertrophy, and to identify the role of GDF11 in modulating these processes. Results: GDF11 delivery (1mg/kg every other day) in AngII mice (AngII+GDF11) induced earlier expression of NF-kB target genes (IL-1beta, IL-6, TNF-alpha – p>0.05) compared to AngII+Veh control group at 3d ppi, revealing accelerated activation of NF-kB response pathways in AngII+GDF11 group. This is associated with faster recruitment of the M1 macrophage population observed by an increased expression of CD86 or iNOS at 3d ppi. We also observed decreased markers of fibroblast activation after GDF11 supplementation, with less collagen deposition (F-CHP) and fibrosis (Masson Trichrome) at 28 days. Conclusions: Our data suggest that exogenous GDF11 changes the dynamics of NF-kB inflammatory mechanisms while reducing later fibrosis. This new finding could suggest that the dynamics of the inflammatory response early after pressure overload is a critical factor in cardiac fibrosis.

Curcumin loaded hydrogel with double ROS-scavenging effect regulates microglia polarization to promote poststroke rehabilitation

Cyclodextrins are used to include curcumin to form complex, which is subsequently loaded into a reactive oxygen species (ROS) responsive hydrogel (Cur gel). This gel exhibits a dual ROS scavenging effect. The gel can neutralize extracellular ROS to lead to a ROS-sensitive curcumin release. The released curcumin complex can eliminate intracellular ROS. Furthermore, the Cur gel effectively downregulates the expression of CD16 and IL-1β while upregulating CD206 and TGF-β in oxygen and glucose-deprived (OGD) BV2 cells. Additionally, it restores the expression of synaptophysin and PSD95 in OGD N2a cells. Upon injection into the stroke cavity, the Cur gel reduces CD16 expression and increases CD206 expression in the peri-infarct area of stroke mice, indicating an in vivo anti-inflammatory polarization of microglia. Colocalization studies using PSD95 and VGlut-1 stains, along with Golgi staining, reveal enhanced neuroplasticity. As a result, stroke mice treated with the Cur gel exhibit the most significant motor function recovery. Mechanistic investigations demonstrate that the released curcumin complex scavenges ROS and suppresses the activation of the ROS-NF-κB signaling pathway by inhibiting the translocation of p47-phox and p67-phox to lead to anti-inflammatory microglia polarization. Consequently, the Cur gel exhibits promising potential for promoting post-stroke rehabilitation in clinics.