Peripheral Mononuclear Cells Research Articles

Three Months of Strength Training Changes the Gene Expression of Inflammation-Related Genes in PBMC of Older Women: A Randomized Controlled Trial.

Here, we investigate changes in inflammation-related gene-expression in peripheral mononuclear blood cells (PBMC) by strength training. A total of 14 women aged ≥65 years were randomized into 3 months of either 3×/week intensive strength training (IST: 3×10 rep at 80% 1RM), strength endurance training (SET: 2×30 reps at 40% 1RM) or control (CON: 3×30 sec stretching). Differentially expressed genes (fold change ≤0.67 or ≥1.5) were identified by targeted RNA-sequencing of 407 inflammation-related genes. A total of 98 genes (n = 61 pro-inflammatory) were significantly affected. IST and SET altered 14 genes in a similar direction and 19 genes in the opposite direction. Compared to CON, IST changed the expression of 6 genes in the same direction, and 17 genes in the SET. Likewise, 18 and 13 genes were oppositely expressed for, respectively, IST and SET compared to CON. Changes in gene expression affected 33 canonical pathways related to chronic inflammation. None of the altered pathways overlapped between IST and SET. Liver X Receptor/Retinoid X Receptor Activation (LXR/RXR) and Triggering Receptor Expressed On Myeloid Cells 1 (TREM1) pathways were enriched oppositely in both training groups. We conclude that three months IST and SET can induce changes in CLIP-related gene expression in PBMC, but by affecting different genes and related pathways.

The aberrant expression of CD45 isoforms and levels of sex hormones in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a common autoimmune disease with significant gender bias in women, and sex hormones are considered to play an important role in the regulation of immune activity. The CD45 isoforms generated through alternative splicing of mRNA identify different functional status of lymphocytes and also are suggested as a biomarker for assessing the progression of SLE, while the modulation of CD45 expression in SLE patients is not clear. In this study, the peripheral blood sera of 46 SLE patients and 15 health individuals were collected for detecting the levels of sex hormones and immune associated factors. The expression of CD45 isoforms and the status of CD45 DNA methylation of the peripheral mononuclear blood cells were detected by flow cytometry and bisulfite sequencing PCR, respectively. The levels of complement C3 and IgA decreased, especially decline of the serum IgA to the level of selective immunoglobulin A deficiency, and the C-reactive protein increased in SLE patients when compared with healthy controls, which manifested the abnormal immune activity of the SLE patients. Sex hormones detection showed a decreased testosterone and increased prolactin in SLE. An accelerated expression of CD45RO, reduced CD45RA and CD45RB, and a relative hypermethylation of CD45 DNA in SLE were also identified that provided a clue to explain the possible regulatory mechanism for the immune function in SLE. The results indicated that the aberrant CD45 isoforms, DNA methylation and hormone levels might be correlated with the imbalanced immune activity of SLE patients. Key Points • Selective immunoglobulin A deficiency was significantly higher in SLE than in healthy individuals. • SLE patients had decreased testosterone and increased prolactin in the sera. • An aberrant expression of CD45 isoforms and CD45 DNA methylation were identified in SLE.

Subfornical organ interleukin 1 receptor: A novel regulator of spontaneous and conditioned fear associated behaviors in mice

Impaired threat responding and fear regulation is a hallmark of psychiatric conditions such as post-traumatic stress disorder (PTSD) and Panic Disorder (PD). Most studies have focused on external psychogenic threats to study fear, however, accumulating evidence suggests a primary role of homeostatic perturbations and interoception in regulating emotional behaviors. Heightened reactivity to interoceptive threat carbon dioxide (CO2) inhalation associates with increased risk for developing PD and PTSD, however, contributory mechanisms and molecular targets are not well understood. Previous studies from our group suggested a potential role of interleukin 1 receptor (IL-1R1) signaling within BBB-devoid sensory circumventricular organ, the subfornical organ (SFO) in CO2-evoked fear. However, the necessity of SFO-IL-1R1 in regulating CO2-associated spontaneous fear as well as, long-term fear potentiation relevant to PD/PTSD has not been investigated. The current study tested male mice with SFO-targeted microinfusion of the IL-1R1 antagonist (IL-1RA) or vehicle in a recently developed CO2-startle-fear conditioning-extinction paradigm. Consistent with our hypothesis, SFO IL-1RA treatment elicited significant attenuation of freezing and increased rearing during CO2 inhalation suggesting SFO-IL1R1 regulation of spontaneous fear to CO2. Intriguingly, SFO IL-1RA treatment normalized CO2-associated potentiation of conditioned fear and impaired extinction a week later suggesting modulation of long-term fear by SFO-IL-1R1 signaling. Post behavior FosB mapping revealed recruitment of prefrontal cortex-amygdala-periaqueductal gray (PAG) areas in SFO-IL-1RA mediated effects. Additionally, we localized cellular IL-1R1 expression within the SFO to blood vessel endothelial cells and observed CO2-induced alterations in IL-1β/IL-1R1 expression in peripheral mononuclear cells and SFO. Lastly, CO2-evoked microglial activation was attenuated in SFO-IL-1RA treated mice. These observations suggest a peripheral monocyte-endothelial-microglia interplay in SFO-IL-1R1 modulation of CO2-associated spontaneous fear and delayed fear memory. Collectively, our data highlight a novel, “bottom-up” neuroimmune mechanism that integrates interoceptive and exteroceptive threat processing of relevance to fear-related pathologies.

Detection of α-Synuclein-Specific T Cells in Parkinson's Disease.

Parkinson's disease (PD) is a widely prevalent chronic neurodegenerative disease. The disease is characterized by loss of dopaminergic neurons with abnormal aggregation of α-synuclein (α-syn). The misfolded deposition of α-syn is known to mount robust adaptive immune response by activating T cells. Here, we show that peripheral mononuclear cells when stimulated with a α-syn-derived peptide pool activate α-syn-specific T cells that produce cytokines.

Zinc supplementation prior to heat shock enhances HSP70 synthesis through HSF1 phosphorylation at serine 326 in human peripheral mononuclear cells

Acute alteration of the functional zinc status increases the HSP70 protein level after heat shock in peripheral blood mononuclear cells (PBMC), while zinc deficiency, but not zinc supplementation, further augments heat shock-induced cell death.

Kinetics of Mononuclear Cell Subpopulations in the Peripheral Blood of Patients with Giant Cell Arteritis During the Acute Phase of the Disease: The Role of Steroids.

Background/Aim:Giant cell arteritis (GCA) represents the most prevalent form of systemic vasculitis in the elderly, primarily affecting the temporal artery, the extracranial branches of carotid arteries, and the aorta. GCA is a highly heterogeneous disease in terms of clinical and histological findings, pathophysiology, and treatment selection strategies. The disease is highly responsive to glucocorticosteroids (GCs), but almost half of patients may relapse following GCs tapering. The main hypothesis of GCA pathogenesis includes altered immune responses and changes in the vascular microenvironment, leading to a dynamic interplay between innate and adaptive immunity. The aim of this study is to explore the effect of GCs on the phenotype of peripheral mononuclear cell subpopulations and on the major inflammatory molecules detected in the peripheral blood of patients during the acute phase of the disease.Methods:Patient PBMCs will be studied using Cytometry by time of flight (CyTOF). Following the CyTOF analysis, Luminex Assay will be performed on the same patient samples to identify the kinetics of the most prominent inflammatory mediators correlating with the subpopulations detected. Patient population consists of 8 patients with GCA, 6 with polymyalgia rheumatica, as disease control group and 5 healthy controls (sex and age matched) at 3 time points: disease diagnosis, 48 and 96 hours after treatment administration.Conclusion:The identification of potential alterations in cell subpopulations and the kinetics of inflammatory mediators are expected to lead to the production of new knowledge regarding the role of corticosteroids in the phase of acute inflammatory response.

The Secretome of Irradiated Peripheral Mononuclear Cells Attenuates Hypertrophic Skin Scarring

The Secretome of Irradiated Peripheral Mononuclear Cells Attenuates Hypertrophic Skin Scarring

Mitochondrial Dysfunction Associated with Low Expression of Bcl-2: an Inflammatory Mediator in Diabetic Patients with Atherosclerosis

Objective: To investigate mitochondrial dysfunction and pattern of expression of B-cell lymphoma 2 (Bcl-2) and correlate these with cardiovascular diseases (CVD) in type 2 diabetes mellitus (T2DM).   Background: Diabetes mellitus (DM) raises the risk of CVD, which contributes significantly to the high mortality rates associated with the disease. The role of mitochondrial dysfunction and apoptosis in the development of CVD has been suggested, however the mechanisms involved are unknown.   Methods: Type 2 diabetic mellitus patients with atherosclerosis (T2DM + ATHER, n=40), T2DM patients (n=40) and a control group of age-matched non-diabetic individuals (n=35) were enrolled in this study. Blood samples were collected and analysed for the presence of anti-apoptotic-related factors, Bcl-2 in Peripheral mononuclear cells by qPCR as well as mitochondrial dysfunction by immunofluorescence analysis. Serum TNF-α and antioxidant enzymes (SOD) were measured by ELISA.   Result: Bcl-2 expression was significantly decreased in patients with T2DM + ATHER and T2DM (P < 0.05) compared to the control group. MitoSpy Red staining revealed nuclear condensation and fragmentation in T2DM + ATHER and T2DM. There was a significant increase in pro-inflammatory cytokine TNF-α while the levels of SOD were significantly decreased in the T2DM + ATHER and T2DM patient group (P<0.05) compared to the control group.   Conclusion: These results suggest that mitochondrial dysfunction was associated with oxidative stress and inflammation in patients with T2DM +ATHER.

Hypoxia-Induced Intracellular and Extracellular Heat Shock Protein gp96 Increases Paclitaxel-Resistance and Facilitates Immune Evasion in Breast Cancer.

BackgroundsHypoxia contributes to cancer progression, drug resistance and immune evasion in various cancers, including breast cancer (BC), but the molecular mechanisms have not been fully studied. Thus, the present study aimed to investigate this issue.MethodsThe paclitaxel-sensitive BC (PS-BC) cells were administered with continuous low-dose paclitaxel treatment to establish paclitaxel-resistant BC (PR-BC) cells. Exosomes were isolated/purified by using the commercial kit, which were observed by Transmission electron microscopy (TEM). Cell viability was measured by MTT assay, cell apoptosis was determined by flow cytometer (FCM). Gene expressions were respectively measured by Real-Time qPCR, Western Blot and immunofluorescence staining assay. The peripheral mononuclear cells (PBMCs) derived CD8+ T cells were obtained and co-cultured with gp96-containing exosomes, and cell proliferation was evaluated by EdU assay. ELISA was employed to measure cytokine secretion in CD8+ T cells’ supernatants.ResultsHSP gp96 was significantly upregulated in the cancer tissues and plasma exosomes collected from BC patients with paclitaxel-resistant properties. Also, continuous low-dose paclitaxel treatment increased gp96 levels in the descendent PR-BC cells and their exosomes, in contrast with the parental PS-BC cells. Upregulation of gp96 increased paclitaxel-resistance in PS-BC cells via degrading p53, while gp96 silence sensitized PR-BC cells to paclitaxel treatments. Moreover, PR-BC derived gp96 exosomes promoted paclitaxel-resistance in PS-BC cells and induced pyroptotic cell death in the CD8+ T cells isolated from human peripheral blood mononuclear cells (pPBMCs). Furthermore, we noticed that hypoxia promoted gp96 generation and secretion through upregulating hypoxia-inducible factor 1 (HIF-1), and hypoxia increased paclitaxel-resistance and accelerated epithelial-mesenchymal transition (EMT) in PS-BC cells.ConclusionsHypoxia induced upregulation of intracellular and extracellular gp96, which further degraded p53 to increase paclitaxel-sensitivity in BC cells and activated cell pyroptosis in CD8+ T cells to impair immune surveillance.

M6A Regulators in Human Adipose Tissue - Depot-Specificity and Correlation With Obesity.

BackgroundN6-methyladenosine (m6A) is one of the most abundant post-transcriptional modifications on mRNA influencing mRNA metabolism. There is emerging evidence for its implication in metabolic disease. No comprehensive analyses on gene expression of m6A regulators in human adipose tissue, especially in paired adipose tissue depots, and its correlation with clinical variables were reported so far. We hypothesized that inter-depot specific gene expression of m6A regulators may differentially correlate with clinical variables related to obesity and fat distribution.MethodsWe extracted intra-individually paired gene expression data (omental visceral adipose tissue (OVAT) N=48; subcutaneous adipose tissue (SAT) N=56) of m6A regulators from an existing microarray dataset. We also measured gene expression in another sample set of paired OVAT and SAT (N=46) using RT-qPCR. Finally, we extracted existing gene expression data from peripheral mononuclear blood cells (PBMCs) and single nucleotide polymorphisms (SNPs) in METTL3 and YTHDF3 from genome wide data from the Sorbs population (N=1049). The data were analysed for differential gene expression between OVAT and SAT; and for association with obesity and clinical variables. We further tested for association of SNP markers with gene expression and clinical traits.ResultsIn adipose tissue we observed that several m6A regulators (WTAP, VIRMA, YTHDC1 and ALKBH5) correlate with obesity and clinical variables. Moreover, we found adipose tissue depot specific gene expression for METTL3, WTAP, VIRMA, FTO and YTHDC1. In PBMCs, we identified ALKBH5 and YTHDF3 correlated with obesity. Genetic markers in METTL3 associate with BMI whilst SNPs in YTHDF3 are associated with its gene expression.ConclusionsOur data show that expression of m6A regulators correlates with obesity, is adipose tissue depot-specific and related to clinical traits. Genetic variation in m6A regulators adds an additional layer of variability to the functional consequences.

Circulating CD4+ TEMRA and CD4+ CD28- T cells and incident diabetes among persons with and without HIV.

A higher proportion of circulating memory CD4+ T cells is associated with prevalent diabetes mellitus in persons with HIV (PWH) and HIV-negative persons. We assessed whether circulating T-cell subsets could also identify individuals who will subsequently develop diabetes. This is a longitudinal follow-up study of PWH and similar HIV-negative individuals from the Veterans Aging Cohort Study who provided peripheral mononuclear blood cells between 2005 and 2007. We quantified T-cell subsets using flow cytometry and functional assays to identify CD4+ and CD8+ naive, activated, senescent, memory (central, effector, and effector RA+), and TH1, TH2, and TH17-phenotype cells. The occurrence of an incident diabetes diagnosis (i.e. after baseline blood draw) was adjudicated by a two-physician chart review. Cox proportional hazards models adjusted for traditional risk factors, cytomegalovirus serostatus, and plasma inflammatory biomarkers assessed the relationship between T-cell subsets and incident diabetes. One thousand, eight hundred and thirty-seven participants (1259 PWH) without diabetes at baseline were included; 69% were black, 95% were men, and median follow-up was 8.6 years. Higher baseline frequencies of CD4+ T effector memory RA+ (TEMRA) cells defined as CD45RA+ CD27- (P = 0.04) and senescent T cells defined as CD4+ CD28- (P = 0.04) were associated with incident diabetes in PWH only. Higher frequencies of CD4+ TEMRA and CD4+ CD28- T cells were associated with incident diabetes in PWH only after adjustment for other factors. Additional studies are necessary to assess whether these cells act in blood via inflammatory mediators or reflect T-cell populations in metabolically active tissues.

5-Dodecanolide, a Compound Isolated from Pig Lard, Presents Powerful Anti-Inflammatory Properties.

Background: Pork lard (PL) is traditionally used as an anti-inflammatory agent. We propose to demonstrate the anti-inflammatory properties of PL, and elucidate which compounds could be responsible for the anti-inflammatory effects. Methods: The anti-inflammatory effects of PL were tested in a rat model of zymosan-induced hind paw inflammation. Further, the hydroalcoholic extract from PL was obtained, the composition analyzed, and the anti-inflammatory activity of the extracts and isolated components assayed using immune cells stimulated with lipopolysaccharide (LPS). Results: Applying the ointment on the inflamed rat feet reduced the foot diameter, foot weight, and activities of antioxidant enzymes and inflammatory markers of circulating neutrophils. The main components of the hydroalcoholic extract were 5-dodecanolide, oleamide, hexadecanoic acid, 9-octadecenoic acid, hexadecanamide, and resolvin D1. Conclusions: PL reduces the immune response in an animal model stimulated with zymosan. Hydroalcoholic PL extract and its components (5-Dodecanolide, Oleamide, and Resolvin D1) exerted an anti-inflammatory effect on LPS-stimulated neutrophils and peripheral mononuclear cells reducing the capability to produce TNFα, as well as the activities of antioxidant and pro-inflammatory enzymes. These effects are attributable to 5-dodecanolide, although the effects of this compound alone do not reach the magnitude of the anti-inflammatory effects observed by the complete hydroalcoholic extract.

Using the co-expression network of T cell-activation-related genes to assess the disease activity in Takayasu\u2019s arteritis patients

BackgroundThere have been lacking reliable serum biomarkers in assessing the disease activity of Takayasu’s arteritis (TAK). This study aimed to assess the disease activity of TAK by assayed gene expression levels in peripheral mononuclear cells (PBMCs).MethodsThe expression level of genes that essential in T cell activation in PBMCs in active TAK patients, inactive TAK patients, and healthy controls were detected by real-time fluorescence quantitative polymerase chain reaction, including TCR, CD28, CD40, CD40L, PD-1, PD-L1, PD-L2, CTLA4, TIGIT, TIM3, LAG3, CCL5, T-bet, RORC, and FOXP3. Gene co-expression network was established, and the signature of the topology structure in active TAK patients compared to the inactive TAK patients were extracted and described by formulas. Respectively, the disease activity was assessed by the routine serum biomarkers, including ESR, CRP, IL-6, and TNF-α, the gene expression level of TCR, CD28, T-bet, and RORC, as well as the signature of the topology structure, and the diagnostic efficacies were compared.ResultsCompared with the inactive TAK patient group, the active TAK patient group had a greater clustering coefficient in the network consisting of genes that essential in T cell activation. When assessing the disease activity used this signature of topology structure, the sensitivity was 90.9%, the specificity was 100%, and the AUC was 0.98, which was greater than the AUCs of these biomarkers.ConclusionsThe signature of the topology structure could distinguish the active TAK patients from inactive TAK patients. This maybe is a novel evaluation algorithm of disease activity.

Upregulation of toll-like receptors 4 in peripheral mononuclear cells of chronic renal disease patients on hemodialysis is associated with poor cardiac function and increased mortality

Upregulation of toll-like receptors 4 in peripheral mononuclear cells of chronic renal disease patients on hemodialysis is associated with poor cardiac function and increased mortality

Prevalence of Candida species in Psoriasis.

Psoriasis patients are more frequently colonised with Candida species. The correlation between fungal colonisation and clinical severity is unclear, but may exacerbate psoriasis and the impact of antipsoriatic therapies on the prevalence of Candida is unknown. To examine the prevalence of Cspecies in psoriasis patients compared to an age- and sex-matched control population, we investigated the influence of Candida colonisation on disease severity, immune cell activation and the interplay on psoriatic treatments. The prevalence of Cspecies was examined in 265 psoriasis patients and 200 control subjects by swabs and stool samples for fungal cultures. Peripheral mononuclear blood cells (PBMCs) were collected from 20 fungal colonised and 24 uncolonised patients and stimulated. The expression of interferon (IFN)-γ, IL-17A, IL-22 and tumour necrosis factor (TNF)-α from stimulated PBMCs was measured by quantitative real-time polymerase chain reaction (qPCR). A significantly higher prevalence for Candida was detected in psoriatic patients (p≤.001) compared to the control subjects; most abundant in stool samples, showing Candida albicans. Older participants (≥51years) were more frequent colonised, and no correlation with gender, disease severity or systemic treatments like IL-17 inhibitors was found. Although Candida colonisation is significantly more common in patients with psoriasis, it does not influence the psoriatic disease or cytokine response. Our study showed that Candida colonisation is particularly more frequent in patients with psoriasis ≥51years of age. Therefore, especially this group should be screened for symptoms of candidiasis during treatment with IL-17 inhibitors.

Phosphodiesterase-4 Inhibition Reduces Cutaneous Inflammation and IL-1β Expression in a Psoriasiform Mouse Model but Does Not Inhibit Inflammasome Activation.

Apremilast (Otezla®) is an oral small molecule phosphodiesterase 4 (PDE4) inhibitor approved for the treatment of psoriasis, psoriatic arthritis, and oral ulcers associated with Behçet’s disease. While PDE4 inhibition overall is mechanistically understood, the effect of apremilast on the innate immune response, particularly inflammasome activation, remains unknown. Here, we assessed the effect of apremilast in a psoriasis mouse model and primary human cells. Psoriatic lesion development in vivo was studied in K5.Stat3C transgenic mice treated with apremilast for 2 weeks, resulting in a moderate (2 mg/kg/day) to significant (6 mg/kg/day) resolution of inflamed plaques after 2-week treatment. Concomitantly, epidermal thickness dramatically decreased, the cutaneous immune cell infiltrate was reduced, and proinflammatory cytokines were significantly downregulated. Additionally, apremilast significantly inhibited lipopolysaccharide- or anti-CD3-induced expression of proinflammatory cytokines in peripheral mononuclear cells (PBMCs). Notably, inflammasome activation and secretion of IL-1β were not inhibited by apremilast in PBMCs and in human primary keratinocytes. Collectively, apremilast effectively alleviated the psoriatic phenotype of K5.Stat3 transgenic mice, further substantiating PDE4 inhibitor-efficiency in targeting key clinical, histopathological and inflammatory features of psoriasis. Despite lacking direct effect on inflammasome activation, reduced priming of inflammasome components upon apremilast treatment reflected the indirect benefit of PDE4 inhibition in reducing inflammation.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Peripheral Mononuclear Cells in Patients With Ankylosing Spondylitis.

ObjectiveGenetic studies on ankylosing spondylitis (AS) have identified more than 100 pathogenic genes. Building a bridge between these genes and biologically targeted therapies is the current research hotspot.MethodsWe integrated single-cell assaying transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) to explore the key genes and related mechanisms associated with AS pathogenesis.ResultsWe identified 18 cell types in peripheral mononuclear cells from patients with AS and normal controls and summarized the cell-type-specific abnormal genes by scRNA-seq. Interestingly, we found that the pathogenic gene NFKB involved in AS progression originated from CD8+ T cells. Moreover, we observed an abnormal tumor TNF pathway mediated by abnormal expression of TNF, NFKB, FOS, JUN, and JUNB, and scATAC-seq results confirmed the abnormal accessible binding sites of transcriptional factors FOS, JUN, and JUNB. The final magnetic bead sorting and quantitative real-time PCR(RT-qPCR) confirmed that NFKB, FOS, JUN, and JUNB in CD8+ T cells differed in the AS group.ConclusionsOur results revealed a possible mechanism by which NFKB abnormally regulates FOS, JUN, and JUNB and drives AS progression, providing a novel perspective from a single cell point of view in AS.

Abstract 10241: Single-Cell RNA-Sequencing Reveals Hyperacute NK Cell and Monocyte Cell States Correlating with Poor Neurological Outcomes After Cardiac Arrest

Background: Lymphopenia and elevated cytokine levels suggest a role for the systemic immune response after cardiac arrest (CA). However, little is known about the phenotypes and interactions of immune cells after CA. This study aimed to investigate the immunological network after CA and identify cell states correlating with poor neurological outcomes. Methods: Peripheral mononuclear blood cells of 11 post-out-of-hospital CA patients and 3 healthy subjects were analyzed by single-cell RNA-sequencing (scRNA-seq), with validation by flow cytometry, bulk RNA-seq of sorted cell subsets, plasma levels of cytokines, and the murine model of CA. Good and poor neurological outcomes at hospital discharge were defined by Cerebral Performance Category 1-2 and 3-5, respectively. Results: The scRNA-seq analysis of 96,179 cells revealed 6 major cell lineages with several subclusters. At 6h post-CA, cells of patients with good outcomes co-clustered with healthy subjects, while patients with poor outcomes formed distinct transcriptomic clusters. This distinction was lost by 48h post-CA. At 6h post-CA, we identified pro-inflammatory and hypoxia-responsive Tim-3 + NK cell and Nectin-2 + monocyte cell states specific to poor outcomes. In a validation cohort of post-CA patients, Tim-3 + NK cells and Nectin-2 + monocyte cell states were validated by flow cytometry. Bulk RNA-seq analysis of these sorted cells reproduced the transcriptomic profile determined by scRNA-seq. Interactome analysis identified NK cell-monocyte axes in poor outcomes, including IFNG-IFNGR2. Measurement of plasma cytokine levels revealed increased IFNγ levels at 6h post-CA that were associated with poor outcomes, but both poor and good outcomes had similarIFNγ levels by 48h. A murine model of CA demonstrated that IFNγ drives poor outcomes and mortality after CA. Conclusions: In clinical CA, transcriptomic analysis at single-cell resolution revealed hyperacute cell states of NK cells and monocytes associated with poor neurological outcomes. These hyperacute cell states interact by IFNγ-IFNγR2 axis, which is pathogenic in the murine model of CA. The immunophenotypes that distinguish patients with poor or good neurological outcomes are present at 6h but are no longer detectable by 48h post-CA.

Abstract 10926: Importance of Mitochondrial DNA Damage in Cigarette Smoke Extract-Induced Activation of Innate Immune System

Objective: Smoking is a well-known risk factor for atherosclerosis, yet the mechanism by which smoking causes atherosclerosis remains unclear. It has been reported that inflammation and DNA damage are involved in the formation and development of atherosclerosis. We have previously reported that nuclear DNA damage was increased in peripheral mononuclear cells of smokers compared to those of non-smokers, and that DNA damage was accumulated in atherosclerotic lesions. In this study, to clarify the mechanism by which smoking induces inflammation in vascular endothelial cells, we investigated the effect of cigarette smoke extract (CSE) on both nuclear and mitochondrial DNA damage and the subsequent cellular response in human umbilical vein endothelial cells (HUVEC). Methods and Results: CSE increased double-strand breaks in HUVEC. Notably, this increase was detected relatively late (after 3 days of CSE exposure) compared to other genotoxic agents. CSE also increased oxidative DNA damage both in the nucleus and mitochondria and induced mitochondrial dysfunction. Mitochondrial dysfunction decreased cytosolic protein levels of ICAD, an inhibitor of caspase-activated DNase (CAD), which causes nuclear DNA fragmentation, and increased CAD in the nucleus, suggesting that this is one of the mechanisms of nuclear DNA double-strand breaks. In addition, continuous stimulation by CSE induced the accumulation of cytosolic DNA. Interestingly, real-time PCR analysis revealed that the accumulated DNA in the cytosol was derived not only from the nucleus but also from mitochondria. We further examined whether DNA accumulated in the cytosol activated cytosolic DNA-sensing pathways. The production of cGAMP, a second messenger in cGAS (a DNA-sensing receptor) signaling, was increased by CSE. We also found that the mRNA expression of IL-6 was increased by CSE, and that the increase was suppressed by si-cGAS. Conclusions: This study showed that continuous exposure to CSE induces not only nuclear but also mitochondrial DNA damage, which leads to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS-STING pathway.

Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.