mRNA In Peripheral Blood Mononuclear Cells Research Articles

The effect of myo‐inositol supplementation on AMPK/PI3K/AKT pathway and insulin resistance in patients with NAFLD

AbstractInsulin resistance (IR) is the pivotal pathological hit in non‐alcoholic fatty liver disease (NAFLD). There is specific attention to combination/conjugated therapies for NAFLD management. As myo‐inositol (MI) has been shown to improve IR in animal and human trials, this study aimed to investigate the influence of MI supplementation on glycemic response and IR through AMPK/PI3K/AKT signaling pathway in obese patients with NAFLD. This double‐blinded placebo‐controlled randomized clinical trial was conducted on 48 obese (BMI = 30–40 kg/m2) patients with NAFLD who were randomly assigned to receiving either MI (4 g/day) or placebo (maltodextrin 4 g/day) group for 8 weeks. Before and after the trial, weight, height, serum glycemic parameters (inc. fasting glucose and insulin) as well as IR indices were assessed. Moreover, the mRNA expression levels of AMPK, AKT, and PDK‐1 in peripheral blood mononuclear cells (PBMCs) were determined. MI supplementation resulted in significant increases in the fold changes of AMPK, AKT, and PDK‐1 genes (p = .019, p = .049, and p = .029, respectively). Indeed, IR improved in terms of all IR indices in MI group (p < .05) after adjusting for the confounders, apart from quantitative insulin sensitivity check index (QUICKI). The results showed that MI supplementation not only upregulated AMPK, AKT, and PDK‐1 mRNA in PBMCs but also improved IR in obese patients with NAFLD.

The Role of Notch1 Pathway Regulating CD4+CD25+Foxp3+ T Cells in Renovascular Hypertension

Abstract OBJECTIVE To explore the role and mechanism of the Notch1 pathway in regulating CD4+CD25+Foxp3+ Treg cells in renal vascular hypertension (RVH). METHODS According to the diagnostic criteria of RVH in the 8th edition of Internal Medicine, 220 patients treated in the Department of Nephrology of Sichuan Provincial People’s Hospital from January 2019 to January 2022 were enrolled in RVH group, and 220 patients with essential hypertension (EH) in the same period were selected as EH group; another 220 healthy examinees were selected as control group (NC). The proportion of CD4+CD25+ Treg cells in peripheral blood mononuclear cells (PBMCs) and the expression levels of interleukin-10 (IL-10), transforming growth factor-β (TGF-β), and cell proliferation nuclear antigen-67 (Ki-67) in PBMC of the 3 groups were compared by real-time fluorescence quantitative PCR and western blotting; the relative expression levels of Notch1 receptor and ligand mRNA in PBMC of the 3 groups were compared by real-time fluorescence quantitative PCR; the expression of downstream proteins Hes1 and Hes5 of Notch1 signaling pathway, the proportion of CD4+CD25+Foxp3+ Treg cells, and the effect on TGF-β expression were compared by western blotting after adding Notch1 signaling pathway inhibitor (DAPT). Real-time quantitative PCR and western PCR were used. The proportion of CD4+CD25+ Treg cells in PBMCs and the expression levels of IL-10, TGF-β, and cell proliferating nuclear antigen-67 (Ki-67) in PBMC were compared between the 2 groups. Real-time quantitative PCR was used to compare the mRNA expression levels of the Notch1 receptor and ligand in PBMC between the 2 groups. RESULTS Flow cytometry results showed that compared with the NC group, the RVH group had a significantly lower proportion of CD4+CD25+Foxp3+ Treg cells, TGF-β and Ki-67 expression levels, and higher IL-10 expression levels, with significant differences (P < 0.001). Compared with the RVH group, the EH group had a significantly higher proportion of CD4+CD25+Foxp3+ Treg cells, TGF-β and Ki-67 expression levels, and lower IL-10 expression levels, with significant differences (P < 0.001). Real-time quantitative PCR results showed that the RVH group also had significantly higher relative expression levels of Notch receptor Notch1 mRNA, Notch ligand DLL1, DLL4, Jagged1, and Jagged2 mRNA in PBMC than the NC group and EH group, with significant differences (P < 0.001). Western blot results showed that after adding Notch1 signaling pathway inhibitor (DAPT), Hes1 and Hes5 expression in peripheral CD4+CD25+Foxp3+ Treg cells of patients decreased significantly (P < 0.001), while CD4+CD25+Foxp3+ Treg proportion and TGF-β expression level increased significantly (P < 0.001). CONCLUSIONS The Notch1 signaling pathway plays an immunoregulatory role in RVH regulated by CD4+CD25+Foxp3+ Treg cells. Regulation of the Notch1 signaling pathway can significantly promote the proliferation of CD4+CD25+Foxp3+ Treg cells and the ability to secrete anti-inflammatory factors.

WNT16 as a promising biomarker for systemic lupus erythematosus and its role in regulating cell proliferation and apoptosis.

To investigate the expression and function of WNT16, a member of the WNT family protein, in the context of systemic lupus erythematosus (SLE). WNT16 expression was assessed in peripheral blood mononuclear cells (PBMCs) from 35 SLE patients and 25 healthy individuals using quantitative polymerase chain reaction. Additionally, serum WNT16 protein levels were quantified via enzyme-linked immunosorbent assay in 162 SLE patients, 96 healthy controls (HC), and disease controls comprised 154 individuals with rheumatoid arthritis (RA) and Sjögren's syndrome (SS). We investigated the associations between WNT16 protein levels and clinical manifestations, laboratory indices, and disease activity in SLE patients. Receiver operating characteristic (ROC) curve analysis was employed to evaluate the diagnostic potential of serum WNT16 for SLE. Furthermore, we performed a knockdown assay on Jeko-1 cells and assessed cell proliferation and apoptosis using Cell Counting Kit-8 and flow cytometry. WNT16 mRNA in SLE patients' PBMCs were significantly lower than those in HC. Furthermore, serum WNT16 in SLE patients were markedly reduced compared to HC, RA, and SS cohorts. ROC curve analysis indicated that plasma WNT16 levels could serve as a potential biomarker for SLE identification (AUC=0.809, SLE vs. HC; AUC=0.760, SLE vs. RA; AUC=0.710, SLE vs. SS). Notably, a weak positive correlation was observed between WNT16 protein and both alkaline phosphatase and lymphocyte percentages. Conversely, a weak negative correlation existed between WNT16 and low-density lipoprotein, neutrophil percentage, and the incidence of pleurisy and disease activity. Additionally, our study confirmed that WNT16 knockdown impairs cell proliferation and enhances apoptosis. Serum WNT16 levels effectively differentiate SLE patients from healthy controls and individuals with other autoimmune disorders. WNT16 serves as a potential biomarker with high sensitivity. The diminished expression of WNT16 in SLE may have a significant role in its pathogenesis through the regulation of cell proliferation and apoptosis.

The Role of Notch Signaling Pathway in Adult Patients with Epstein-Barr Virus-induced Infectious Mononucleosis

To investigate the changes of Notch signaling molecules and Th22 cells in adult patients with infectious mononucleosis (IM), and assess the regulatory function of Notch signaling inhibition to Th22 cells. Forty-two IM patients and twenty-one healthy controls were enrolled in this study. Their peripheral blood was collected, from which plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Plasma interleukin (IL)-17 and IL-22 were measured by enzyme-linked immunosorbent assay. The percentages of CD3+ CD4+ IL-17+ Th17 cells and CD3+ CD4+ IL-22+ Th22 cells were investigated by flow cytometry. The mRNA relative levels corresponding to Th17 transcription factor retinoic acid related orphan receptor γt (RORγt), Th22 transcription factor aryl hydrocarbon receptor (AhR), and Notch signaling pathway molecules (including Notch receptors, Notch ligands, Notch downstream molecules) were semi-quantified by real-time PCR. CD4+ T cells were purified and stimulated with γ-secretase inhibitor (GSI). Cellular proliferation, Th17 and Th22 percentage, IL-17 and IL-22 secretion, transcription factor mRNA were measured in response to GSI stimulation. The relative expression levels of Notch1 and Notch2 mRNA in PBMCs of IM group were 13.58±3.18 and 4.73±1.16, respectively, which were significantly higher than 1.09±0.12 and 1.07±0.15 in PBMCs of control group (both P < 0.001). However, there were no significant differences in relative expression levels of Notch3 and Notch4 mRNA between IM group and control group (P >0.05). The relative expression levels of Notch ligands (including DLL1 and Jagged1 ) mRNA and Notch downstream molecules (including Hes1, Hes5, and Hey1 ) were increased in IM group compared with control group (all P < 0.001). In IM group, the Th17 and Th22 percentage were 5.03%±1.15% and 4.48%±1.29%, respectively, which were both higher than 4.36%±0.82% and 3.83%±0.55% in control group (both P < 0.05). In IM group, the IL-17 and IL-22 level were (301.1±53.82) and (101.2±16.45) pg/ml, respectively, which were both higher than (237.2±72.18) and (84.75±11.83) pg/ml in control group (both P < 0.001). In IM group, the relative expression levels of RORγt and AhR mRNA were 1.25±0.22 and 1.21±0.12, respectively, which were both higher than 0.99±0.15 and 1.04±0.11 in control group (both P < 0.001). There were no remarkable differences in CD4+ T cell proliferation, Th17 percentage, IL-17 secretion, and relative expression level of RORγt mRNA between cells with GSI stimulation and without GSI stimulation (P >0.05). GSI stimulation reduced Th22 percentage, IL-22 secretion, and relative expression level of AhR mRNA compared with non-stimulation (all P < 0.05). Notch signaling pathway regulates IL-22 secretion by CD4+ T cells via AhR in IM patients. Notch-AhR-Th22 pathway may take part in the pathogenesis of IM.

Anti-inflammatory and immunoregulatory effects of colistin sulphate on human PBMCs.

In previous studies, CST has been identified as having an immunostimulatory effect on Caenorhabditis elegans and macrophage of rats. Here, we further investigated its immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). LPS-stimulated PBMCs inflammatory model was established. Flow cytometry was applied to measure phagocytosis of PBMCs. Cytokine mRNA and protein expression levels of LPS-stimulated PBMCs with or without CST were measured by qRT-PCR and ELISA. The transcriptomic profile of CST-treated PBMCs was investigated by RNA-sequencing. Gene Ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) were applied to find potential signalling pathways. PBMCs showed a significant increase in phagocytic activity at 6 h after being incubated with CST at the concentration of 10 μg/mL. In the presence of LPS, CST maintained and promoted the expression of TNF-α and chemokine CCL24. The content of pro-inflammatory cytokines, such as IL-1β, IL-6 and IFN-γ, which were released from LPS-stimulated PBMCs, was reduced by CST at 6 h. Anti-inflammatory cytokines, such as IL-4, IL-13 and TGF-β1, were significantly increased by CST at 24 h. A total of 277 differentially expressed immune-related genes (DEIRGs) were detected and cytokine-cytokine receptor interaction was highly enriched. CST presented obvious anti-inflammatory and immunoregulatory effects in LPS-induced PBMCs inflammatory model not only by improving the ability of PBMCs to clear pathogens but also by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. And the mechanism may be related to cytokine-cytokine receptor interaction.

Novel peripheral blood mononuclear cell mRNA signature for IFN-beta therapy responsiveness prediction in multiple sclerosis

Interferon-beta (IFN- β ) is one of the classical drugs for immunomodulatory therapy in relapsing-remitting multiple sclerosis (RRMS) patients, but the drug responsiveness of different patients varies. Currently, there is no valid model to predict IFN- β responsiveness. This research attempted to develop an IFN- β responsiveness prediction model based on mRNA expression in RRMS patient peripheral blood mononuclear cells. Peripheral blood mononuclear cell mRNA expression datasets including 50 RRMS patients receiving IFN- β treatment were obtained from GEO. Among the datasets, 24 cases from GSE24427 were included in a training set, and 18 and 9 cases from GSE19285 and GSE33464, respectively, were adopted as two independent test sets. In the training set, blood samples were collected immediately before first, second, month 1, 12, and 24 IFN- β injection, and the mRNA expression data at four time points, namely, two days, one month, one year and two years after the onset of IFN- β treatment, were compared with pre-treatment data to identify IFN-stimulated genes (ISGs). The ISGs at the one-month time point were used to construct the drug responsiveness prediction model. Next, the drug responsiveness model was verified in the two independent test sets to examine the performance of the model in predicting drug responsiveness. Finally, we used CIBERSORTx to estimate the content of cell subtypes in samples and evaluated whether differences in the proportions of cell subtypes were related to differences in IFN- β responsiveness. Among the four time points, one month was the time point when the training set GSE24427 and test set GSE33464 had the highest number of ISGs. Functional analysis showed that these one-month ISGs were enriched in biological functions such as the innate immune response, type-I interferon signalling pathway, and other IFN- β -associated functions. Based on these ISGs, we obtained a four-factor prediction model for IFN- β responsiveness including MX1, MX2, XAF1, and LAMP3. In addition, the model demonstrated favourable predictive performance within the training set and two external test sets. A higher proportion of activated NK cells and lower naive CD4/total CD4 ratio might indicate better drug responsiveness. This research developed a polygene-based biomarker model that could predict RRMS patient IFN- β responsiveness in the early treatment period. This model could probably help doctors screen out patients who would not benefit from IFN- β treatment early and determine whether a current treatment plan should be continued.

Abstract 6428: Transcriptome analysis of tissues and peripheral blood revealed potential biomarkers for chemo/radio-immunotherapy in non-small cell lung cancer

Abstract Background: Combination of anti-PD-(L)1 antibody with chemotherapy has shown promising efficacy in advanced/metastatic non-small cell lung cancer (NSCLC). Dynamics of peripheral blood mononuclear cells (PBMC) might have impact on repertore of immune cells further resulting different immune infiltration in tumor microenvironment. Thus, monitoring changes in PBMC during therapy or measuring tumor microenvironment provides the possibility to predict the efficacy of tumor immunotherapy. Methods: Patients diagnosed with NSCLC (Stage III-IV)were enrolled in this study. Anti-PD-L1 and chemo/radio combination therapy was performed as adjuvant therapy. Tumor samples were obtained prior to adjuvant therapy PBMC samples were obtaind at multiple time points, including prior adjuvant therapy, 3 months after adjuvant therapy, and 3 months after adjuvant therapy. Tumor and PBMC mRNA sequencing were performed by Amoy Diagnostics Co Ltd. (Xiamen, China). Patients who achieved CR/PR/SD and PFS &amp;gt; 6 months were defined as well responders. Pathway enrichment and GSEA analyses were performed by clusterProfiler (version 4.0.5) on GO, KEGG, and from six different gene sets. Correlations between immune cellular signature ES scores and gene expression were estimated using Pearson correlation. Results: 28 patients were enrolled; mean age was 60.38±7.86 years, 27 (96.4%) patients were male. 21 (75.0%) patients were identified as responder and 7 (25.0%) were non-responder. In the baseline tumor samples, immune infiltration was more extensive in responders compared to non-responders. Cytotoxic lymphocytes, activated CD8+ T cell, and NK cells were significantly enriched in responders. High expression of CCL17 and down regulation of CTSG/PLK1/PRTN3, a signaling axis that inhibits histone H3 hydrolysis and promotes monocyte to macrophage differentiation, was significant in responders. Dynamic monitoring of PBMC before and after treatment showed significant upregulation of immune infiltration-related pathways in responders, including effector cells, NK cells and CD8+ T cells. In the non-responder group, the up-regulated pathways after treatment were mainly enriched in tumor growth rate, EMT, and matrix remodeling. Multi-time point analysis showed that CEACAM5/MKI67 level and tumor proliferation rate in non-responders were higher than those in responders at each time point after treatment. Conclusion: Baseline tumor transcripton signatures and dynamic changes in PBMC could be applied to develop novel biomarkers for immune-related therapy. Patients with stronger immune infiltration signals in baseline tumor samples and PBMC after treatment had better immune response. The upregulation of proliferation-related signals in PBMC predicts poor immune response. Citation Format: Jing Hu, Jianguo Zhang, Peng Cheng, Yanhua Chen, Changbin Zhu, Feilin Yi, Conghua Xie. Transcriptome analysis of tissues and peripheral blood revealed potential biomarkers for chemo/radio-immunotherapy in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6428.

Role of Ferroptosis in the Progression of COVID-19 and the Development of Long COVID.

To examine the role of ferroptosis on the pathogenesis and progression of COVID-19. A total of 127 patients who were hospitalized for COVID-19 were categorized into two groups according to the intensity of oxygen therapy (high-flow or low-flow). Clinical characteristics, laboratory parameters, plasma markers, and peripheral blood mononuclear cell (PBMC) markers were measured at baseline and one or two weeks after treatment. Telephone follow-up was performed 3 months after discharge to assess long COVID. Patients receiving high-flow oxygen therapy had greater levels of neutrophils; D-dimer; C reactive protein; procalcitonin; plasma protein levels of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), IL-17, and acyl-CoA synthetase long-chain family member 4 (ACSL4); and PBMC mRNA level of TNF-α; but had lower levels of lymphocytes and plasma glutathione peroxidase 4 (GPX4). There were negative correlations of plasma GPX4 and cystine/glutamate transporter-11 (SLC7A11) with TNF-α, IL-6, and IL-17, and positive correlations of ACSL4 with inflammatory markers in plasma and PBMCs. The plasma levels of TNF-α, IL-6, IL-17, and ACSL4 were significantly lower after treatment than at baseline, but there were higher post-treatment levels of lymphocytes, GPX4, and SLC7A11. Patients with long COVID had a lower baseline level of plasma SLC7A11. Ferroptosis is activated during the progression of COVID-19, and a low baseline level of a ferroptosis marker (SLC7A11) may indicate an increased risk for long COVID-19. Ferroptosis has potential as a clinical indicator of long COVID and as a therapeutic target.

Sex-Specific Variations in the mRNA Levels of Candidate Genes in Peripheral Blood Mononuclear Cells from Patients with Diabetes: A Multistep Study

ABSTRACT Background Type 2 diabetes (T2D) is one of the most prevalent diseases that also show sexual dimorphism in many different aspects. Objectives This study aimed to distinguish the mRNA expression of genes in peripheral blood mononuclear cells (PBMCs) in men or women with T2D using a multistep analysis. Methods A total of 95 patients with T2D were compared based on their sex in terms of clinical variables and mRNA expression in their PBMCs. Results Men with T2D had lower LDLC, HDLC, and HbA1c values in their blood, but greater creatinine levels. In men with T2D, TLR4, CCR2, NOX2, and p67phox mRNA expression was greater, but IL6 and NF-κB mRNA expression was lesser in PBMCs. There was a link between fasting plasma glucose (FPG), triglycerides, and hs-CRP, as well as COX1 mRNA in men with T2D. In women with T2D, FPG was associated with the mRNA expression of THBS1 and p67phox, as well as triglycerides and HDLC levels. We found the exclusive effect of FPG on HDLC, HbA1c, as well as p67phox mRNA in PBMCs of women with T2D. Analysis revealed the exclusive effect of FPG on hs-CRP and PAFR mRNA in PBMCs of men with T2D. FPG was shown to be associated with body mass index, hs-CRP, triglycerides, and COX1 mRNA in men with T2D, and with serum triglycerides, THSB1, and p67phox mRNA in women with T2D, according to network analysis. HbA1c was linked with NF-κB mRNA in women with T2D. Conclusions Using a multistep analysis, it was shown that network analysis outperformed traditional analytic techniques in identifying sex-specific alterations in mRNA gene expression in PBMCs of T2D patients. The development of sex-specific therapeutic approaches may result from an understanding of these disparities.

T-Cell Exhaustion in HIV-1/Hepatitis C Virus Coinfection Is Reduced After Successful Treatment of Chronic Hepatitis C.

T-cell responses during chronic viral infections become exhausted, which is reflected by upregulation of inhibitory receptors (iRs) and increased interleukin 10 (IL-10). We assessed 2 iRs-PD-1 (programmed cell death protein 1) and Tim-3 (T-cell immunoglobulin and mucin domain-containing protein 3)-and IL-10 mRNAs in peripheral blood mononuclear cells (PBMCs) and their soluble analogs (sPD-1, sTim-3, and IL-10) in plasma in chronic HIV-1/hepatitis C virus (HCV) coinfection and explored the effect of HCV treatment on these markers. We also aimed to establish whether iR expression may be determined by the HCV CD8+ T-cell immunodominant epitope sequence. Plasma and PBMCs from 31 persons with chronic HIV-1/HCV coinfection from the Swiss HIV Cohort Study were collected before and after HCV treatment. As controls, 45 persons who were HIV-1 negative with chronic HCV infection were recruited. Exhaustion markers were assessed by enzyme-linked immunosorbent assay in plasma and by quantitative reverse transcription polymerase chain reaction in PBMCs. Analysis of an HCV epitope sequence was conducted by next-generation sequencing: HLA-A*02-restricted NS31073-1081 and NS31406-1415 and HLA-A*01-restricted NS31436-1444. The study revealed higher plasma sPD-1 (P = .0235) and IL-10 (P = .002) levels and higher IL-10 mRNA in PBMCs (P = .0149) in HIV-1/HCV coinfection. A decrease in plasma sPD-1 (P = .0006), sTim-3 (P = .0136), and IL-10 (P = .0003) and Tim-3 mRNA in PBMCs (P = .0210) was observed following successful HCV treatment. Infection with the HLA-A*01-restricted NS31436-1444 ATDALMTGY prototype variant was related to higher sTim-3 levels than infection with the ATDALMTGF escape variant (P = .0326). The results underscore the synergistic effect of coinfection on expression of exhaustion markers, their reduction following successful HCV treatment and imply that iR levels may operate on an epitope-specific manner.

Inflammation induces α1-adrenoceptor expression in peripheral blood mononuclear cells of patients with complex regional pain syndrome

Persistent regional and systemic inflammation may promote pain and hyperalgesia in complex regional pain syndrome (CRPS). In this study, we investigated whether stimulation of α1-adrenoceptors (α1-AR) on peripheral blood mononuclear cells (PBMC) might contribute to this inflammatory state. PBMC were isolated from venous blood collected from 21 CRPS patients and 21 sex and age-matched controls. Lipopolysaccharide (LPS), a bacterial toxin, was administered to cultured PBMC for 24 h to trigger inflammation. Exposure to LPS resulted in heightened gene expression of α1-AR subtype B (α1B-AR) in PBMC of CRPS patients relative to controls. Interleukin (IL)-1β and IL-6 levels did not change when the α1-AR agonist phenylephrine was administered to naïve PBMC. However, α1-AR stimulation following LPS treatment increased IL-6 mRNA and protein levels in PBMC of patients and controls. To investigate the possible consequence of heightened IL-6 levels on immunoglobulin G antibody production, PBMC were stimulated with CD40 ligand and IL-21 to generate plasmablasts (B cells that secrete antibodies). This response was similar in patients and controls. Adding IL-6 to the cell culture medium increased plasmablast differentiation in controls and antibody production both in patients and controls. These findings suggest that the inflammatory cascade associated with elevated levels of IL-6 may generate α1B-AR expression in CRPS PBMC. A reciprocal interaction between heightened α1-AR expression in PBMC and IL-6 secretion may contribute to systemic inflammation and antibody production in CRPS.

Profile and clinical significance of interferon gamma-inducible protein-10 (IP-10) and its receptor in patients with hepatocellular carcinoma.

Chemokines play a vital role in tumor progression, metastasis and prognosis; however, the profile and clinical significance of gamma interferon-inducible protein-10 (IP-10) and its receptor (CXCR3) in patients with hepatocellular carcinoma (HCC) have not been well evaluated. Liquid-phase chip technology was used to detect the serum IP-10 in 85 patients with HBV-related HCC, 50 patients with chronic hepatitis B (CHB) and 50 liver cirrhosis subjects (CS); simultaneously, the CXCR3 and Alpha fetoprotein (AFP) were determined. Additionally, their mRNA or protein expression levels in peripheral blood mononuclear cells (PBMC), liver tumor and paracancerous tissues were quantified using qRT-PCR or ELISA. Moreover, the IP-10 and CXCR3 expression was verified by the online data from Gene Expression Omnibus. Furthermore, the relationships of serum IP-10, CXCR3 and AFP levels with their overall survival rate were also analyzed. The levels of IP-10 and CXCR3 in HCC group were significantly higher than those in CHB and CS groups, and their mRNA of PBMC is significantly positive correlation with those in their liver tissues or HBV DNA load (P < 0.0001), respectively. The serum IP-10 and CXCR3 in HCC were significantly correlated with tumor differentiation, metastases staging and distant metastasis (P < 0.05), but not related to gender, age and tumor size (P > 0.05, except IP-10 based on age). The serum IP-10 (142.6 pg/mL) and CXCR3 (241.2 pg/mL) could be differential diagnostic surrogates that distinguish HCC from CS, and the lower IP-10 level may be conducive to the postoperative survival of HCC patients. Moreover, the IP-10 and CXCR3 would be related to anti-tumor immunity in HCC patients and be a potential target for treatment of HCC.

Triple motif proteins 19 and 38 correlated with treatment responses and HBsAg clearance in HBeAg-negative chronic hepatitis B patients during peg-IFN-α therapy

ObjectiveTo investigate whether the expression of triple motif protein 19/38 (TRIM19/38) mRNA in peripheral blood mononuclear cells (PBMCs) of HBeAg-negative chronic hepatitis B virus (HBV) carriers is associated with the response to pegylated interferon alpha (peg-IFN-α) treatment and HBsAg clearance.MethodsIn this prospective study, HBeAg-negative chronic HBV carriers treated with peg-IFN-α completed 48 weeks of follow-up. After treatment with peg-IFN-α, the patients were divided into responders (R group) and nonresponders (NR group) according to the changes in HBV DNA and HBsAg levels at week 48 of treatment. According to whether serum HBsAg loss or seroconversion occurred, the patients were divided into a serological response group (SR group) and a nonserological response group (NSR group). The level of TRIM19/38 mRNA in PBMCs was detected by real-time fluorescence quantitative PCR. The diagnostic performance of TRIM19/38 was analysed by calculating the receiver operating characteristic (ROC) curve and area under the ROC curve (AUC).Results43 HBeAg-negative chronic HBV carriers, 35 untreated CHB patients and 19 healthy controls were enrolled in this study. We found that TRIM19/38 mRNA levels were significantly lower in untreated CHB patients than in healthy controls. In HBeAg-negative chronic HBV carriers who underwent prospective follow-up, TRIM19/38 mRNA levels were negatively correlated with HBV DNA and ALT at baseline. Among the patients treated with peg-IFN-α, 16 patients achieved a treatment response (R group) and 27 patients did not achieve a treatment response (NR group). Compared with baseline, HBsAg levels in the R group decreased significantly at 12 and 24 weeks of treatment; at the early stage of peg-IFN-α treatment, the dynamic changes in TRIM19/38 mRNA levels in the R and NR groups were different, and the TRIM19/38 mRNA levels in the R group were significantly higher than those in the NR group, especially at 24 weeks of treatment. ROC curve analysis showed that the changes in mRNA levels of TRIM19 and TRIM38 predicted the treatment response, with AUCs of 0.694 and 0.757, respectively. Among the patients treated with peg-IFN-α, 11 patients achieved a serological response (SR group) and 32 patients did not achieve a serological response (NSR group). Compared with baseline, HBsAg levels in the SR group decreased significantly at 12 and 24 weeks of treatment; TRIM19/38 mRNA levels were significantly higher in the SR group than in the NSR group at week 24.ConclusionThe higher level of TRIM19/38 mRNA in PBMCs of HBeAg-negative chronic HBV carriers may be related to the early treatment effect of peg-IFN-α and HBsAg clearance. TRIM19 and TRIM38 have clinical significance in predicting virological response and guiding treatment regimens.

The Expression of Fibrogenic Cytokines by Human Peripheral Blood Mononuclear Cells in Response to SARS-CoV-2 Spike Protein

SARS-CoV-2, a single stranded positive RNA-virus, emerged in 2019 and caused a global pandemic. Some infected patients develop severe complications, such as acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. The mechanisms leading to lung fibrosis in the context of COVID-19 are still unclear. However, there are correlations between this condition and certain inflammatory mediators that are elevated in the plasma of patients. This study addressed the question of whether SARS-CoV-2 spike protein was able to directly activate immune cells to produce inflammatory and fibrogenic cytokines, independent of viral infection. By stimulating peripheral blood mononuclear cells (PBMC) from healthy blood donors through different formulations of SARS-CoV-2 spike protein, the expression of fibrogenic and inflammatory cytokines was measured. The spike protein induced a significantly increased expression of IL-1β and IL-6 mRNA in PBMC. Both cytokines are important players in the COVID-19 cytokine storm, in ARDS and in the development of pulmonary fibrosis. Different receptors with an affinity to the spike protein may be involved. However, the exact mechanism on how the spike protein leads to a higher cytokine expression in PBMC needs further investigation.

#5607 EFFECTS OF COMMON OSMOLYTES ON THE EXPRESSION OF FOXP3 SPLICE VARIANTS IN HUMAN PBMCS

Abstract Background and Aims Regulatory T cells modulate autoimmune and allogenic responses in glomerulonephritis and transplantation. The two most abundant fork-head box P3 (FOXP3) splice variants, full-length FOXP3 (FOXP3-FL) and FOXP3 lacking exon 2 (FOXP3-Δ 2), confer suppressive ability to regulatory T cells. Some studies showed a weaker suppressive ability of FOXP3-Δ 2. Renal organic osmolytes, urea and Trimethylamine N-oxide (TMAO), play critical roles in the hyperosmotic renal environment with high concentrations of Na+ and glucose. However, the effects of these osmolytes on FOXP3 splice variants in peripheral blood mononuclear cells (PBMCs) remain unclear. The aim of this study was to evaluate the effects of common osmolytes on the expression of FOXP3 splice variants in human PBMCs. Method PBMCs were isolated from the blood of healthy donors using Ficoll density gradients and cultured in RPMI-1640 with or without additional osmolytes at 37°C. A part of PBMCs were activated with 25ng/ml Phorbol-12-myristate-13-acetate (PMA) and 1ug/ml ionomycin (PMA+IONO). FOXP3-FL and FOXP3-Δ 2 mRNA were detected by qPCR after 4hrs’ incubation, using specific primers. The relative expression value was calculated by the formula of (1+E0)∧ Ct0(β-actin)/(1+E)∧ Ct (target gene). Values are given as the mean±S.E.M. Statistical significance was determined by One-Way ANOVA with Post Hoc Test, using Graphpad Prism 9. A P-value &amp;lt;0.05 was considered significant. Results The cell viabilities after 4 hours’ incubation were all above 90%. PMA+IONO significantly increased the relative expression of both FOXP3-Δ 2 (from 0.001051 to 0.001609, P = 0.0289, n = 5) and FOXP3-FL (from 0.001024 to 0.001684, P = 0.0101, n = 5) (Figure 1). However, increased concentrations of urea (from 5mM to 50mM) had no effect on either of the two FOXP3 splice variants (P &amp;gt; 0.05, n = 5, Figure 1). TMAO of 4um significantly reduced FOXP3-FL in the PMA+IONO-activated PBMCs from 0.001605 to 0.0007208 (P = 0.0051, Figure 2). However, TMAO didn’t affect FOXP3-FL expression in non-activated PBMCs and had little effect on FOXP3-Δ 2 expression either (P &amp;gt; 0.05, n = 4). On the other hand, compared with control group, the high concentration of glucose (25mM) significantly increased the expression of FOXP3-Δ 2 from 0.002815 to 0.003461 (P = 0.0375, n = 3) in non-activated PBMCs (Figure 2). The high concentration of NaCl (147mM) had no significant effect on either of the FOXP3 splice variants (P &amp;gt; 0.05, n = 3, Figure 2). Furthermore, the equal osmolarity of mannitol didn’t affect the mRNA expressions of FOXP3 splice variants, either (P &amp;gt; 0.05, n = 3, Figure 2). But compared with glucose group (0.003461), the expression of FOXP3-Δ 2 was significantly lower in mannitol group (0.002785) (P = 0.0479, n = 3, Figure 2). It suggested that the effect of high glucose in non-activated PBMCs was independent of the osmolarity change. Conclusion In isolated human PBMCs, PMA+IONO increases both FOXP3-Δ 2 and FOXP3-FL expressions. PMA+IONO-induced FOXP3-FL increase can be inhibited by TMAO. Urea and NaCl had little effect on FOXP3 splice expressions. High concentration of glucose increases FOXP3-Δ 2 mRNA in non-activated PBMCs, which was independent of the osmolarity change.

Decreased SIRT1 mRNA expression in peripheral blood mononuclear cells from patients with neuromyelitis optica spectrum disorders.

Sirtuin (SIRT)1, as a molecular link between immunity and metabolic pathways, is a key immune response regulator. The significance of SIRT1 in peripheral blood mononuclear cells (PBMCs) of neuromyelitis optica spectrum disorder (NMOSD) has not been investigated. Here, we aimed to evaluate the SIRT1 mRNA level in PBMCs of patients with NMOSD and its clinical relevance and explore the potential mechanism of SIRT1 action. A total of 65 patients with NMOSD and 60 normal controls from North China were enrolled. Using real-time fluorescence quantitative-polymerase chain reaction, mRNA levels were detected in PBMCs, and protein levels were detected using western blotting. Compared to the healthy controls and chronic-phase patients with NMOSD, SIRT1 mRNA and protein levels in PBMCs of NMOSD patients with acute attack were significantly downregulated (p < 0.0001). ∆EDSS scores (EDSS scores in the acute phase-EDSS scores before the recent attack) were higher in NMOSD patients with low SIRT1 mRNA level than in patients with high SIRT1 expression (p = 0.042). SIRT1 mRNA level in patients with acute-phase NMSOD was positively correlated with lymphocyte and monocyte counts and negatively correlated with neutrophil counts and the neutrophil-to-lymphocyte ratio. Furthermore, the transcription factor FOXP3 mRNA level was significantly positively correlated with the SIRT1 mRNA level in PBMCs of patients with acute-phase NMOSD. Our study indicated that SIRT1 mRNA expression was downregulated in the PBMCs of patients with acute-phase NMOSD, and its level was correlated with the clinical parameters of the patients, suggesting a potential role of SIRT1 in NMOSD.

T Lymphocyte Subsets Profile and Toll-Like Receptors Responses in Patients with Herpes Zoster.

Herpes zoster (HZ) is caused by the varicella-zoster virus (VZV), and 20% of healthy humans and 50% of people with immune dysfunction have a high probability of suffering from HZ. This study aimed to screen dynamic immune signatures and explore the potential mechanism during HZ progression. Peripheral blood samples from 31 HZ patients and 32 age-sex-matched healthy controls were collected and analyzed. The protein levels and gene levels of toll-like receptors (TLRs) were detected in peripheral blood mononuclear cells (PBMCs) by flow cytometry and quantitative real-time PCR. Further, the characteristics of T cell subsets and cytokines were detected via a cytometric bead array. Compared to healthy controls, the mRNA levels of TLR2, TLR4, TLR7, and TLR9 mRNA in PBMCs were significantly increased in HZ patients. The protein level of TLR4 and TLR7 was significantly increased in HZ patients, but the levels of TLR2 and TLR9 were dramatically decreased. The CD3+ T cells were constant in HZ and healthy controls. CD4+ T cells were decreased in HZ patients, while CD8+ T cells were increased, resulting in an improved CD4+/CD8+ T cells ratio. Further, it was found that Th2 and Th17 were not changed, but the decreased Th1 and upregulated Treg cells were found in HZ. The Th1/Th2 and Th17/Treg ratios were significantly decreased. Last, the levels of IL-6, IL-10, and IFN-γ were significantly increased, but IL-2, IL-4, and IL-17A had no significant changes. The dysfunction of host's lymphocytes and activation of TLRs in PBMCs were the important mechanism in varicella-zoster virus induced herpes zoster. TLRs might be the core targets for the therapy drug development in treating HZ.

Association of interleukin 33 gene polymorphisms with susceptibility and regulation of inflammatory mediators in Systemic lupus erythematosus patients.

Studies have indicated the involvement of interleukin (IL)-33 in the pathogenesis of Systemic lupus erythematosus (SLE). This research intended to evaluate the association of IL33 gene rs1929992 and rs7044343 Single nucleotide polymorphisms (SNPs) with risk of SLE. In addition, the association between these SNPs and inflammatory cytokines was determined. In this study, 200 SLE cases and 200 healthy subjects were recruited. Using allelic discrimination Real-time PCR, IL33 gene rs1929992 and rs7044343 SNPs were genotyped. The mRNA expression levels of IL-1β, IL-6, IL-33, TNF-α were determined in the peripheral blood mononuclear cells (PBMCs). The serum levels of cytokines were also measured. The G allele (OR = 1.57, CI: 1.18-2.08, P = 0.0017), GG genotype (OR = 2.52, CI: 1.33-4.77, P = 0.0043), and GA genotype (OR = 2.12, CI: 1.34-3.34, P = 0.0011) of rs1929992 SNP was significantly associated with an increased SLE risk. The C allele (OR = 1.44, CI: 1.08-1.90; P = 0.0105), CC genotype (OR = 2.07, CI: 1.15-3.71; P = 0.0146), and CT genotype (OR = 1.61, CI: 1.02-2.53, P = 0.0395) of rs7044343 was significantly associated with increased SLE risk. The PBMC mRNA expression and serum levels of IL-1β, IL-6, IL-33, TNF-α were significantly increased in the SLE patients compared to controls. However, there was no significant difference in the mRNA expression and serum levels of IL-1β, IL-6, IL-33, and TNF-α among the SLE patients with three genotypes for both rs1929992 and rs7044343 polymorphisms. IL33 gene rs1929992 and rs7044343 SNPs are involved in SLE pathogenesis but they might not influence on the inflammatory pathway.

TGF-β is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells

BackgroundSoluble urate leads to a pro-inflammatory phenotype in human monocytes characterized by increased production of IL-1β and downregulation of IL-1 receptor antagonist, the mechanism of which remains to be fully elucidated. Previous transcriptomic data identified differential expression of genes in the transforming growth factor (TGF)-β pathway in monocytes exposed to urate in vitro. In this study, we explore the role of TGF-β in urate-induced hyperinflammation in peripheral blood mononuclear cells (PBMCs).MethodsTGF-β mRNA in unstimulated PBMCs and protein levels in plasma were measured in individuals with normouricemia, hyperuricemia and gout. For in vitro validation, PBMCs of healthy volunteers were isolated and treated with a dose ranging concentration of urate for assessment of mRNA and pSMAD2. Urate and TGF-β priming experiments were performed with three inhibitors of TGF-β signalling: SB-505124, 5Z-7-oxozeaenol and a blocking antibody against TGF-β receptor II.ResultsTGF-β mRNA levels were elevated in gout patients compared to healthy controls. TGF-β-LAP levels in serum were significantly higher in individuals with hyperuricemia compared to controls. In both cases, TGF-β correlated positively to serum urate levels. In vitro, urate exposure of PBMCs did not directly induce TGF-β but did enhance SMAD2 phosphorylation. The urate-induced pro-inflammatory phenotype of monocytes was partly reversed by blocking TGF-β.ConclusionsTGF-β is elevated in individuals with hyperuricemia and correlated to serum urate concentrations. In addition, the urate-induced pro-inflammatory phenotype in human monocytes is mediated by TGF-β signalling. Future studies are warranted to explore the intracellular pathways involved and to assess the clinical significance of urate-TGF-β relation.

P66Shc is increased in peripheral blood mononuclear cells of the patients with obstructive sleep apnea.

Objective: Obstructive sleep apnea (OSA) is characterized by nocturnal intermittent hypoxemia and linked to oxidative stress. Evidence demonstrated that p66Shc plays a key role in regulating oxidative stress. This study aimed to investigate the expression of p66Shc in peripheral blood mononuclear cells (PBMCs) of patients with OSA and the association with polysomnographic parameters. Methods: Fifty-four OSA subjects and 19 no OSA controls were enrolled in this study. All the subjects underwent standard polysomnography. P66Shc mRNA and protein levels in the PBMCs were detected by quantitative real-time polymerase chain reaction and western blotting. Plasma 3-nitrotyrosine (3-NT), oxidized low density lipoprotein (oxLDL), and advanced oxidation protein products (AOPP) were measured by ELISA method. Results: P66Shc mRNA and protein levels in PBMCs were significantly higher in OSA patients than in controls. P66Shc mRNA was positively correlated with plasma 3-NT, oxLDL, AOPP, hypopnea index (AHI), oxygen desaturation index (ODI), percentage of total sleep time with oxygen saturation (SaO2) below 90% (CT90), epworth sleepiness scale (ESS) and lymphocytes; negatively correlated with lowest SaO2 (LSaO2) and mean SaO2 (MSaO2). Further multivariate linear regression analysis showed that p66Shc mRNA levels were independently associated with AHI, MSaO2 and CT90. Conclusions: Oxidative stress regulator p66Shc may play a role in the pathophysiology of OSA and might serve as a potential biomarker for this disease.