Control Peripheral Blood Mononuclear Cells Research Articles

Expression of HMGB1, TGF-β1, BIRC3, ADAM17, CDKN1A, and FTO in Relation to Left Ventricular Remodeling in Patients Six Months after the First Myocardial Infarction: A Prospective Study.

Background: After myocardial infarction (MI), adverse left ventricular (LV) remodeling may occur. This is followed by LV hypertrophy and eventually heart failure. The remodeling process is complex and goes through multiple phases. The aim of this study was to investigate the expression of HMGB1, TGF-β1, BIRC3, ADAM17, CDKN1A, and FTO, each involved in a specific step of LV remodeling, in association with the change in the echocardiographic parameters of LV structure and function used to assess the LV remodeling process in the peripheral blood mononuclear cells (PBMCs) of patients six months after the first MI. The expression of selected genes was also determined in PBMCs of controls. Methods: The study group consisted of 99 MI patients, who were prospectively followed-up for 6 months, and 25 controls. Cardiac parameters, measured via conventional 2D echocardiography, were evaluated at two time points: 3-5 days and 6 months after MI. The mRNA expression six-months-post-MI was detected using TaqMan® technology (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Results:HMGB1 mRNA was significantly higher in patients with adverse LV remodeling six-months-post-MI than in patients without adverse LV remodeling (p = 0.04). HMGB1 mRNA was significantly upregulated in patients with dilated LV end-diastolic diameter (LVEDD) (p = 0.03); dilated LV end-diastolic volume index (LVEDVi) (p = 0.03); severely dilated LV end-systolic volume index (LVESVi) (p = 0.006); impaired LV ejection fraction (LVEF) (p = 0.01); and LV enlargement (p = 0.03). It was also significantly upregulated in PBMCs from patients compared to controls (p = 0.005). TGF-β1 and BIRC3 mRNA were significantly lower in patients compared to controls (p = 0.02 and p = 0.05, respectively). Conclusions: Our results suggest that HMGB1 is involved in adverse LV remodeling six-months-post-MI, even on the mRNA level. Further research and validation are needed.

Immunomodulatory Effect of Adipose Stem Cell-Derived Extra-Cellular Vesicles on Cytokine Expression and Regulatory T Cells in Patients with Asthma.

Although mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are as effective as MSCs in the suppression of allergic airway inflammation, few studies have evaluated the immunomodulatory capacity of MSC-derived EVs in patients with asthma. Thus, we assessed the effects of adipose stem cell (ASC)-derived EVs on cytokine expression and regulatory T cells (Tregs) in peripheral blood mononuclear cells (PBMCs) of asthmatic patients. PBMCs (1 × 106 cells/mL) were isolated from asthmatic patient and healthy controls and co-cultured with 1 μg/mL of ASC-derived EVs. Th (T helper) 1-, Th2-, and Treg-related cytokine expression, fluorescence-activated cell sorting analysis of CD4+CD25+FOXP3+ T cells, and co-stimulatory molecules were analyzed before and after ASC-derived EV treatment. The expression levels of IL-4 and costimulatory molecules such as CD83 and CD86 were significantly higher in PBMCs of asthmatic patients than in control PBMCs. However, ASC-derived EV treatment significantly decreased the levels of interleukin (IL)-4 and co-stimulatory molecules such as CD83 and CD86 in the phytohemagglutinin (PHA)-stimulated PBMC of asthmatic patients. Furthermore, ASC-derived EVs remarkably increased the transforming growth factor-β (TGF-β) levels and expression of Tregs in the PBMC of asthmatic patients. ASC-derived EVs induce Treg expansion and have immunomodulatory effects by downregulating IL-4 and upregulating TGF-β in PBMCs of asthmatic patients.

Influenza Vaccination Mediates SARS-CoV-2 Spike Protein Peptide-Induced Inflammatory Response via Modification of Histone Acetylation.

The effectiveness of coronavirus disease 2019 (COVID-19) vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain rapidly wanes over time. Growing evidence from epidemiological studies suggests that influenza vaccination is associated with a reduction in the risk of SARS-CoV-2 infection and COVID-19 severity. However, the underlying mechanisms remain elusive. Here, we investigate the cross-reactive immune responses of influenza vaccination to SARS-CoV-2 spike protein peptides based on in vitro study. Our data indicate enhanced activation-induced-marker (AIM) expression on CD4+ T cells in influenza-vaccination (IV)-treated peripheral blood mononuclear cells (PBMCs) upon stimulation with spike-protein-peptide pools. The fractions of other immune cell subtypes, including CD8+ T cells, monocytes, NK cells, and antigen-presenting cells, were not changed between IV-treated and control PBMCs following ex vivo spike-protein-peptide stimulation. However, the classical antiviral (IFN-γ) and anti-inflammatory (IL-1RA) cytokine responses to spike-protein-peptide stimulation were still enhanced in PBMCs from both IV-immunized adult and aged mice. Decreased expression of proinflammatory IL-1β, IL-12p40, and TNF-α is associated with inhibited levels of histone acetylation in PBMCs from IV-treated mice. Remarkably, prior immunity to SARS-CoV-2 does not result in modification of histone acetylation or hemagglutinin-protein-induced cytokine responses. This response is antibody-independent but can be mediated by manipulating the histone acetylation of PBMCs. These data experimentally support that influenza vaccination could induce modification of histone acetylation in immune cells and reveal the existence of potential cross-reactive immunity to SARS-CoV-2 antigens, which may provide insights for the adjuvant of influenza vaccine to limit COVID-19-related inflammatory responses.

In vitro cell killing proficiency of autologous immune cells educated with allogenic multi-cancer tumor cell lysate.

e14675 Background: Allogenic tumor lysate therapies offer a novel approach in oncology, to initiate a measured immune system response to combat cancer. Loaded with tumor antigens, these lysates stimulate the patient's immune system for targeted cancer cell destruction. In vitro data suggesting efficacy is pivotal prior to clinical trials. Per-C-Vax is a novel, organ-agnostic, allogenic tumor lysate immuno-stimulant, and is intended to target solid organ malignancies via subcutaneous injection. It is derived from a multi-organ cell-line cocktail, which has been induced into apoptosis to capture diverse Tumor Associated Molecular Patterns (TAMPs) and Damage Associated Molecular Patterns (DAMPs). Our study assesses Per-C-Vax-educated patient-specific immunocytes' in-vitro cell-killing activity against their autologous tumor cells. Methods: We collected simultaneous blood and tumor tissue samples from 22 patients with solid organ tumors under approved protocols. The cohort included 10 head and neck (45%), 4 breast (18%), and 8 other cancers (36%) comprising colorectal, ovary, lung, gallbladder, prostate, uterine, cervix, and gastric cancers. Peripheral Blood Mononuclear Cells (PBMCs) from a 10 ml venous draw were split into control and Per-C-Vax incubated aliquots for each patient. Approximately 1,000 tumor cells, stained with Calcein AM, were co-cultured with about 20,000 cells from each of the Per-C-Vax and control PBMC aliquots (autologous). Imaging-based pre- and post-co-culture counts determined the cell-kill rate for each aliquot. Results: PBMCs not treated with Per C Vax showed a significantly reduced cancer cell killing activity against autologous tumor cells compared to Per-C-Vax -treated PBMCs, suggesting the tolerogenic characteristic of immunocytes to the malignancy (0%-32%, 95% CI 10.16 to 16.65). On the other hand, PBMCs treated with Per-C-Vax showed significant cell-kill activity ranging from 46% to 84% in head and neck (mean 62.1%, 95% CI 52.67 to 71.53), 51% to 73% in breast (mean 62%, 95% CI 44.33 to 79.67), 38% to 87% in other cancers (colorectal, ovary, lung, gallbladder, prostate, uterine, cervix, gastric) (mean 50.87%, 95% CI 37.71 to 64.04). A comparative analysis between the untreated group and the group treated with Per-C-Vax revealed a statistically significant result with a p-value <0.005. Conclusions: Patient-specific PBMCs incubated with Per-C-Vax tumor lysate immuno-stimulant show significant autologous cancer cell killing activity against multiple cancers in vitro. Human trials to establish safety and efficacy are planned.

Circular RNA Expression of Peripheral Blood Mononuclear Cells Associated with Risk of Acute Exacerbation in Smoking Chronic Obstructive Pulmonary Disease.

Circular RNAs (circRNAs) are newly identified endogenous non-coding RNAs that function as crucial gene modulators in the development of several diseases. By assessing the expression levels of circRNAs in peripheral blood mononuclear cells (PBMCs) from patients with chronic obstructive pulmonary disease (COPD), this study attempted to find new biomarkers for COPD screening. We confirmed altered circRNA expression in PBMCs of COPD (n=41) vs controls (n=29). Further analysis focused on the highest and lowest circRNA expression levels. The T-test is used to assess the statistical variances in circRNAs among COPD patients in the smoking and non-smoking cohorts. Additionally, among smokers, the Spearman correlation test assesses the association between circRNAs and clinical indicators. Two circRNAs, hsa_circ_0042590 and hsa_circ_0049875, that were highly upregulated and downregulated in PBMCs from COPD patients were identified and verified. Smokers with COPD had lower hsa_circ_0042590 and higher hsa_circ_0049875, in comparison to non-smokers. There was a significant correlation (r=0.52, P<0.01) between the number of acute exacerbations (AEs) that smokers with COPD experienced in the previous year and the following year (r=0.67, P<0.001). Moreover, hsa_circ_0049875 was connected to the quantity of AEs in the year prior (r=0.68, P<0.0001) as well as the year after (r=0.72, P<0.0001). AUC: 0.79, 95% CI: 0.1210-0.3209, P<0.0001) for hsa_circ_0049875 showed a strong diagnostic value for COPD, according to ROC curve analysis. Hsa_circ_0042590 showed a close second with an AUC of 0.83 and 95% CI: -0.1972--0.0739 (P <0.0001). This research identified a strong correlation between smoking and hsa_circ_0049875 and hsa_circ_0042590 in COPD PBMCs. The number of AEs in the preceding and succeeding years was substantially linked with the existence of hsa_circ_0042590 and hsa_circ_0049875 in COPD patients who smoke. Additionally, according to our research, hsa_circ_0049875 and hsa_circ_0042590 may be valuable biomarkers for COPD diagnosis.

F-box protein 43 promoter methylation as a novel biomarker for hepatitis B virus-associated hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a high prevalence and poor prognosis worldwide. Therefore, it is urgent to find effective and timely diagnostic markers. The objective of this study was to evaluate the diagnostic value of F-box protein 43 promoter methylation in peripheral blood mononuclear cells (PBMCs) for HCC. A total of 247 participants were included in this study, comprising individuals with 123 hepatitis B virus-associated HCC, 79 chronic hepatitis B, and 45 healthy controls. F-box protein 43 methylation and mRNA levels in PBMCs were detected by MethyLight and quantitative real-time PCR. F-box protein 43 promoter methylation levels were significantly lower in HCC PBMCs than the chronic hepatitis B (P < 0.001) and healthy control PBMCs (P < 0.001). Relative mRNA expression levels of F-box protein 43 in HCC PBMCs were significantly higher than those in chronic hepatitis B (P < 0.001) and healthy control PBMCs (P < 0.001). Receiver operating characteristic analysis of F-box protein 43 promoter methylation levels yielded an area under curve (AUC) of 0.793 with 76.42% sensitivity and 68.35% specificity when differentiating HCC from chronic hepatitis. These values for the F-box protein 43 promoter methylation level were superior to those of the alpha-fetoprotein serum (AFP) level (AUC: 0.780, sensitivity: 47.97%, and specificity: 96.20%), with increments in values for the combination of F-box protein 43 promoter methylation AFP levels (AUC: 0.888, sensitivity: 76.42%, and specificity: 86.08%). Hypomethylation of the F-box protein 43 promoter in PBMCs is a promising biochemical marker for HBV-associated HCC.

Proteomic analysis of lysine 2-hydroxyisobutyryl in SLE reveals protein modification alteration in complement and coagulation cascades and platelet activation Pathways

BackgroundPost-translational modifications (PTMs) are considered to be an important factor in the pathogenesis of Systemic lupus erythematosus (SLE). Lysine 2-hydroxyisobutyryl (Khib), as an emerging post-translational modification of proteins, is involved in some important biological metabolic activities. However, there are poor studies on its correlation with diseases, especially SLE.ObjectiveWe performed quantitative, comparative, and bioinformatic analysis of Khib proteins in Peripheral blood mononuclear cells (PBMCs) of SLE patients and PBMCs of healthy controls. Searching for pathways related to SLE disease progression and exploring the role of Khib in SLE.MethodsKhib levels in SLE patients and healthy controls were compared based on liquid chromatography tandem mass spectrometry, then proteomic analysis was conducted.ResultsCompared with healthy controls, Khib in SLE patients was up-regulated at 865 sites of 416 proteins and down-regulated at 630 sites of 349 proteins. The site abundance, distribution and function of Khib protein were investigated further. Bioinformatics analysis showed that Complement and coagulation cascades and Platelet activation in immune-related pathways were significantly enriched, suggesting that differentially modified proteins among them may affect SLE.ConclusionKhib in PBMCs of SLE patients was significantly up- or down-regulated compared with healthy controls. Khib modification of key proteins in the Complement and coagulation cascades and Platelet activation pathways affects platelet activation and aggregation, coagulation functions in SLE patients. This result provides a new direction for the possible significance of Khib in the pathogenesis of SLE patients.

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.

IAPs and RIPK1 mediate LPS-induced cytokine production in healthy subjects and Crohn's disease.

Innate immune activity fuels intestinal inflammation in Crohn's disease (CD), an inflammatory bowel disease. Identification and targeting of new molecular regulators of the innate activity are warranted to control the disease. Inhibitor of apoptosis proteins (IAPs) regulate both cell survival and inflammatory signaling. We investigated the effects of IAP inhibition by second mitochondria-derived activator of caspases (SMAC) mimetics (SMs) on innate responses and cell death to pathogen-associated molecular patterns in peripheral blood mononuclear cells (PBMCs) and monocytes. IAPs inhibited lipopolysaccharide (LPS)-induced expression of proinflammatory interleukin (IL)-1β, IL-6. Likewise, LPS (but not muramyl dipeptide or Escherichia coli) induced TNF-α was inhibited in CD and control PBMCs. The SM effect was partially reversed by inhibition of receptor-interacting serine/threonine-protein kinase 1 (RIPK1). The effect was mainly cell death independent. Thus, IAP inhibition by SMs leads to reduced production of proinflammatory cytokines and may be considered in the efforts to develop new therapeutic strategies to control CD.

G-CSF-mobilized peripheral blood mononuclear cells combined with platelet-rich plasma restored the ovarian function of aged rats

BackgroundRegenerative medicine with peripheral blood mononuclear cell (PBMC) transplantation sheds light on the issue of premature ovarian insufficiency (POI). However, the efficiency of PBMC treatment in natural ovarian aging (NOA) remains unclear. MethodsThirteen-month-old female Sprague–Dawley (SD) rats were used to verify the NOA model. Seventy-two NOA rats were randomly divided into three groups: the NOA control group, PBMC group, and PBMC+platelet-rich plasma (PRP) group. PBMCs and PRP were transplanted by intraovarian injection. The effects on ovarian function and fertility were measured after transplantation. ResultsTransplantation of PBMCs could restore the normal estrous cycle, consistent with the recovery of serum sex hormone levels, increased follicle numbers at all stages, and restoration of fertility by facilitating pregnancy and live birth. Moreover, when combined with PRP injection, these effects were more significant. The male-specific SRY gene was detected in the ovary at all four time points, suggesting that PBMCs continuously survived and functioned in NOA rats. In addition, after PBMC treatment, the expression of angiogenesis-related and glycolysis-related markers in the ovaries was upregulated, which indicated that these effects were associated with angiogenesis and glycolysis. ConclusionsPBMC transplantation restores the ovarian functions and fertility of NOA rats, and PRP could enhance the efficiency. Increased ovarian vascularization, follicle production, and glycolysis are likely the major mechanisms.

Interleukin family (IL-2 and IL-1β) as predictive biomarkers in Indian cancer patients: A proof of concept study

The immune system protects and defends its host against disease through immune cells. Immune cells such as T lymphocytes play a critical role in disease and health by producing key cytokines (pro-inflammatory TH1 or anti-inflammatory TH2). The inflammatory cytokines produced by immune cells such as peripheral blood mononuclear cells (PBMCs) may suppress the immune state or can lead to disease progression. In our study, we evaluated the functionality of resting PBMCs as well as phytohaemagglutinin mitogen (PHA-M) stimulated PBMCs in cancer patients and healthy controls in the Indian population. PHA-M was used as an activator of PBMCs and served as an index to assess the ability of PBMCs in both cohorts to respond to a superantigen stimulus. We hypothesized that key cytokines (TH1 and TH2 cytokines) could serve as biomarkers to assess the immune status of cancer patients based on the functionality of PBMCs primarily in resting conditions and their ability to respond to PHA-M. In our study, we found that PBMCs from cancer patients showed a marked difference in their resting and activated cytokine profiles with respect to Interleukin-2 (IL-2) and Interleukin-1β (IL-1β) as compared to their healthy counterparts. The study also demonstrated changes in Interleukin-10 (IL-10) profiles which require further study. These cytokines may serve as a potential predictive biomarker and provide the basis to form an immune index for each individual – a tool that could be used to prescribe treatment and therapies for cancer patients.

The regulatory function of tumor-infiltrating Th9 cells to anti-tumor activity of CD8(+) T cells in patients with gastric cancer

Objective: To investigate the levels of Th9 cells and interleukin-9 (IL-9), and to assess the regulatory activity of Th9/IL-9 to anti-tumor immune response in patients with gastric cancer. Methods: Thirty-four patients with gastric cancer who received operation in the First Affiliated Hospital of Xinxiang Medical University between October 2018 and August 2019 were included. Twenty individuals who received physical examination in the same period were also enrolled. Peripheral blood was collected, and then plasma and peripheral blood mononuclear cells (PBMCs) were isolated. Tumor-infiltrating lymphocytes (TILs) and autologous gastric cancer cells were isolated from resected gastric cancer tissues. CD4(+) T cells, CD8(+) T cells, and CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells were purified from PBMCs and TILs. Plasma IL-9 level was measured by enzyme linked immunosorbent assay (ELISA). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs and TILSs was assessed by flow cytometry. The mRNA levels of IL-9 and transcriptional factors purine-rich nucleic acid binding protein 1 (PU.1) were semi-quantified by real-time quantitative polymerase chain reaction (RT-qPCR). PBMCs and TILs from gastric cancer patients were stimulated with recombinant human IL-9. Cellular proliferation was measured by cell counting kit-8. The phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and STAT6 were investigated by western blot. Cytokine production was measured by ELISA. Purified CD8(+) T cells from TILs of gastric cancer patients were stimulated with recombinant human IL-9. CD8(+) T cells and autologous gastric cancer cells were cocultured in direct contact and indirect contact manner. The percentage of target cell death was calculated by measuring the lactate dehydrogenase (LDH) level. These cretion of γ-Interferon (γ-IFN) and tumor necrosis factor-α (TNF-α) was measured by ELISA. CD4(+) CCR4(-)CCR6(-)CXCR3(-)cells, CD8(+) T cells, and autologous gastric cancer cells were directly cocultured, and anti-IL-9 neutralizing antibody was added. The target cell death was measured. Results: The percentages of CD3(+) CD4(+) IL-9(+) Th9 cells in PBMCs of control group and PBMCs of gastric cancer group were (1.21±0.25)% and (1.14±0.19)%, respectively. The difference was not statistically significant (P=0.280). The percentage of CD3(+) CD4(+) IL-9(+) Th9 cells in TILs of gastric cancer group was (2.30±0.55)%, which was higher than those in PBMCs of control group and PBMCs of gastric cancer group (P<0.001). The plasma IL-9 level in control group and gastric cancer group were (5.04±1.51) and (4.93±1.25) ng/ml. The difference was not statistically significant (P=0.787). The relative levels of IL-9 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.33±0.39 and 1.36±0.27. The difference was not statistically significant (P=0.691). The relative level of IL-9 mRNA in TILs of gastric cancer group was 2.90±0.75, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). The relative levels of PU.1 mRNA in PBMCs of control group and PBMCs of gastric cancer group were 1.21±0.12 and 1.20±0.11. The difference was not statistically significant (t=0.21, P=0.833). PU.1 mRNA relative level in TILs of gastric cancer group was 2.81±0.65, which was higher than those in PBMCs of control group (P<0.001) and PBMCs of gastric cancer group (P<0.001). Recombinant human IL-9 stimulation did not affect the proliferation of PBMCs and TILs of gastric cancer patients (P>0.05), but elevated the phosphorylation level of STAT6 and induced the secretions of γ-IFN, IL-17, and IL-22 by TILs (P<0.05). In direct contact culture system, IL-9 stimulation promoted tumor-infiltrating CD8(+) T cells-induced autologous gastric cancer cell death [(20.62±2.27)% vs. (16.08±2.61)%, P<0.01)]. In indirect contact culture system, IL-9 stimulation did not increase CD8(+) T cell-induced autologous gastric cancer cell death [(5.21±0.70)% vs. (5.31±1.22)%, P=0.998)]. However, the secretion levels of γ-IFN were elevated in response to IL-9 stimulation in both culture systems [direct contact culture system: (100.40±12.05) pg/ml vs. (76.45±8.56) pg/ml; indirect contact culture system: (78.00±9.98) pg/ml vs. (42.09±10.71) pg/ml; P<0.01]. The TNF-α secretion level did not significantly changed (P>0.05). In direct contact culture system, the percentage of target cells was (22.01±3.05) % and γ-IFN secretion level was (104.5±12.84) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group, which was higher than (16.08±2.61)% and (76.45±8.56) pg/ml in CD8(+) T cells+ gastric cancer cells group (P<0.01). However, the percentage of target cells was (14.47±3.14)% and γ-IFN secretion level was (70.45±19.43) pg/ml in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells+ anti-IL-9 neutralizing antibody group, which were lower than those in CD4(+) CCR4(-)CCR6(-)CXCR3(-) cells+ CD8(+) T cells+ gastric cancer cells group (P<0.01). Conclusion: Tumor-infiltrating Th9 cells and the secreting IL-9 promote the activity of CD8(+) T cells in gastric cancer patients, and enhance anti-tumor immune response.

Ex Vivo Treatment with Allogenic Mesenchymal Stem Cells of a Healthy Donor on Peripheral Blood Mononuclear Cells of Patients with Severe Alopecia Areata: Targeting Dysregulated T Cells and the Acquisition of Immunotolerance.

Alopecia areata (AA) is an autoimmune condition related to the collapse of the immune privilege of hair follicles. Certain AA populations present severe clinical manifestations, such as total scalp hair or body hair loss and a treatment refractory property. The aim of this study was to assess the effects of allogenic human mesenchymal stem cells (hMSCs) from healthy donors on the peripheral blood mononuclear cells (PBMCs) of severe AA patients, with a focus on the change in the cell fraction of Th1, Th17, and Treg cells and immunomodulatory functions. PBMCs of 10 AA patients and eight healthy controls were collected. Levels of Th17, Th1, and Treg subsets were determined via flow cytometry at baseline, activation status, and after co-culturing with hMSCs. All participants were severe AA patients with SALT &gt; 50 and with a long disease duration. While the baseline Th1 and Treg levels of AA patients were comparable to those of healthy controls, their Th17 levels were significantly lower than those of the controls. When stimulated, the levels of CD4+IFN-γ+ T cells of the AA patients rose sharply compared to the baseline, which was not the case in those of healthy controls. The cell fraction of CD4+Foxp3+ regulatory T cells also abruptly increased in AA patients only. Co-culturing with allogenic hMSCs in activated AA PBMCs slightly suppressed the activation levels of CD4+INF-γ+ T cells, whereas it significantly induced the differentiation of CD4+Foxp3+ regulatory T cells. However, these changes were not prominent in the PBMCs of health controls. To examine the pathomechanisms, PBMCs of healthy donors were treated with IFN-γ to induce AA-like environment and then treated with allogenic grants and compared with ruxolitinib as a positive treatment control. hMSC treatment was shown to significantly inhibit the mRNA levels of proinflammatory cytokines, such as IFN-γ, TNF-α, IL-1α, IL-2R, IL-15, and IL-18, and chemokines, such as CCR7 and CCR10, in IFN-treated PBMCs. Interestingly, hMSCs suppressed the activation of JAK/STAT signaling by IFN in PBMCs with an effect that was comparable to that of ruxolitinib. Furthermore, the hMSC treatment showed stronger efficacy in inducing Foxp3, IL-10, and TGF-β mRNA transcription than ruxolitinib in IFN-treated PBMCs. This study suggests that allogenic hMSC treatments have therapeutic potential to induce immune tolerance and anti-inflammatory effects in severe AA patients.

Intrauterine interventions for women with two or more implantation failures: A systematic review and network meta-analysis.

ObjectiveTo compare the effectiveness of different intrauterine interventions for women with two or more unexplained implantation failures.DesignA systematic review and network meta-analysis of randomized controlled trials (RCTs).Patient(s)Women with two or more implantation failures undergoing fresh or frozen embryo transfer (ET).Intervention(s)An electronic search of the following databases: Pubmed, Cochrane Central Register of Controlled Trials (CENTRAL), and Embase.Main Outcome Measure(s)Clinical pregnancy, live birth/ongoing pregnancy, and miscarriage.Result(s)We included 21 RCTs(3079 women) in the network meta-analysis. The network meta-analysis showed that compared with control treatment, platelet-rich plasma(PRP), peripheral blood mononuclear cells (PBMC), granulocyte colony-stimulating factor(G-CSF), human chorionic gonadotropin(HCG), and endometrial scratch(ES) significantly increased clinical pregnancy(OR 3.78, 95% CI 2.72 to 5.25; 2.79, 95% CI 1.75 to 4.45; 1.93, 95% CI 1.37 to 2.72; 1.80, 95% CI 1.18 to 2.72; 1.75, 95% CI 1.29 to 2.36, respectively). PRP ranked the highest in improving clinical pregnancy, followed by PBMC, G-CSF, HCG, and ES. Compared with control treatment, PRP, PBMC, and ES significantly increased live birth/ongoing pregnancy (OR 5.96, 95% CI 3.38 to 10.52; OR 2.55, 95% CI 1.27 to 5.11; OR 1.70, 95% CI 1.07 to 2.69, respectively). PRP ranked the highest in improving live birth/ongoing pregnancy, followed by PBMC, and ES.Conclusion(s)PRP is the most effective intrauterine intervention in improving pregnancy outcome in women with two or more implantation failures.

Phosphatase control of cytokine-mediated overproduction of galactose-deficient IgA1, the main autoantigen in IgA nephropathy

IgA nephropathy (IgAN) is an autoimmune disease characterized by the deposition of galactose-deficient IgA1 (Gd-IgA1)-containing immune complexes in the kidneys. Elevated serum levels of Gd-IgA1, the main autoantigen in IgAN, are associated with mucosal infections and poor renal outcome in IgAN patients, but little is known about the activation of IgA1-secreting cells overproducing this autoantigen. We found that in peripheral blood mononuclear cells (PBMCs), cytokine stimulation elevated Gd-IgA1 production in B cells from IgAN patients but not in those from healthy controls (p < 0.01). These results were replicated in immortalized B cells derived from PBMCs of IgAN patients and healthy controls. Using single-cell transcriptomics, we identified subsets of IgA1-secreting cells from IgAN patients, but not from healthy controls, with decreased expression of C1GALT1 in response to cytokine stimulation. The C1GALT1-encoded glycosyltransferase is responsible for addition of galactose to IgA1 O-glycans, and its reduced activity is associated with elevated serum levels of Gd-IgA1. These newly identified subsets of IgA1-secreting cells with reduced C1GALT1 expression exhibited reduced expression of several genes related to cytokine-mediated signaling, including those encoding phosphatases, such as SOCS1. siRNA knock-down of SOCS1, and the related SOCS3, increased Gd-IgA1 production in cells derived from PBMCs of healthy controls, indicating a role of these regulators in abnormal cytokine signaling and Gd-IgA1 overproduction. These results revealed that specific subsets of IgA1-secreting cells may be responsible for autoantigen production in IgAN due to abnormal regulation of cytokine-mediated signaling, a process that may occur in inflammatory responses in IgAN patients.

Regulatory T cells in erythema nodosum leprosum maintain anti-inflammatory function

The numbers of circulating regulatory T cells (Tregs) are increased in lepromatous leprosy (LL) but reduced in erythema nodosum leprosum (ENL), the inflammatory complication of LL. It is unclear whether the suppressive function of Tregs is intact in both these conditions. A longitudinal study recruited participants at ALERT Hospital, Ethiopia. Peripheral blood samples were obtained before and after 24 weeks of prednisolone treatment for ENL and multidrug therapy (MDT) for participants with LL. We evaluated the suppressive function of Tregs in the peripheral blood mononuclear cells (PBMCs) of participants with LL and ENL by analysis of TNFα, IFNγ and IL-10 responses to Mycobacterium leprae (M. leprae) stimulation before and after depletion of CD25+ cells. 30 LL participants with ENL and 30 LL participants without ENL were recruited. The depletion of CD25+ cells from PBMCs was associated with enhanced TNFα and IFNγ responses to M. leprae stimulation before and after 24 weeks treatment of LL with MDT and of ENL with prednisolone. The addition of autologous CD25+ cells to CD25+ depleted PBMCs abolished these responses. In both non-reactional LL and ENL groups mitogen (PHA)-induced TNFα and IFNγ responses were not affected by depletion of CD25+ cells either before or after treatment. Depleting CD25+ cells did not affect the IL-10 response to M. leprae before and after 24 weeks of MDT in participants with LL. However, depletion of CD25+ cells was associated with an enhanced IL-10 response on stimulation with M. leprae in untreated participants with ENL and reduced IL-10 responses in treated individuals with ENL. The enhanced IL-10 in untreated ENL and the reduced IL-10 response in prednisolone treated individuals with ENL was abolished by addition of autologous CD25+ cells. The findings support the hypothesis that the impaired cell-mediated immune response in individuals with LL is M. leprae antigen specific and the unresponsiveness can be reversed by depleting CD25+ cells. Our results suggest that the suppressive function of Tregs in ENL is intact despite ENL being associated with reduced numbers of Tregs. The lack of difference in IL-10 response in control PBMCs and CD25+ depleted PBMCs in individuals with LL and the increased IL-10 response following the depletion of CD25+ cells in individuals with untreated ENL suggest that the mechanism of immune regulation by Tregs in leprosy appears independent of IL-10 or that other cells may be responsible for IL-10 production in leprosy. The present findings highlight mechanisms of T cell regulation in LL and ENL and provide insights into the control of peripheral immune tolerance, identifying Tregs as a potential therapeutic target.

Trained Immunity in Primary Sjögren's Syndrome: Linking Type I Interferons to a Pro-Atherogenic Phenotype.

BackgroundTrained immunity – or innate immune memory – can be described as the long-term reprogramming of innate immune cells towards a hyperresponsive state which involves intracellular metabolic changes. Trained immunity has been linked to atherosclerosis. A subgroup of patients with primary Sjögren’s syndrome (pSS) exhibits systemic type I interferon (IFN) pathway activation, indicating innate immune hyperactivation. Here, we studied the link between type I IFNs and trained immunity in an in vitro monocytic cell model and peripheral blood mononuclear cells (PBMCs) from pSS patients.MethodsThe training stimuli heat killed Candida albicans, muramyl dipeptide, IFNβ, and patient serum were added to THP-1 cells for 24 hours, after which the cells were washed, rested for 48 hours and subsequently re-stimulated with LPS, Pam3Cys, poly I:C, IFNβ or oxLDL for 4-24 hours. PBMCs from pSS patients and healthy controls were stimulated with LPS, Pam3Cys, poly I:C or IFNβ for 0.5-24 hours.ResultsTraining with IFNβ induced elevated production of pro-atherogenic cytokines IL-6, TNFα and CCL2, differential cholesterol- and glycolysis-related gene expression, and increased glucose consumption and oxLDL uptake upon re-stimulation. Type I IFN production was increased in Candida albicans- and IFNβ-trained cells after LPS re-stimulation, but was reduced after poly I:C re-stimulation. Training with muramyl dipeptide and IFNβ, but not Candida albicans, affected the IFN-stimulated gene expression response to IFNβ re-stimulation. PBMCs from pSS patients consumed more glucose compared with healthy control PBMCs and tended to produce more TNFα and type I IFNs upon LPS stimulation, but less type I IFNs upon poly I:C stimulation.ConclusionsType I IFN is a trainer inducing a trained immunity phenotype with pro-atherogenic properties in monocytes. Conversely, trained immunity also affects the production of type I IFNs and transcriptional response to type I IFN receptor re-stimulation. The phenotype of pSS PBMCs is consistent with trained immunity. This connection between type I IFN, trained immunity and cholesterol metabolism may have important implications for pSS and the pathogenesis of (subclinical) atherosclerosis in these patients.

Mechanism of Mycobacterium tuberculosis on interleukin-6 receptor 3'-untranslated region methylation in CD4+T cells

Objective: To investigate the role and mechanism of DNA methylation in Mycobacterium tuberculosis (MTB lysate) -induced downregulation of interleukin-6 receptor(IL-6R) expression in CD4+T cells. Methods: A prospective study was conducted. Bisulfite sequencing (BSP) was applied to determine the methylation levels of CpG island in IL-6R promoter region and 3'untranslated region (3'UTR) region in CD4+T cells from peripheral blood mononuclear cells (PBMC) of control group (healthy person, n=10) and TB group (tuberculosis patients, n=10) in Shenzhen Third People's Hospital between 2019 and 2020. Quantitative reverse transcription-PCR (RT-qPCR) and Western blotting were used to detect the expression of IL-6R, DNMT1, DNMT3A and DNMT3B in MTB lysate-stimulated CD4+T cells and Jurkat E6-1 cells. Furthermore, PBMC in control group and Jurkat E6-1 cells activated by anti-CD3/CD28 antibody were stimulated by MTB lysates to detect the methylation levels of CpG island and IL-6R and DNMT expression. Transcriptional activity of differently methylation regions of IL-6R 3'UTR was detected by using luciferase reporter gene system. Results: IL-6R expression in TB group was lower than that in control group, but DNMT1 and DNMT3B expressions were higher than those in control group in CD4+T cells isolated from PBMC. There was no significant difference in the methylation rate of IL-6R promoter CpG island of CD4+T cells between control and TB group. However, the methylation rates of CpG island in 3'UTR region were significantly higher (P<0.001) in TB (69.5%±3.4%), compared with control (54.3%±4.7%). Besides, IL-6R expression was lower than unstimulated, while DNMT1 and DNMT3B expression was higher than unstimulated after MTB lysate-stimulation of activated control PBMC in vitro. The methylation rate of CpG island in IL-6R 3'UTR region of CD4+T cells increased from 58.8%±11.6% to 79.4%±10.9% (P<0.001) after MTB lysate-stimulated PBMC of the control. The same results were observed in the MTB lysate-stimulated CD4+T cells isolated from PBMC in control and Jurkat E6-1 cell line. Furthermore, IL-6R expression after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysate was higher than that stimulated by MTB lysate alone. In addition, the methylation levels of CpG islands in the 3' UTR region of IL-6R were lower than those stimulated by MTB lysates alone after co-treatment of the DNA methyltransferase inhibitor decitabine (5-aza) with MTB lysates. The transcriptional activity of the fully unmethylated IL-6R 3'UTR CpG island reporter gene was higher than that of the fully methylated IL-6R 3'UTR CpG island. Conclusions: MTB lysates stimulation inhibited IL-6R expression transcriptionalely as well as on the protein level by inducing hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells. The hypermethylation of CpG island in IL-6R 3'UTR region of CD4+T cells induced by MTB may be related to the increased expression of DNMT1 and DNMT3B.

CFTR-mediated monocyte/macrophage dysfunction revealed by cystic fibrosis proband-parent comparisons.

Cystic fibrosis (CF) is an inherited disorder caused by biallelic mutations of the CF transmembrane conductance regulator (CFTR) gene. Converging evidence suggests that CF carriers with only 1 defective CFTR copy are at increased risk for CF-related conditions and pulmonary infections, but the molecular mechanisms underpinning this effect remain unknown. We performed transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) of CF child-parent trios (proband, father, and mother) and healthy control (HC) PBMCs or THP-1 cells incubated with the plasma of these participants. Transcriptomic analyses revealed suppression of cytokine-enriched immune-related genes (IL-1β, CXCL8, CREM), implicating lipopolysaccharide tolerance in innate immune cells (monocytes) of CF probands and their parents. These data suggest that a homozygous as well as a heterozygous CFTR mutation can modulate the immune/inflammatory system. This conclusion is further supported by the finding of lower numbers of circulating monocytes in CF probands and their parents, compared with HCs, and the abundance of mononuclear phagocyte subsets, which correlated with Pseudomonas aeruginosa infection, lung disease severity, and CF progression in the probands. This study provides insight into demonstrated CFTR-related innate immune dysfunction in individuals with CF and carriers of a CFTR mutation that may serve as a target for personalized therapy.

Chicken Peripheral Blood Mononuclear Cells Response to Avian Leukosis Virus Subgroup J Infection Assessed by Single-Cell RNA Sequencing.

Chicken peripheral blood mononuclear cells (PBMCs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Here we performed single-cell RNA sequencing (scRNA-seq) of PBMCs in Avian leukosis virus subgroup J (ALV-J) infected and control chickens at 21 days post infection (DPI) to determine chicken PBMCs subsets and their specific molecular and cellular characteristics. Eight cell populations and their potential marker genes were identified in PBMCs. T cell populations had the strongest response to (ALV-J) infection, based on the detection of the largest number of differentially expressed genes (DEGs), and could be further grouped into four subsets: activated CD4+ T cells, Th1-like cells, Th2-like cells, and cytotoxic CD8+ T cells. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection activated CD4+ T cell was probably inclined to differentiate into Th1-like cells. Compared to the control PBMCs, ALV-J infection also had an obvious impact on PBMCs composition. B cells showed inconspicuous response and their numbers decreased in PBMCs from ALV-J infected chicken. Proportions of cytotoxic Th1-like cells and CD8+ T cells increased in the T cell population of PBMCs from ALV-J infected chicken, which were potentially key mitigating effectors against ALV-J infection. More importantly, our results provide a rich resource of gene expression profiles of chicken PBMCs subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.