Peripheral Blood Mononuclear Cells Of Healthy Controls Research Articles

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.

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.

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.

Ex vivo generation of regulatory T cells from liver transplant recipients using costimulation blockade

The potential of adoptive cell therapy with regulatory T cells (Tregs) to promote transplant tolerance is under active exploration. However, the impact of specific transplant settings and protocols on Treg manufacturing is not well-delineated. Here, we compared the use of peripheral blood mononuclear cells (PBMCs) from patients before or after liver transplantation to the use of healthy control PBMCs to determine their suitability for Treg manufacture using ex vivo costimulatory blockade with belatacept. Despite liver failure or immunosuppressive therapy, the capacity for Treg expansion during the manufacturing process was preserved. These experiments did not identify performance or quality issues that disqualified the use of posttransplant PBMCs-the currently favored protocol design. However, as Treg input correlated with output, significant CD4-lymphopenia in both pre- and posttransplant patients limited Treg yield. We therefore turned to leukapheresis posttransplant to improve absolute yield. To make deceased donor use feasible, we also developed protocols to substitute splenocytes for PBMCs as allostimulators. In addition to demonstrating that this Treg expansion strategy works in a liver transplant context, this preclinical study illustrates how characterizing cellular input populations and their performance can both inform and respond to clinical trial design and Treg manufacturing requirements.

Hyperresponsive cytosolic DNA-sensing pathway in monocytes from primary Sjögren's syndrome.

ObjectivesCytosolic DNA-sensing pathway stimulation prompts type I IFN (IFN-I) production, but its role in systemic IFN-I pathway activation in primary SS (pSS) is poorly studied. Here we investigate the responsiveness of pSS monocytes and plasmacytoid dendritic cells (pDCs) to stimulator of interferon genes (STING) activation in relation to systemic IFN-I pathway activation and compare this with SLE.MethodsExpression of DNA-sensing receptors cGAS, IFI16, ZBP-1 and DDX41, signalling molecules STING, TBK1 and IRF3, positive and negative STING regulators, and IFN-I-stimulated genes MxA, IFI44, IFI44L, IFIT1 and IFIT3 was analysed in whole blood, CD14+ monocytes, pDCs, and salivary glands by RT-PCR, monocyte RNA sequencing data, flow cytometry and immunohistochemical staining. Peripheral blood mononuclear cells (PBMCs) from pSS, SLE and healthy controls (HCs) were stimulated with STING agonist 2′3′-cGAMP. STING phosphorylation (pSTING) and intracellular IFNα were evaluated using flow cytometry.ResultsSTING activation induced a significantly higher proportion of IFNα-producing monocytes, but not pDCs, in both IFN-low and IFN-high pSS compared with HC PBMCs. Additionally, a trend towards more pSTING+ monocytes was observed in pSS and SLE, most pronounced in IFN-high patients. Positive STING regulators TRIM38, TRIM56, USP18 and SENP7 were significantly higher expression in pSS than HC monocytes, while the dual-function STING regulator RNF26 was downregulated in pSS monocytes. STING was expressed in mononuclear infiltrates and ductal epithelium in pSS salivary glands. STING stimulation induced pSTING and IFNα in pSS and SLE pDCs.ConclusionpSS monocytes and pDCs are hyperresponsive to stimulation of the STING pathway, which was not restricted to patients with IFN-I pathway activation.

Preservation of antigen-specific responses in cryopreserved CD4+ and CD8+ T cells expanded with IL-2 and IL-7

ObjectivesWe sought to develop medium throughput standard operating procedures for screening cryopreserved human peripheral blood mononuclear cells (PBMCs) for CD4+ and CD8+ T cell responses to potential autoantigens. MethodsDendritic cells were loaded with a peptide cocktail from ubiquitous viruses or full-length viral protein antigens and cocultured with autologous T cells. We measured expression of surface activation markers on T cells by flow cytometry and cytometry by time of flight 24–72 h later. We tested responses among T cells freshly isolated from healthy control PBMCs, cryopreserved T cells, and T cells derived from a variety of T cell expansion protocols. We also compared the transcriptional profile of CD8+ T cells rested with interleukin (IL)7 for 48 h after 1) initial thawing, 2) expansion, and 3) secondary cryopreservation/thawing of expanded cells. To generate competent antigen presenting cells from PBMCs, we promoted differentiation of PBMCs into dendritic cells with granulocyte macrophage colony stimulating factor and IL-4. ResultsWe observed robust dendritic cell differentiation from human PBMCs treated with 50 ng/mL GM-CSF and 20 ng/mL IL-4 in as little as 3 days. Dendritic cell purity was substantially increased by magnetically enriching for CD14+ monocytes prior to differentiation. We also measured antigen-dependent T cell activation in DC-T cell cocultures. However, polyclonal expansion of T cells with anti-CD3/antiCD28 abolished antigen-dependent upregulation of CD69 in our assay despite minimal transcriptional differences between rested CD8+ T cells before and after expansion. Furthermore, resting these expanded T cells in IL-2, IL-7 or IL-15 did not restore the antigen dependent responses. In contrast, T cells that were initially expanded with IL-2 + IL-7 rather than plate bound anti-CD3 + anti-CD28 retained responsiveness to antigen stimulation and these responses strongly correlated with responses measured at initial thawing. SignificanceWhile screening techniques for potential pathological autoantibodies have come a long way, comparable full-length protein target assays for screening patient T cells at medium throughput are noticeably lacking due to technical hurdles. Here we advance techniques that should have broad applicability to translational studies investigating cell mediated immunity in infectious or autoimmune diseases. Future studies are aimed at investigating possible CD8+ T cell autoantigens in MS and other CNS autoimmune diseases.

Targeting MyD88 Downregulates Inflammatory Mediators and Pathogenic Processes in PBMC From DMARDs-Naïve Rheumatoid Arthritis Patients

MyD88-dependent intracellular signalling cascades and subsequently NF-kappaB-mediated transcription lead to the dynamic inflammatory processes underlying the pathogenesis of rheumatoid arthritis (RA) and related autoimmune diseases. This study aimed to identify the effect of the MyD88 dimerization inhibitor, ST2825, as a modulator of pathogenic gene expression signatures and systemic inflammation in disease-modifying antirheumatic drugs (DMARDs)-naïve RA patients. We analyzed bulk RNA-seq from peripheral blood mononuclear cells (PBMC) in DMARDs-naïve RA patients after stimulation with LPS and IL-1β. The transcriptional profiles of ST2825-treated PBMC were analyzed to identify its therapeutic potential. Ingenuity Pathway Analysis was implemented to identify downregulated pathogenic processes. Our analysis revealed 631 differentially expressed genes between DMARDs-naïve RA patients before and after ST2825 treatment. ST2825-treated RA PBMC exhibited a gene expression signature similar to that of healthy controls PBMC by downregulating the expression of proinflammatory cytokines, chemokines and matrix metalloproteases. In addition, B cell receptor, IL-17 and IL-15 signalling were critically downregulated pathways by ST2825. Furthermore, we identified eight genes (MMP9, CXCL9, MZB1, FUT7, TGM2, IGLV1-51, LINC01010, and CDK1) involved in pathogenic processes that ST2825 can potentially inhibit in distinct cell types within the RA synovium. Overall, our findings indicate that targeting MyD88 effectively downregulates systemic inflammatory mediators and modulates the pathogenic processes in PBMC from DMARDs-naïve RA patients. ST2825 could also potentially inhibit upregulated genes in the RA synovium, preventing synovitis and joint degeneration.

T-cells of invasive candidiasis patients show patterns of T-cell-exhaustion suggesting checkpoint blockade as treatment option

Recent data imply that strengthening host immunity by checkpoint inhibition improves outcome in invasive fungal infections (IFI), particularly in candidiasis. To assess T-cell exhaustion in this context, we compared peripheral blood mononuclear cells (PBMCs) and serum samples of patients with invasive Candida albicans infection (IC, n=21) to PBMCs or tumor-infiltrating lymphocytes (TILs) from cancer patients (n=14) and PBMCs of healthy controls (n=20). Type and differentiation of lymphocytes and expression of 29 immune-regulatory molecules were analyzed by flow cytometry. C. albicans specific responses were assessed by FluoroSpot (n=8) and antibody measurement (n=14). Fractions and phenotypes of lymphocyte subsets in PBMCs of IC patients were similar compared to PBMCs of controls, while they were different in TILs. PBMCs of patients with IC showed increased expression of immune-checkpoint molecules. The pattern of upregulated molecules was similar to TILs, but not present in PBMCs of control cancer patients. Fractions of T-cells expressing PD-1 and TIGIT were higher in IC patients that died. FluoroSpot analysis showed a Candida-specific IFN-y or IL-2 response in 5/8 patients, enhanced by addition of nivolumab in vitro. Together with preclinical data and preliminary evidence of clinical efficacy in mucormycosis, our results support clinical evaluation of immune-checkpoint inhibition in IFI treatment. NCT04533087; retrospectively registered on August 31, 2020.

Prospective Evaluation of Thrombo-Hemorrhagic Events and Hypercoagulation Markers in Patients with Acute Promyelocytic Leukemia (APL) Receiving All-Trans Retinoic Acid (ATRA) Plus Chemotherapy

Introduction: Patients with APL typically present with a life-threatening thrombo-hemorrhagic syndrome (THS), due to the consumption of coagulation factors and platelets, secondary to clotting activation. Presently, all- trans retinoic acid (ATRA) has greatly improved the management of APL, but has not significantly changed the rate of early hemorrhagic deaths and may actually promote thrombosis.Characterizing the coagulopathy and identifying predictive markers of bleeding/clotting complications remain critical issues. Aim: In newly diagnosed APL patients, in the first month after diagnosis, we wanted prospectively to: 1. register thrombo/hemorrhagic events; 2. evaluate, the profiles of circulating hypercoagulation markers and the molecular expression of Tissue Factor (TF), before and during ATRA therapy; and 3. verify whether changes in the biomarker profiles correlate with the occurrence of thrombo-hemorragic events. Methods: Sixty consecutive APL patients receiving ATRA+Idarubicin for remission induction (GIMEMA protocol AIDA2000) were enrolled from January 2005 to March 2017. Blood samples from a subgroup of 30 patients (F/M= 11/19) were obtained at the onset (T0), and on days 7, 14 and 28 (T7, T14, T28) of remission induction therapy. Plasma levels of activated factor VII-antithrombin complex (FVIIa-AT), thrombin-antithrombin complex (TAT), and D-dimer were measured by ELISA; TF mRNA levels in peripheral blood mononuclear cells (PBMC) was evaluated in 16 APL patients by real time-polymerase chain reaction. Results: At enrolment (T0), before starting ATRA, 13.3% of APL patients presented with early major hemorrhages, including 3 (5%) fatal intracranial bleeding and 5 (8.3%) non-fatal major bleeding (i.e. gengivorrhagia, gastrointestinal bleeding, epistaxis, menorrhagia). At T0, the prevalence of thrombosis in the total group of 60 APL patients was 6.6%, including 1 fatal pulmonary embolism (PE), 2 non-fatal venous thrombosis (1 PE, 1 lower limb deep vein thrombosis [DVT]), and 1 non-fatal arterial thrombosis (acute myocardial infarction [AMI]). During ATRA therapy, 8.3% of APL patients developed major bleeding: including 2 fatal intracranial hemorrhage after 3 days of ATRA treatment and 3 non-fatal major bleeding (i.e. gengivorrhagia, epistaxis, menorrhagia) after 7, 10 and 17 days of ATRA. Two more patients developed non-fatal thrombosis (i.e. 1 AMI and 1 DVT) after 9 and 20 days of ATRA treatment, respectively. The laboratory sub-study of 30 subjects showed that, at T0, before starting therapy, the plasma levels of FVIIa-AT, TAT and D-dimer were significantly higher in APL patients compared to controls (p<0.05). During induction therapy with ATRA, the levels of TAT and D-dimer significantly (p<0.05) and progressively decreased at T7 and T14, reaching the lowest value at 28 days. Differently, FVIIa-AT levels were unchanged at T7 and T14, and dropped significantly only at T28 (p<0.05 vs T0). In addition, in APL PBMC from 16 patients, the levels of TF mRNA, initially elevated compared to healthy control PBMC (p<0.05), during induction therapy were significantly decreased by 68% and 90%, at T7 and T28, respectively. In these 16 APL patients, a statistically significant correlation (p<0.05) was found between decrease in TF mRNA and FVIIa-AT levels during ATRA therapy. Of the 30 patients included in the laboratory sub-study, 3 had non-fatal thrombosis at diagnosis, no major hemorrhages were recorded. The predictive value of THS of thrombotic markers could not be calculated. Conclusions: Our data show a significant rate (31%) of severe (fatal and non-fatal) thrombo/hemorrhagic events in our cohort of APL patients in the first 3 weeks of diagnosis, with 4 fatal events (6.6%) occurring early, before starting ATRA. Laboratory data show for the first time that, while TAT and D-dimer confirm to be sensitive to ATRA downregulation, differently FVIIa-AT levels persist at high level over time, showing the perseverance of TF-dependent clotting activation. Future large prospective clinical trials should include FVIIa-AT to evaluate persistent clotting activation during ATRA. DisclosuresFalanga:Advisory Board: Bayer, Janssen Speakers' bureau: Rovi, BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rambaldi:Novartis, Roche/Genentech, Amgen, Italfarmaco: Consultancy; Novartis, Amgen, Celgene, Sanofi: Other: Travel, Accomodations, Expenses. Lo Coco:Lundbeck: Honoraria, Speakers Bureau; TEVA: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.

Two distinct subsets of LDGs (low density granulocytes) in ANCA-associated vasculitis

Objectives and methods Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is an autoimmune disorder that causes vasculitis in small blood vessels throughout the body. Low-density granulocytes (LDGs) in autoimmune diseases, such as SLE and AAV, might play a critical role in the pathogenesis of these diseases. Here, we aimed to determine the characteristics of LDGs in patients with AAV. We assessed the number of whole white blood cells, neutrophil extracellular traps (NETs) productivity, proportion of cell surface markers (e.g. CD10), responses to immunosuppressants, and proteomics of LDGs in patients with AAV. Results We found more LDGs in peripheral blood mononuclear cells (PBMCs) of patients with AAV than PBMCs of healthy controls (HCs) and confirmed that these LDGs in AAV produced more NETs than normal density granulocytes (NDGs) in HCs. We identified CD10-positive LDGs with mature neutrophil features and CD10-negative LDGs with immature granulocyte properties; the proportion of the two LDG types decreased and increased, respectively, in the patients during treatment. Proteomic analysis revealed that the two LDG groups shared protein expression that differed from those of NDGs. Conclusion We identified distinct CD10-positive and CD10-negative LDGs in patients with AAV. The roles of these LDGs in AAV pathology will require further investigation.

Helios serves as a suppression marker to reduce regulatory T cell function in pancreatic cancer patients.

Destabilizing and reprogramming regulatory T (Treg) cells have become a potential strategy to treat tumor. Mounting evidence indicates that the transcription factor Helios is required for the stable differentiation of Treg lineage. Hence, we investigated whether Helios suppression could be a potential treatment option for pancreatic cancer patients. We found that Helios+ cells were predominantly in Foxp3+ Treg cells. By contrast, Foxp3+ Treg cells can be Helios+ or Helios-, but the level of Foxp3 expression was significantly higher in Helios+Foxp3+ Treg cells than in Helios-Foxp3+ Treg cells. Resected pancreatic tumors were highly enriched with both Helios+Foxp3+ Treg cells and Helios-Foxp3+ Treg cells. Also, the proportion of Helios+ cells in total Foxp3+ Treg cells was significantly higher in peripheral blood mononuclear cells (PBMCs) of patients than in PBMCs of healthy controls and further increased in patient tumors. Using shRNA, we knocked down Helios expression without significant downregulation of Foxp3. After Helios knockdown, CD4+CD25+CD127- Treg cells presented significantly lower levels of TGF-β secretion, lower levels of IL-10 secretion, and higher levels of IFN-γ secretion. In addition, Helios shRNA-transfected CD4+CD25+CD127- Treg cells presented lower capacity to inhibit CD4+CD25-CD127+ T conventional cell proliferation than control shRNA-transfected CD4+CD25+CD127- Treg cells. Of note, CD4+CD25+CD127- Treg cells from pancreatic cancer patients demonstrated higher TGF-β expression and higher suppression capacity than the cells from healthy controls. Overall, these results suggest that in pancreatic cancer patients, Helios may serve as a candidate to suppress Treg function, which could be used as a target to treat pancreatic cancer.

Elevated MicroRNA-326 Levels Regulate the IL-23/IL-23R/Th17 Cell Axis in Hashimoto's Thyroiditis by Targeting a Disintegrin and Metalloprotease 17.

Background: MicroRNAs (miRNAs) are a class of critical epigenetic regulators involved in several autoimmune diseases. Our previous study reported an miR-326-induced increase in T helper (Th) 17 cells in a mouse model of Hashimoto's thyroiditis (HT), but the pathogenic effect of miR-326 in HT patients has not been verified. The goal of the present study was to explore the pathogenic role of miR-326 and its underlying molecular mechanism in HT patients. Methods: A total of 58 HT patients and 55 normal controls were enrolled in this study. We examined whether Th17 cells and miR-326 were aberrantly altered in the peripheral blood mononuclear cells (PBMCs) of HT patients with flow cytometry and real-time polymerase chain reaction. Levels of membrane interleukin (IL)-23R (mIL-23R) were determined by flow cytometry and Western blot to explore the critical role of mIL-23R in the development of Th17 cells. Isolated CD3+ T cells were used to further investigate the ectodomain shedding of mIL-23R by a disintegrin and metalloprotease (ADAM17). Furthermore, miR-326 inhibitor and mimics were transfected into PBMCs derived from HT patients and healthy controls to verify the regulation of ADAM17 by miR-326. Results: We observed elevated miR-326 levels in the PBMCs of HT patients compared with those in the PBMCs of healthy controls. Consistent with IL-23-induced STAT3 overactivation, substantially more HT patient-derived PBMCs differentiated into Th17 cells under polarization culture conditions, which may, at least in part, have resulted from enhanced mIL-23R levels. Furthermore, ADAM17, an ectodomain sheddase of mIL-23R, was targeted and negatively regulated by miR-326. Inhibiting ADAM17 might attenuate the ectodomain shedding of mIL-23R. Conclusions: Our findings suggest that the effect of miR-326 on the IL-23/IL-23R/Th17 cell axis in HT patients might be partially due to the targeting of ADAM17.

Ex vivo immunological evaluation of stable mixed chimeric patients after matched related donor allogeneic transplantation in sickle cell disease

Background aimsAllogeneic hematopoietic stem cell transplantation is curative for sickle cell disease, and the use of matched related donors, non-myeloablative conditioning and sirolimus immunosuppression results in stable mixed chimerism without graft-versus-host disease (GVHD). However, the time to terminate sirolimus while maintaining mixed chimerism is unclear. MethodsIn this study, we developed a two-way mixed lymphocyte reaction (MLR) to evaluate ex vivo immunoreaction in mixed chimeric patients. ResultsIn co-culture of peripheral blood mononuclear cells (PBMCs) from two healthy controls (without irradiation), we detected proliferation at various ratios of PBMC mixtures (1:9 to 9:1) as well as various concentrations of sirolimus, suggesting that two-way MLR is applicable to patients (having >10% chimerism) undergoing sirolimus treatment. In two-way MLR using PBMCs (including donor and recipient cells) from mixed chimeric patients (n = 28), greater ex vivo proliferation was observed <6 months compared with >6 months post-transplant and healthy control PBMC monoculture. Robust ex vivo proliferation was observed in a patient with acute GVHD, and persistent ex vivo proliferation (until 2 years) was observed in a patient with decreasing donor chimerism. ConclusionsIn summary, we demonstrated that in two-way MLR, ex vivo immunoreaction decreases to low levels ~6 months post-transplant. These findings suggest a rationale to continue immunosuppression for 6 months.

Gene Regulatory Networks in Peripheral Mononuclear Cells Reveals Critical Regulatory Modules and Regulators of Multiple Sclerosis

Multiple sclerosis (MS) is a complex, demyelinating disease with the involvement of autoimmunity and neurodegeneration. Increasing efforts have been made towards identifying the diagnostic markers to differentiate the classes of MS from other similar neurological conditions. Using a systems biology approach, we constructed four types of gene regulatory networks (GRNs) involved in peripheral blood mononuclear cells (PBMCs). The regulatory strength of each GRN across primary progressive MS (PPMS), relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), and control were evaluated by an integrity algorithm. Among the constructed GRNs (referred as TF_gene_miRNA), POU3F2_CDK6_hsa-miR-590-3p, MEIS1_CASC3_hsa-miR-1261, STAT3_OGG1_hsa-miR-298, and TCF4_FMR1_hsa-miR-301b were top-ranked and differentially regulated in all classes of MS compared to control. These GRNs showed potential involvement in regulating various molecular pathways such as interleukin, integrin, glypican, sphingosine phosphate, androgen, and Wnt signaling pathways. For validation, the qPCR analysis of the GRN components (TFs, gene, and miRNAs) in PBMCs of healthy controls (n = 30), RRMS (n = 14), PPMS (n = 13) and SPMS (n = 12) were carried out. Real-time expression analysis of GRNs showed a similar regulatory pattern as derived from our systems biology approach. Also, our study provided several novel GRNs that regulate unique and common molecular mechanisms between MS conditions. Hence, these regulatory components of GRNs will help to understand the disease mechanism across MS classes and further insight may though light towards diagnosis.

ADAM17-Mediated Reduction in CD14++CD16+ Monocytes ex vivo and Reduction in Intermediate Monocytes With Immune Paresis in Acute Pancreatitis and Acute Alcoholic Hepatitis.

Impaired immune responses and increased susceptibility to infection characterize acute inflammatory conditions such as pancreatitis and alcoholic hepatitis and are major causes of morbidity and mortality. However, the mechanisms that drive this apparent immune paresis remain poorly understood. Monocytes mediate host responses to damage and pathogens in health and disease, and three subsets of monocytes have been defined based on CD14 and CD16 expression. We sought to determine the changes in monocyte subsets in acute pancreatitis (AP) and acute alcoholic hepatitis (AAH), together with functional consequences and mechanisms that underlie this change. Peripheral blood mononuclear cells (PBMCs) from patients with AP or AAH were compared with healthy controls. Monocyte subsets were defined by HLA-DR, CD14, and CD16 expression. Changes in surface and intracellular protein expression and phosphorylation were determined by flow cytometry. Phenotype and function were assessed following stimulation with lipopolysaccharide (LPS) or other agonists in the presence of specific inhibitors of TNFα and a disintegrin and metalloproteinase 17 (ADAM17). Patients with AP and AAH had reduced CD14++CD16+ intermediate monocytes compared to controls. Reduction of intermediate monocytes was recapitulated ex vivo by stimulating healthy control PBMCs with Toll-like receptor (TLR) agonists LPS, flagellin or polyinosilic:polycytidylic acid (poly I:C). Stimulation caused shedding of CD14 and CD16, which could be reversed using the ADAM17 inhibitor, TMI005 but not direct inhibitors of TNFα, a known ADAM17-target. Culturing PBMCs from healthy controls resulted in expansion of intermediate monocytes, which did not occur when LPS was in the culture medium. Cultured intermediate monocytes showed reduced expression of CX3CR1, CCR2, TLR4, and TLR5. We found reduced migratory responses, intracellular signaling and pro-inflammatory cytokine production, and increased expression of IL-10. Stimulation with TLR agonists results in ADAM17-mediated shedding of phenotypic markers from CD16+ monocytes, leading to apparent “loss” of intermediate monocytes. Reduction in CD14++CD16− monocytes and increased CD14++CD16+ is associated with altered responses in functional assays ex vivo. Patients with AP and AAH had reduced proportions of CD14++CD16+ monocytes and reduced phosphorylation of NFκB and IL-6 production in response to bacterial LPS. Together, these processes may contribute to the susceptibility to infection observed in AP and AAH.

A new T helper 17 cytokine in hidradenitis suppurativa: antimicrobial and proinflammatory role of interleukin-26.

Interleukin (IL)-26 is a signature T helper 17 cytokine described as a proinflammatory and antimicrobial mediator. So far, IL-26 has been reported in several immune-mediated inflammatory diseases, but its involvement in inflammatory skin disorders is poorly known. To investigate the role of IL-26 in hidradenitis suppurativa (HS), through its involvement in antimicrobial activity. IL-26 was assessed in patients with HS through gene expression and protein analysis at skin and circulating levels. Ex vivo HS organ skin cultures, together with IL-26 antibody treatment, were performed to determine the IL-26 activity. Peripheral blood mononuclear cells (PBMCs) from patients with HS and healthy controls were either silenced or not with IL-26 small interfering (si)RNA in order to measure its antimicrobial, cytotoxic and phagocytic activities against Staphylococcus aureus. Firstly, we observed that IL-26 is able to modulate the proinflammatory response at the immune cell level. IL-26 was increased in the plasma of patients with HS compared with healthy controls. Subsequently, we explored the bactericidal, cytotoxic and phagocytic activities of PBMCs against S. aureus in patients with HS and healthy controls. These activities were lower in patients with HS than in controls. Remarkably, the killing activities were reduced when healthy control PBMCs were transfected with IL-26 siRNA. However, the transfection did not affect the killing activity of HS PBMCs, supporting the idea that IL-26 lacks efficacy in HS. Our findings suggest that infection susceptibility in HS might be related to IL-26. Although the role of bacteria remains controversial in HS, this paper supports that there is a defect of antimicrobial response in these patients. What's already known about this topic? Interleukin (IL)-26 is a T helper 17 cytokine described as an antimicrobial and proinflammatory mediator. IL-26 has been reported in immune-mediated inflammatory diseases, but its involvement in inflammatory skin disorders remains unclear. Hidradenitis suppurativa (HS) is a chronic inflammatory skin disorder characterized by deficiency of IL-20 and IL-22 (a close homologue of IL-26), which causes antimicrobial peptide pauperization leading to severe and recurrent skin infections. What does this study add? IL-26 plasma levels are higher in patients with HS than in healthy control individuals. The antimicrobial activity of IL-26 might be ineffective in patients with HS. What is the translational message? Cutaneous antimicrobial incompetence in HS could be related to IL-26.

Klotho Ameliorates Cellular Inflammation via Suppression of Cytokine Release and Upregulation of miR-29a in the PBMCs of Diagnosed Alzheimer's Disease Patients.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by neural inflammation and oxidative stress. In the current study, the protective effects of klotho and linagliptin treatment on human peripheral blood mononuclear cells (PBMCs) of AD patients and healthy controls (HCs) are assessed through measurement of inflammatory cytokines, signaling proteins, and miRNA expression. Sixteen diagnosed AD patients and sixteen HCs were enrolled in the study. Blood samples were obtained and PBMCs were isolated. PBMCs were treated with klotho at different concentrations (0.5, 1, and 2nM) and linagliptin (50μM). The concentration of interleukin-1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), epsilon isoform of protein kinase C (PKCε), phosphorylated cyclic AMP response element binding (pCREB), and Wnt1 were measured by ELISA. The expression of miR-29a and miR-195 was detected by real-time PCR. The results showed that klotho significantly reduced IL-1β, IL-6, and TNF-α levels in both groups of the experiment. Linagliptin also remarkably reduced TNF-α levels in the AD group. Moreover, klotho caused the downregulation of Wnt1 in the PBMCs of both groups and the upregulation of the pCREB in HCs. Meanwhile, klotho induced miR-29a expression in the PBMCs of HCs, while miR-29a expression was induced in the AD group by klotho and linagliptin. The current findings revealed that klotho alleviates inflammation in human PBMCs, probably through the suppression of inflammatory cytokines and the upregulation of miR-29a, and part of its beneficial effect is mediated through appropriate modulation of the Wnt1/pCREB signaling cascade. In addition, linagliptin exerts protective effects by reducing TNF-α and inducing miR-29a expression in PBMCs.

Aberrant Expression of a Proliferation-Inducing Ligand Underlies Autoimmune Mechanisms in Immune Thrombocytopenia

Background: Immunological thrombocytopenia (ITP) is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction and suboptimal platelet production. The proliferation-inducing ligand (APRIL or TNFSF13), a member of the TNF superfamily, is structurally and functionally related to the TNF family of B cell activating factors (BAFF, TNFSF13b) and has been shown to regulate lymphocyte survival by interacting with its receptors. And activation. Transmembrane activators and calcium regulate cyclophilin ligand interactors (TACI) and B cell mature antigens (BCMA). APRIL is secreted by various cells as soluble factors, including inactive B cells, T cells, monocytes, neutrophils, macrophages and dendritic cells, as well as epithelial cells, osteoclasts and megakaryocytes. Recent studies have shown that APRIL not only participates in normal immune responses, but also plays an important role in the establishment and/or maintenance of autoimmune and inflammatory diseases.Aims: Based on the relationship between APRIL, which promotes proliferation and regulates immunity, and the development of autoimmunity, we hypothesize that APRIL may play a role in the pathogenesis of ITP.Methods:1. The EDTA anticoagulated whole blood was collected, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation. The APRIL levels on the surface of T cells, B cells, DC cells and platelets were detected by flow cytometry.Detection of plasma APRIL levels in patients with ITP by ELISA.Real time quantitative PCR were used for detecting the level of APRIL and its receptors BCMA and TACI from PBMC of healthy controls and ITP patients.Use soluble APRIL or BLyS protein and APRIL inhibitors to examine the effect of APRIL inhibition on IL-10 secretion by B cells. Flow cytometry and intracellular staining were used to evaluate B10 cells.Resoult: 1. The APRIL on the platelet surface of patients with ITP was significantly lower than that of the normal control group (p<0.01). In the ITP patients of 10 patients with complete remission, the content of APRIL on the platelet surface was significantly increased after treatment (p=0.02), and there was no significant change in the treatment-ineffective group. .The levels of APRIL and its receptors BCMA and TACI on B cells and DC cells in ITP patients were higher than those in normal controls, and the difference was statistically significant. APRIL is not expressed on CD4 + T cells, CD8 + T cells.The expression of APRIL mRNA in PBMNCs was significantly higher in ITP patients than in the normal control group (p <0.01). There was no difference in BCMA and TACI expression in PBMNC of ITP patients compared to normal controls.Plasma APRIL levels were significantly higher in ITP patients than in the normal control group, p = 0.04, and negatively correlated with platelet count, p = 0.029.In 10 patients with ITP, the percentage of CD19 + B cells remained similar between patients, and the results showed that the amount of B10 cells in the medium supplemented with APRIL was greater than that of B10 cells containing BLyS and control medium (p<0.01; p= 0.01), and the use of APRIL inhibitors resulted in a decrease in B10 cells.Conclusion: Our study shows that aberrant expression of APRIL is involved in the autoimmune response of ITP, and the effect of treatment can be assessed by measuring changes in the level of APRIL on the platelet surface. We also speculate that APRIL inhibits, rather than promotes, an immune-mediated inflammatory response in the pathogenesis of ITP. Our current observations support that the immunomodulatory effects of APRIL may be due, at least in part, to stimulation of IL-10 producing B cells. DisclosuresNo relevant conflicts of interest to declare.

Toll-like receptors pathway in common variable immune deficiency (CVID) and X-linked agammaglobulinemia (XLA).

Common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA) are two major humoral immunodeficiencies, causing a high rate of early age mortality in children. In order to identifiy the possible factors involved in the pathogenesis of CVID and XLA, recent studies have focused on Toll-like receptors (TLRs) and demonstrate the defects in different TLR pathways in immune cells of CVID and XLA patients. Herein, we measured TLR-4 and TLR-9 RNA levels and consequently TNF-α and IFN-α production in peripheral blood mononuclear cells (PBMCs) of patients with CVID and XLA. Contrary to healthy individuals, TLR-9 expression was not significantly increased after ligand stimulation, whereas ligand-induced TLR-4 expression was not significantly different from that in healthy control PBMCs. Lipopolysaccharide (LPS)-stimulated TNF-α production was significantly reduced in patients compared to controls, whereas IFN-α production was increased in all groups after CpG stimulation without any significant inter-group difference. Our data suggest that defects in TLR-9 activated pathways may be a result of the decreased TLR-9 expression, although TLR-9 is not the only modulator of IFN-α production in these patients. On the other hand, impaired signaling in TLR-4 activated pathways which results in significant reduction in TNF-α production are not related to a defect in TLR-4 expression.