P234 Characterisation of human synovial tissue dendritic cells in rheumatoid arthritis patients

Abstract Background Dendritic cells (DCs) direct immune responses against pathogens while maintaining tolerance to self-antigens. However, their aberrant activation can lead to autoimmunity and inflammation. DCs consist of two subtypes: plasmacytoid and myeloid DCs. Recently, single-cell sequencing has revealed complex heterogeneity within myeloid DCs. These cells can be divided into CD141pos (DC1), DC2 defined as CD1highCD163neg and DC3 defined as CD1clowCD163high. In addition, a population of CD1cnegCD141negCD16pos (DC4), which share some gene expression with CD16pos monocytes, has been identified. To date, most studies of RA investigated DC precursors in circulation or synovial fluid (SF). However, DCs perform their functions within lymphoid or peripheral tissue. Therefore, in this study, we sought to characterise myeloid DC subsets in synovial tissue (ST) with the prospect of better understanding their role in driving autoimmunity in RA. Methods We developed a flow sorting strategy to characterise the phenotype of distinct myeloid DC subsets in multiple biological compartments (PB, SF, and ST). Ultrasound-guided ST biopsies (RA n = 9; Psoriatic arthritis n = 3 with active disease) were digested with liberase prior to analysis. PB DCs (RA n = 19, Psoriatic arthritis n = 16, healthy donors n = 12), and SF DC (n = 3) were used as comparators. ST, SF and PB DCs were sorted then microRNA, pro-inflammatory and regulatory cytokine expression analysed by amplified qPCR. To evaluate the role of miR-155 in RA CD1cpos DC activation, CD1cpos cells were computationally sub-setted from a scRNAseq synovial dataset comparing synovial tissues of healthy individuals (n = 4) with patients with active RA (n = 7) and those in sustained clinical and ultrasound remission (n = 7). To elucidate the role of miR-155, CD1cpos DC from RA patients (n = 10) were sorted then transfected with miR-155 mimic or control mimic and the cytokines expression and produced were evaluated using quantitative RT-PCR and Luminex, respectively. Results Our data revealed that whilst all myeloid DCs subsets can be present in inflamed RA synovium they occur with variable frequencies. CD1cpos (DC2/3) being the most abundant, followed by CD16pos DCs (DC4), whilst CD141pos DCs (DC1) occur in low numbers or are absent. CD1cpos DC sorted from RA ST express high levels of epigenetic regulators of inflammatory response miR-155, IL-6, TNF-a and IL-23 as compared to circulating cells.Unsupervised clustering of synovial CD1c cells identified 4 separate clusters including a distinctive miR155 positive CD1c cluster in RA patients. These cells showed significantly higher expression of pro-inflammatory cytokines and co-stimulatory molecules compared to other synovial CD1c clusters. Overexpression of miR-155 in CD1cpos leads to increased production of TNF-a and IL-23. Conclusion Our data show that miR-155 is strongly implicated as an epigenetic regulator of the pro-inflammatory synovial CD1cpos DCs sub-cluster and contributes to exacerbating RA. Disclosures A. Elmesmari None. L. MacDonald None. D. Vaughan None. B. Tolusso None. L. Bui None. M. Gigante None. F. Federico None. G. Ferraccioli None. E. Gremese None. I. B McInnes None. S. Alivernini None. M. Kurowska-Stolarska None.

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Biological Sequelae of TRAF2 Downregulation in Waldenstrom Macroglobulinemia Cells.

Blood ◽

10.1182/blood.v110.11.3526.3526 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3526-3526

Author(s):

Xavier Leleu ◽

Lian Xu ◽

Zachary R. Hunter ◽

Sophia Adamia ◽

Evdoxia Hatjiharissi ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Cell Growth ◽

Cell Lines ◽

Genomic Alteration ◽

Rt Pcr ◽

Growth And Survival ◽

Healthy Donors ◽

Cell Growth And Survival

Abstract Background. Several TNF family members (CD40L and BAFF/BLYS) have been implicated in Waldenstrom’s Macroglobulinemia (WM) cell growth and survival. More recently, abnormalities in the APRIL-TACI pathway have been demonstrated by us in WM cells (Hunter, ASH2006, #228). TRAFs (TNFR-associated factor) are a family of adaptor proteins that mediate signal transduction from multiple members of the TNF receptor superfamily. In particular, TRAFs facilitate pro-apoptotic signaling from the TACI receptor, and TRAF2 is of importance among the TRAF adapter proteins since this protein is required for TNF-alpha-mediated activation of SAPK/JNK MAPK known to be involved in drug-induced death of tumor B cells. We therefore examined the role of TRAF2 in WM growth and survival. Method. We investigated TRAF2, 3 and 5 gene expression in WM patient bone marrow (BM) CD19+ cells and cell lines (BCWM.1, WSU-WM) and compared their expression to BM CD19+ cells from healthy donors. Expression of human TRAF transcripts were determined using real time quantitative RT-PCR (qPCR) based on TaqMan fluorescence methodology. To evaluate the role of TRAF2, a knockdown model was prepared in BL2126 B-cells and BCWM.1 WM cells using electroporation, with resulted ≥50% knockdown efficiency using RT-PCR and immunoblotting. Results. We found that TRAF3 and 5 gene expression was higher in WM versus healthy donors, while TRAF2 expression was lower in 8/13 (60%) patients, using qPCR. TRAFs gene expression did not correlate with tumor burden or WM prognostic markers. We next sought to understand the biological sequelae of TRAF2 deficiency in BL2126 and BCWM.1 cells and found that TRAF2 knockdown induced increased survival at 72 hours in both cell lines. We next studied sequence analysis of 20 WM patients CD19+ BM cells to determine whether there was a TRAF2 genomic alteration, and found heterozygous early termination mutation in exon 5 in 1 (5%) patient. Conclusion. Our data demonstrate that TRAF2 is a commonly dysregulated TNF family adapter protein in patients with WM, with important consequences in WM cell growth and survival.

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BCR-ABL-Induced Transcriptional Repression of the Interferon Regulatory Factor 8 (IRF-8/ICSBP) Leads to Depletion of Plasmocytoid Dendritic Cells (PDC), Which May Contribute to Leukemogenesis in a Murine Model of Chronic Myeloid Leukemia

Blood ◽

10.1182/blood.v120.21.36.36 ◽

2012 ◽

Vol 120 (21) ◽

pp. 36-36 ◽

Cited By ~ 1

Author(s):

Sabrina Inselmann ◽

Simone Liebler ◽

Cornelia A Brendel ◽

Steffen Koschmieder ◽

Andreas Neubauer ◽

...

Keyword(s):

Dendritic Cells ◽

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Cpg Odn ◽

Toll Like Receptor ◽

Rt Pcr ◽

Healthy Donors ◽

Functional Relevance

Abstract Abstract 36 Introduction: Chronic myeloid leukemia (CML) is caused by the BCR-ABL oncogene. CML patients lack expression of IRF-8 - an interferon-regulated transcription factor that has been shown to exert tumor suppressor functions. IRF-8 is also critical for the development of a rare dendritic cell population, so called plasmocytoid dendritic cells (PDC). PDC are quantitatively significantly reduced or absent in the peripheral blood of first diagnosis CML patients. PDC are also the major producers of IFN-alpha (IFNa) in man. IFNa is a cytokine that has significant therapeutic efficiency in the treatment of CML patients. We here wished to experimentally test, whether BCR-ABL expression and loss of IRF-8 may be causally linked to a reduction of PDC in murine CML and whether there could be any functional relevance for PDC loss in CML development or treatment. Methods: PDC counts were studied from peripheral blood samples of primary CML patients at diagnosis, at the time of remission or from healthy donors. PDC function was assessed in vitro by treatment of magnetic bead-enriched PDC with Toll-like receptor 9-specific oligos (ODN 2216) and subsequent assessment of the intracellular IFNa expression in stimulated PDC. A supposed link between BCR-ABL expression, IRF-8 repression and loss of PDC counts was studied in vivo using a murine CML transduction-transplantation model (C57/Bl6 mice, 7Gy sub-lethal irradiation for conditioning). Multiparameter flow cytometry and cell sorting were performed to analyze and enrich, BCR-ABL-positive (GFP+) hematopoietic subpopulations and PDC in order to then quantitate their IRF-8 and BCR-ABL transcript level by RT-PCR. In order to also test the functional relevance of PDC during CML leukemogenesis, CML mice were injected intravenously, weekly from day +5 after transplantation with in vitro generated PDC. Mice were simultaneously also s.c.-injected weekly with ODN 2216 to stimulate IFNα secretion in adoptively transferred PDC in vivo. Results: As previously reported, newly diagnosed CML patients displayed a significantly reduced PDC count when compared to healthy donors (p<0.001). Upon remission induction with imatinib, PDC counts restored partially, but to a much lesser extend in patients successfully treated with IFNa therapy. Importantly, albeit significantly reduced in number, BCR-ABL-positive first diagnosis CML PDC seem to be functionally intact: CML and healthy donor PDC produced comparable amounts of IFNa in response to Toll-like receptor 9 -specific CpG ODN 2216 stimulation. This suggested that BCR-ABL may compromise PDC function by quantitative rather than qualitative dysregulation. CML mice developed a fatal, BCR-ABL-positive myeloproliferation within 13 to 29 days with 88% penetrance. Compared to control mice (n=8), CML mice (n=14) showed a 7-fold and 3-fold reduction of the frequency of B220+mPDCA-1+ PDC in bone marrow and spleen, respectively. This was associated with a statistically significant (4-fold) suppression of IRF8 mRNA expression in sorted BCR-ABL(GFP)-positive PDC relative to BCR-ABL-negative PDC from the same mice (n=3) or from control transplantations (n=5). By RT-PCR, there was a trend also for lower IRF8 expression in CML progenitor cells (Lin− c-Kit+ Sca-1- GFP+), but not in the stem cell enriching population (Lin− c-Kit+ Sca-1+ GFP+). This implied that IRF8 expression is lost during BCR-ABL-induced leukemogenesis in more mature compartments, which supposedly include PDC precursors. Intriguingly, a once weekly adoptive transfer of in vitro generated (to > 30% enriched) PDC for three successive weeks combined with a once weekly subcutaneous injection of CpG ODN 2216 for three weeks was sufficient to almost double survival of CML mice. Conclusions: Using a murine model of CML, we provide first experimental evidence that BCR-ABL induced myeloproliferation is causally linked to a quantitative suppression of PDC, and that this is associated with a BCR-ABL-mediated suppression of IRF8 transcription. Since adoptively transferred PDC were capable of counteracting murine CML development, BCR-ABL may facilitate leukemogenesis in part by obstructing PDC maturation. PDC could thus be a novel immunological effector cell population that exerts and/or integrates anti-leukemic immune responses in CML. Disclosures: No relevant conflicts of interest to declare.

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Investigate the role of GPR15/BOB in the SLE patients

American Journal of BioMedicine ◽

10.18081/2333-5106/018-19-32 ◽

2018 ◽

Vol 6 (1) ◽

pp. 19-32

Author(s):

Caroline G. Jackson ◽

Donald R. Kwan

Keyword(s):

Peripheral Blood ◽

Messenger Rna ◽

Viral Envelope ◽

Orphan Receptor ◽

Blood Monocytes ◽

Rt Pcr ◽

Peripheral Blood Monocytes ◽

Healthy Donors ◽

Hiv 1

GPR15 functions as a cellular co-receptor for some isolates of HIV-1, HIV-2, and SIV through interactions with several viral envelope proteins. The objective of this study was to investigate the expression of orphan receptor GPR15/BOB in the serum of SLE patients and non-SLE healthy people. GPR15/BOB expression was analysed by flow cytometry while, GPR15/BOB messenger RNA was examined in peripheral blood monocytes by RT-PCR. GPR15/BOB mRNA was detected in all periphral blood of SLE patients examined. Further, a significant increase in GPR15/BOB expression as measured by mean fluorescence intensity was observed on SLE PB neutrophils compared to these cell populations from healthy donors. We concluded that GPR15/BOB is expressed in monocytes and neutrophils in peripheral blood, and expression is up-regulated in SLE patients compared to controls. GPR15/BOB may play a role in SLE pathogenesis.

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Interleukin-35 inhibited production of histamine and pro-inflammatory cytokines through suppression MAPKs pathway in HMC-1 cells

10.21203/rs.3.rs-41258/v1 ◽

2020 ◽

Author(s):

Tao Chen ◽

Lixin Fu ◽

Qiaomei Sun ◽

Peimei Zhou ◽

Zai-pei Guo

Keyword(s):

Immune Response ◽

Inflammatory Cytokine ◽

Human Mast Cell ◽

Rt Pcr ◽

Effector Cells ◽

Response System ◽

Mast Cell Line ◽

Anti Inflammatory Cytokine ◽

Pro Inflammatory Cytokines

Abstract Background: IL-35 is a newly anti-inflammatory cytokine which belong to the IL-12 family. Mast cells, as one of the major effector cells in the immune response system, play important roles in the pathogenesis of chronic spontaneous urticarial (CSU). The aim of our study is to explore the inhibited role of IL-35 in HMC-1. Methods: The effects of IL-35 on cell proliferation, cytokine expression and histamine release in human mast cell line (HMC1) were investigated by CCK8, ELISA or RT-PCR. The phosphorylation of ERK1/2, p38 and JNK1/2, in PMA and A23187 induced HMC-1 cells were detected by Western Blot.Results: We found that IL-35 significantly inhibited the proliferation of HMC-1 cells stimulated by PMA and A23187. IL-35 also down-regulates the released of histamine and the mRNA expression of IL-6 and IL-17 in activated HMC-1. Furthermore, IL-35 markedly inhibited the phosphorylation of ERK1/2, p38 and JNK1/2, in PMA and A23187 induced HMC-1 cells. Conclusions: This study provides first observations on the inhibitory and anti-inflammtory effect of IL-35 on activated HMC-1 cells. We suggest that IL35 may play an inhibited role in the pathogenesis of CSU.

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The Role of IL-36 in the Pathophysiological Processes of Autoimmune Diseases

Frontiers in Pharmacology ◽

10.3389/fphar.2021.727956 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wen-jian Chen ◽

Xiao Yu ◽

Xin-Rong Yuan ◽

Bang-jie Chen ◽

Na Cai ◽

...

Keyword(s):

Immune Response ◽

Dendritic Cells ◽

Inflammatory Response ◽

Autoimmune Diseases ◽

Future Research ◽

Dependent Manner ◽

Receptor Complexes ◽

Multiple Organs ◽

Pro Inflammatory Cytokines

A member of the interleukin (IL)-1 superfamily was IL-36, which contained IL-36α, IL-36β, IL-36γ, and IL-36Ra. Heterotrimer complexes, consisting of heterodimeric receptor complexes and IL-36 agonist, gave signals through intracellular functional domains, so as to bind to downstream proteins and induce inflammatory response. IL-36 agonists upregulated mature-associated CD80, CD86, MHCII, and inductively produced several pro-inflammatory cytokines through the IL-36R-dependent manner in dendritic cells (DCs). Besides, DCs had the ability to initiate the differentiation of helper T (Th) cells. Up to date, the role of IL-36 in immunity, inflammation and other diseases is of great importance. Additionally, autoimmune diseases were characterized by excessive immune response, resulting in damage and dysfunction of specific or multiple organs and tissues. Most autoimmune diseases were related to inflammatory response. In this review, we will conclude the recent research advances of IL-36 in the occurrence and development of autoimmune diseases, which may provide new insight for the future research and the treatment of these diseases.

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SAT0003 Synovial tissue cd1c+ dendritic cells in rheumatoid arthritis express high levels of the epigenetic regulator of inflammation, microrna-155 and inflammatory cytokines

10.1136/annrheumdis-2018-eular.6478 ◽

2018 ◽

Author(s):

A. Elmesmari ◽

S. Alivernini ◽

B. Tolusso ◽

L. Bui ◽

D. Vaughan ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Dendritic Cells ◽

Synovial Tissue ◽

Inflammatory Cytokines ◽

Epigenetic Regulator

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The Function and Molecular Mechanism of MicroRNA-429 in Rheumatoid Arthritis

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2352 ◽

2020 ◽

Vol 10 (7) ◽

pp. 945-950

Author(s):

Pengdong Zhang ◽

Bailong Yu ◽

Bin Lei ◽

Changlin Li ◽

Xiaoqiang Yuan

Keyword(s):

Rheumatoid Arthritis ◽

Gene Expression ◽

Molecular Mechanism ◽

Synovial Tissue ◽

Luciferase Assay ◽

Rt Pcr ◽

Tnf Α ◽

Tlr4 Gene ◽

Synovial Tissues ◽

D Model

Objective: To explain the function and molecular mechanism of miRNA-429 in Rheumatoid Arthritis development. Methods: Collecting synovial tissue of 36 RA patients and 36 traumatic amputation patients, the miRNA-429 and TLR4 gene expressions were measured by RT-PCR. The SD rats were divided into NC, 14 d Model and 28 d Model groups. The IL-1β and TNF-α concentrations of serum were measured by Elisa assay in difference rats groups; The synovial tissue pathology was evaluated by HE staining; the miRNA-429 gene expression of rats groups were measured by RT-PCR, the TLR4 and NF-κB proteins expressions of rats groups were evaluated by IHC staining; the correlation between miRNA-429 and TLR4 were evaluated by Double luciferase assay. Results: Compared with normal synovial tissues, the miRNA-429 and TLR4 gene expression of synovial tissues were significantly difference in RA patients. In rats vivo study, we found that IL-1 and TNF-α concentrations were significantly up-regulation with time increasing (P < 0 05, respectively); inflammation degree was serious by HE staining and miRNA-429 gene expression was significantly reduced (P < 0.05, respectively); TLR4 and NF-κB proteins expressions were significantly up-regulation (P < 0.05, respectively) with time increasing; TLR4 was the target gene of miRNA-429 by Double luciferase assay. Conclusion: miRNA-429 over-expression stimulated RA development.

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MicroRNA target Fc receptors to regulate Ab-dependent Ag uptake in primary macrophages and dendritic cells

Innate Immunity ◽

10.1177/1753425916661042 ◽

2016 ◽

Vol 22 (7) ◽

pp. 510-521 ◽

Cited By ~ 15

Author(s):

Afsar Raza Naqvi ◽

Jezrom B Fordham ◽

Salvador Nares

Keyword(s):

Dendritic Cells ◽

Immune Responses ◽

Innate Immune Responses ◽

Microrna Target ◽

Adaptive Immune ◽

Latex Beads ◽

Tnf Α ◽

Particle Internalization ◽

Pro Inflammatory Cytokines

Phagocytosis commences with particle internalization and culminates with the activation of innate and adaptive immune responses. However, the role of miRNAs in phagocytosis remains largely unknown. In this study, we examined the role of miR-24, miR-30b and miR-142-3p in Ab Fc receptor (FcR)-mediated phagocytosis by macrophages (MΦ) and dendritic cells (DC). The expression of these miRNAs was reduced following phagocytosis of both IgG-opsonized beads and Escherichia coli, indicating their regulatory role in the process. Further, overexpression of these miRNAs impaired the uptake of IgG-coated latex beads, which corroborated the reduced secretion of the pro-inflammatory cytokines TNF-α and IL-8 and down-regulation of PKC-α, as well as superoxide-generating enzyme NADPH oxidase 2 expression level. Mechanistically, MΦ and DC transfected with miRNA mimics show marked reduction in expression of FcRs including FCGR2A, FcɛR1G and FCER2. We show that FcɛR1G expression is not affected at the transcription level, rather it is post-transcriptionally regulated by miR-30b. Finally, we demonstrate that siRNA-mediated knockdown of FcɛR1G leads to reduced uptake of IgG-opsonized beads, indicating its involvement on Ab-mediated phagocytosis. These results uncover miR-24, miR-30b and miR-142-3p as an essential component of FcR-mediated phagocytosis and associated innate immune responses.

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Urocortin in the synovial tissue of patients with rheumatoid arthritis

Clinical Science ◽

10.1042/cs1000577 ◽

2001 ◽

Vol 100 (6) ◽

pp. 577-589 ◽

Cited By ~ 21

Author(s):

Miwa UZUKI ◽

Hironobu SASANO ◽

Yasunari MURAMATSU ◽

Kazuhito TOTSUNE ◽

Kazuhiro TAKAHASHI ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Synovial Fluid ◽

Synovial Tissue ◽

High Power ◽

Rt Pcr ◽

Active Stage ◽

High Power Field ◽

Mrna In Situ Hybridization ◽

Synovial Tissues

Urocortin is a newly identified member of the corticotropin-releasing factor (CRF) neuropeptide family, and is known to be involved in the modulation of the inflammatory process. We examined the expression of urocortin, CRF and their receptors (CRF receptor; CRF-R) in the synovial tissue of patients with rheumatoid arthritis (RA) in order to study the possible biological roles of urocortin. Synovial tissues/fluids were obtained from 38 patients with RA, nine patients with osteoarthritis and four with trauma. We studied the concentration of urocortin in the synovial fluid using RIA, and the expression of urocortin in synovial tissue using immunohistochemistry, mRNA in situ hybridization and reverse transcriptase–PCR (RT-PCR). In addition, we examined the immunolocalization of CRF and the expression of CRF-R1, -R2-α and -R2-β mRNAs utilizing RT-PCR in these synovial tissues. Urocortin concentrations in synovial fluid were higher in RA patients (79.8±154 pg/ml) than in control patients (12.3±4.8 pg/ml; P ≤ 0.05). Urocortin immunoreactivity and mRNA signals were both detected in synovial cells, lymphocytes, fibroblasts and macrophages. The number of urocortin-positive cells in the synovium was significantly higher in RA (73.1±32.1 cells per high-power field) than in control (18.4±10.4 cells per high-power field) patients. In addition, both urocortin immunoreactivity and mRNA signals in the synovium reached maximum levels in the active stage of RA inflammation. Moreover, the number of immunoreactive urocortin-positive cells was significantly correlated with the urocortin concentration in synovial fluid (r = 0.705; P < 0.001) and with histologically defined local inflammatory activity (r = 0.641; P < 0.001). The distribution and number of immunoreactive CRF-positive cells in synovial tissue were similar to those of urocortin-positive cells (r = 0.701; P < 0.001). Urocortin, CRF-R1 and CRF-R2-α mRNAs detected by RT-PCR were expressed in in the synovium of 10/10, 10/10 and 2/10 RA patients respectively, but CRF-R2-β was not expressed. Urocortin was actively synthesized in the synovium of RA patients. The present study suggests that urocortin may play an important role as an autocrine and/or paracrine regulator of synovial inflammation in RA.

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