P1800RITUXIMAB ENHANCES THE EXPRESSION OF INTERLEUKIN-6 VIA TOLL-LIKE RECEPTOR 3 SIGNALING IN HUMAN PODOCYTE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Shojiro Watanabe ◽  
Koji Hirono ◽  
Tomomi Aizawa ◽  
Koji Tsugawa ◽  
Hiroshi Tanaka
Keyword(s):  
Nephrotic Syndrome ◽  
B Cell ◽  
Peripheral Blood ◽  
Sustained Remission ◽  
Dependent Manner ◽  
Toll Like Receptor ◽  
Cell Recovery ◽  
Viral Dsrna ◽  
Polycytidylic Acid ◽  
Tlr3 Signaling

Abstract Background and Aims The etiology of idiopathic nephrotic syndrome (INS) in children remains unclear, but it is well known that viral infections often cause relapses of INS. Since toll-like receptor 3 (TLR3), a ligand of viral dsRNA exists in podocytes, dysregulation of TLR3 signaling in podocytes may be involved in the pathogenesis of INS. Recently, rituximab (RTX) a specific antibody to human CD20, has been successfully used to treat children with intractable nephrotic syndrome (frequent relapse nephrotic syndrome, FRNS, and steroid dependent nephrotic syndrome, SDNS). It has been reported that depletion of B cell is main mechanism of RTX treatment. However, some patients experienced recurrence before peripheral blood B cell recovery, whereas selected patients have sustained remission even after peripheral blood B cell recovery. Considering that dramatic effects of RTX in the treatment of INS, it is speculated that some B cell-independent mechanisms of RTX exist. In this context, some recent studies have reported a direct effect of RTX on podocyte, targeting sphingomyelinase phosphodiesterase acid-like 3b (SMPDL3b), but detailed mechanisms remain to be determined. In this study, we examined whether RTX influences TLR3 signaling in cultured human podocyte. Method Immortalized human podocytes in culture were treated with polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of viral dsRNA. RTX were added before or after the treatment of poly IC. Then, expression of interleukin (IL)-6 were measured using real time RT-PCR and ELISA. Direct binding of RTX to podocyte were confirmed by immunofluorescence. Results Poly IC induced the expression of IL-6 in cultured human podocyte in a concentration and time-dependent manner. IL-6 expression induced by poly IC were enhanced in both mRNA and protein level by pretreatment of RTX. RTX itself were apparently stained on podocyte by immunofluorescence. Conclusion RTX binds directly to cultured human podocyte and enhances the IL-6 expression induced by poly IC. Since IL-6 from podocyte was reported to be engaged in regulating glomerular inflammation by cross-talk with endothelial cells, exploring the role of enhanced IL-6 from podocyte by RTX, at least in a part, suggests a direct mechanism of RTX in treating intractable nephrotic syndrome in children.

scholarly journals Interleukin-6 via Toll-Like Receptor 3 Signaling Attenuates the Expression of Proinflammatory Chemokines in Human Podocytes

2021 ◽  
Vol 46 (2) ◽  
pp. 207-218
Author(s):  
Hidenori Umetsu ◽  
Shojiro Watanabe ◽  
Tadaatsu Imaizumi ◽  
Tomomi Aizawa ◽  
Koji Tsugawa ◽  
...  
Keyword(s):  
Rna Interference ◽  
Molecular Mechanisms ◽  
Kidney Diseases ◽  
Enzyme Linked Immunosorbent Assay ◽  
Toll Like Receptor ◽  
Induced Expression ◽  
Inflammatory Reactions ◽  
Monocyte Chemoattractant Protein 1 ◽  
Polycytidylic Acid ◽  
Tlr3 Signaling

<b><i>Background:</i></b> Although toll-like receptor 3 (TLR3) signaling is involved in the development of certain chronic kidney diseases, the specific molecular mechanisms underlying inflammatory reactions via activation of TLR3 signaling in human podocytes remain unclear. Interleukin (IL)-6 is a pleiotropic cytokine associated with innate and adaptive immune responses; however, little is known about the implication of IL-6 via the activation of regional TLR3 signaling in the inflammatory reactions in human podocytes. <b><i>Methods:</i></b> We treated immortalized human podocytes with polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, and assessed the expression of IL-6, monocyte chemoattractant protein-1 (MCP-1), and C-C motif chemokine ligand 5 (CCL5) using quantitative real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against IFN-β and IL-6. <b><i>Results:</i></b> We found that the activation of TLR3 induced expression of IL-6, MCP-1, CCL5, and IFN-β in human podocytes. RNA interference experiments revealed that IFN-β was involved in the poly IC-induced expression of IL-6, MCP-1, and CCL5. Interestingly, IL-6 knockdown markedly increased the poly IC-induced expression of MCP-1 and CCL5. Further, treatment of cells with IL-6 attenuated the expression of CCL5 and MCP-1 mRNA and proteins. <b><i>Conclusion:</i></b> IL-6 induced by TLR3 signaling negatively regulates the expression of representative TLR3 signaling-dependent proinflammatory chemokines in human podocytes.

scholarly journals Cytosolic Sensors of Viral RNA Are Involved in the Production of Interleukin-6 via Toll-Like Receptor 3 Signaling in Human Glomerular Endothelial Cells

2019 ◽  
Vol 44 (1) ◽  
pp. 62-71 ◽  
Author(s):  
Qiang Liu ◽  
Tadaatsu Imaizumi ◽  
Tomomi Aizawa ◽  
Koji Hirono ◽  
Shogo Kawaguchi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Signaling Pathway ◽  
Interleukin 6 ◽  
Viral Rna ◽  
Pivotal Role ◽  
Toll Like Receptor ◽  
Polycytidylic Acid ◽  
Glomerular Endothelial Cells ◽  
Human Glomerular Endothelial Cells ◽  
Tlr3 Signaling

Background/Aims: Dysregulation of interleukin-6 (IL-6) production in residual renal cells may play a pivotal role in the development of glomerulonephritis (GN). Given that Toll-like receptor 3 (TLR3) signaling has been implicated in the pathogenesis of some forms of GN, we examined activated TLR3-mediated IL-6 signaling in cultured normal human glomerular endothelial cells (GECs). Methods: We treated GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of IL-6 and the cytosolic viral RNA sensors retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) using reverse transcription quantitative real-time polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assays. To further elucidate the effects of poly IC on this signaling pathway, we subjected the cells to small interfering RNA (siRNA) against TLR3, interferon (IFN)-β, RIG-I, and MDA5. Results: We found that poly IC induced the expression of RIG-I, MDA5 and IL-6 via TLR3/IFN-β signaling in GECs. siRNA experiments revealed that both MDA5 and RIG-I were involved in the poly IC-induced expression of IL-6, with MDA5 being upstream of RIG-I. Conclusion: Interestingly, cytosolic sensors of viral RNA were found to be involved in IL-6 production via TLR3 signaling in GECs. Regional activation of TLR3/IFN-β/ MDA5/RIG-I/IL-6 axis due to viral and “pseudoviral” infections is involved in innate immunity and inflammatory reactions in GECs. We believe this signaling pathway also plays a pivotal role in the development of some forms of GN.

scholarly journals Toll-like receptor 7 (TLR7)–driven accumulation of a novel CD11c+ B-cell population is important for the development of autoimmunity

Blood ◽  
2011 ◽  
Vol 118 (5) ◽  
pp. 1305-1315 ◽  
Author(s):  
Anatoly V. Rubtsov ◽  
Kira Rubtsova ◽  
Aryeh Fischer ◽  
Richard T. Meehan ◽  
Joann Z. Gillis ◽  
...  
Keyword(s):  
B Cells ◽  
Autoimmune Disease ◽  
B Cell ◽  
Cell Population ◽  
Peripheral Blood ◽  
Elderly Women ◽  
Toll Like Receptor ◽  
Direct Role ◽  
Autoreactive Antibodies

Abstract Females are more susceptible than males to many autoimmune diseases. The processes causing this phenomenon are incompletely understood. Here, we demonstrate that aged female mice acquire a previously uncharacterized population of B cells that we call age-associated B cells (ABCs) and that these cells express integrin αX chain (CD11c). This unexpected population also appears in young lupus-prone mice. On stimulation, CD11c+ B cells, both from autoimmune-prone and healthy strains of mice, secrete autoantibodies, and depletion of these cells in vivo leads to reduction of autoreactive antibodies, suggesting that the cells might have a direct role in the development of autoimmunity. We have explored factors that contribute to appearance of ABCs and demonstrated that signaling through Toll-like receptor 7 is crucial for development of this B cell population. We were able to detect a similar population of B cells in the peripheral blood of some elderly women with autoimmune disease, suggesting that there may be parallels between the creation of ABC-like cells between mice and humans.

scholarly journals Posttransplant Cyclophosphamide Promote Naïve-Subset Dominant B Cell Recovery Coordinated with Early Treg Expansion; Implication of Reduced Risk of Pathogenesis into Chronic Gvhd

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4566-4566
Author(s):  
Miki Iwamoto ◽  
Yusuke Meguri ◽  
Takumi Kondo ◽  
Hiroyuki Sugiura ◽  
Shuntaro Ikegawa ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Chronic Gvhd ◽  
Lymphocyte Subset ◽  
Cell Recovery ◽  
Haplo Group

Abstract Posttransplant cyclophosphamide (PTCy) is an effective prophylaxis for both acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT). We recently studied the immune reconstitution dynamics of each lymphocyte subset after PTCy-based transplant using murine haploidentical BMT model and reported that PTCy strongly promoted Treg-dominant T-cell reconstitution and stem cell-derived mature B-cell generation with broad BCR-diversity. We also found that the early reconstitution of Treg could contribute to promote naïve B cell emergence from bone marrow, indicating the T and B cell recovery might be mutually coordinated after PTCy-based transplant (Iwamoto et al, ASH2017). However, the detailed process of immune reconstitution in patients after haploidentical HSCT with PTCy has not been well studied. To address this issue, we here investigated the early dynamics of donor-lymphocyte subset chimerisms in patient after clinical PTCy-based haploidentical HSCT with comparing those in patients after low-dose ATG-based haploidentical HSCT and patients after cord blood transplantation. Laboratory studies were undertaken in 13 adult patients who received HLA-mismatched allogeneic graft; unrelated cord blood (n=5), and haploidentical related peripheral blood after ATG-based conditioning (n=5) and haploidentical related peripheral blood after PTCy-based conditioning (n=5). Blood samples were obtained before and at 1, 2, 4, 6 and 8 weeks after HSCT. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by density gradient centrifugation and cryopreserved before being analyzed. After thawing, to analyze the subset-specific chimerism, PBMCs were stained with anti-HLA monoclonal antibodies and other subset-specific antibodies as follows: Pacific Blue conjugated anti-CD4, eFluor450 conjugated anti-CD3, PE-Cy7 conjugated anti-CD25, anti-CD14, APC conjugated anti-CD127, anti-CD56, and APC-eFluor780 conjugated anti-CD8a, anti-CD19. Gated lymphotes (CD4+Tcons, CD4+Tregs, CD8+T cells, B cells, NK cells, Monocytes) were analyzed their chimerism by flowcytometry. To examine the detailed phenotype of B cells, the expression of CD27, CD24, CD38 and IgD were tested. Flowcytometry-based method enables us to analyze the lymphocyte subset chemerism in the very early phase after HSCT. At 2 weeks after HSCT, our analysis revealed that CD4+Tcons, CD4+Tregs and CD8+T cells had already achieved complete donor chimerisms (>95% in all subsets) in patients after ATG-based SCT and had been approaching complete donor chimerisms (85.8%, 75.4% and 87.2%, respectively) in patients after CBT. In contrast, percentage of donor chimerisms of CD4+Tcons, CD4+Tregs and CD8+T cells after PTCy-based haplo-SCT was 73.5%, 59.6% and 59.2%, respectively, and those remained to be in the lower levels than other 2 groups. However, at 4 weeks after HSCT, all examined patients achieved complete donor chimerism of T cells, NK cells and Monocytes (>90%). At 8 weeks after HSCT, the number of B cells in PTCy-based haplo-group was higher than in ATG-based haplo-group (3494 vs 1901/mm3). Of note, B cell population in PTCy-based haplo-group at 8 weeks contained the significantly higher percentage of CD24+CD27-IgD+CD38+ transitional/naïve subset and the significantly lower percentage of CD24+CD27+IgD-CD38neg/dim activated/switched-memory subset when compared to B cell population in ATG-based haplo-group (59.9% vs 10.2%, 2.6% vs 21.5%, P<0.02 respectively), suggesting PTCy treatment might be associated with the favorable B cell reconstitution with naïve-subset dominant composition. Moreover, in patients after PTCy-based haplo-group, the percentage of activated/switched-memory subsets in B cell population at 8 weeks was inversely correlated with percentage of Treg in CD4 T cells at 4 weeks (P<0.05, r2=0.77). Taken together, consistently with our murine study, the current data from clinical samples again suggest that PTCy-based immune-modulation lead to coordinated T and B cell recovery, especially promoting naïve-subset dominant B cell recovery with help of the early expansion of Treg, which might reduce the risk of subsequent chronic GVHD. These data provide the important information for understanding the immunological reconstitution after PTCy-based haploidentical HSCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Arhgap25 deficiency leads to decreased numbers of peripheral blood B cells and defective germinal center reactions

2020 ◽  
Author(s):  
Silke E. Lindner ◽  
Colt A. Egelston ◽  
Stephanie M. Huard ◽  
Peter P. Lee ◽  
Leo D. Wang
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Peripheral Blood ◽  
Cell Development ◽  
B Cell Development ◽  
Dependent Manner ◽  
B Cell Differentiation ◽  
Nucleotide Exchange

ABSTRACTRho family GTPases are critical for normal B cell development and function and their activity is regulated by a large and complex network of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). However, the role of GAPs in B cell development is poorly understood. Here we show that the novel Rac-GAP ARHGAP25 is important for B cell development in mice in a CXCR4-dependent manner. We show that Arhgap25 deficiency leads to a significant decrease in peripheral blood B cell numbers, as well as defects in mature B cell differentiation. Arhgap25-/- B cells respond to antigen stimulation in vitro and in vivo but have impaired germinal center formation and decreased IgG1 class switching. Additionally, Arhgap25-/- B cells exhibit increased chemotaxis to CXCL12. Taken together, these studies demonstrate an important role for Arhgap25 in peripheral B cell development and antigen response.

scholarly journals Gut microbiota influence B cell function in a TLR5-dependent manner

2019 ◽  
Author(s):  
Sha Li ◽  
William A. Walters ◽  
Benoit Chassaing ◽  
Benyue Zhang ◽  
Qiaojuan Shi ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Hypervariable Region ◽  
Slight Reduction ◽  
Dependent Manner ◽  
Toll Like Receptor ◽  
Low Levels ◽  
Deficient Mice

AbstractToll-like receptor (TLR) 5-deficient mice display aberrantly low levels of flagellin-specific antibodies (Flic-IgA) secreted into the gut, combined with excess bacterial flagellin in the gut, and together these attributes define microbiome dysbiosis (T5-dysbiosis). How TLR5 signaling deficiency results in T5-dysbiosis is unclear. Here, we address the role of B cells in T-dysbiosis. We observed that B cells do not express TLR5, and that B cell transplantation from TLR5−/− mouse donors into B-cell deficient mice resulted in a slight reduction in Flic-IgA levels compared to B-cells from WT donors. Bone marrow transplants from WT and TLR5−/− donors into recipients of both genotypes confirmed that TLR5 signaling by non-hematopoietic cells is required for T5-dysbiosis. We observed TLR5 deficiency was associated with an expanded population of IgA+ B cells. TLR5−/− mice tended to have higher richness for the IgA gene hypervariable region (CDR3 gene) variants. Transplantation of microbiomes from TLR5−/− and WT microbiomes donors into germfree mice resulted in a higher proportion of IgA-secreting B cells, and higher overall fecal IgA and anti-Flic IgA for TLR5−/− microbiome recipients. This observation indicated that the TLR5−/− mouse microbiome elicits an anti-flagellin antibody response that requires TLR5 signaling. Together these results indicate that TLR5 signaling on epithelial cells influences B cell populations and antibody repertoire.

scholarly journals Ibrutinib Disrupts the Metabolic Program of CLL Cells in-Vivo

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4348-4348
Author(s):  
Uri Rozovski ◽  
David M. Harris ◽  
Ping LI ◽  
Zhiming Liu ◽  
Preetesh Jain ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
B Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Metabolic Reprogramming ◽  
Short Exposure ◽  
Dependent Manner ◽  
Ffa Metabolism

Abstract Introduction: Unlike their normal resting B cell counterparts, chronic lymphocytic leukemia (CLL) cells proliferate. Approximately 1% of the total CLL cell clone expands daily. To adjust for the increase in energetic demands imposed by continuous proliferation, CLL cells undergo metabolic reprogramming and, as recently shown (Rozovski U, et al. Mol Cancer Res. 2015; 13:944-53), CLL cells utilize fat in a manner similar to that of adipocytes. The recent introduction of the oral Bruton tyrosine kinase inhibitor (BTK) ibrutinib revolutionized the treatment of CLL. Because the proliferation of CLL cells is driven by lipid metabolism and ibrutinib inhibits the B cell receptor-induced proliferation of CLL cells, we sought to determine whether ibrutinib also disrupts the metabolic program that provides CLL cells with their unique energy requirements. Methods: We prospectively studied serial peripheral blood samples from 16 patients with CLL. The patients' peripheral blood CLL cells were analyzed prior to and during treatment with ibrutinib. All patients received a daily dose of 420 mg ibrutinib. In addition, we performed in vitro studies using CLL cells from 3 ibrutinib-naïve patients. CLL cells were analyzed for free-fatty acids (FFA) consumption and for the rate of cellular apoptosis using propiduim iodide (PI) and annexin V staining analyzed by flow cytometry. Results: To study lipid metabolism of CLL cells we incubated peripheral blood CLL cells from 3 randomly selected ibrutinib-naïve patients in the presence or absence of FFA and measured the concentration of culture media-dissolved O2 (dO2). Like in our previous study (Rozovski U, et al. Mol Cancer Res. 2015; 13:944-53), we found that CLL cells metabolized FFA and, as a result, the levels of dO2 decreased. However when the cells were co-cultured with FFA and ibrutinib, the delta dO2 (dO2 with FFA minus dO2 without FFA) remained unchanged, suggesting that ibrutinib blocked FFA metabolism in CLL cells.Then, to determine whether ibrutinib also inhibited CLL-cell lipid metabolism in patients treated with ibrutinib, we collected 2 to 5 consecutive PB samples (median: 5) from 16 CLL patients prior to and during treatment with ibrutinib. Unlike the 12% reduction in delta dO2 detected in untreated patients' CLL cells incubated with FFA in vitro, a 6% reduction in delta dO2 was detected in CLL cells of patients treated with ibrutinib 4 days into treatment and after a median of 147 days of ibrutinib treatment a change in delta dO2 was no longer detected. These data suggest that ibrutinib-treated cells lost their capacity to utilize FFA or that the number of FFA consuming circulating CLL cells declined until they were no longer detected. In addition, whereas ibrutinib induced apoptosis of CLL cells in a dose-dependent manner in vitro, ibrutinib did not induce apoptosis at the same time points in vivo, suggesting that interruption of FFA metabolism does not lead to apoptotic cell death and that the metabolic and proapoptotic pathways are not linearly intertwined in CLL cells. In conclusion: Treatment with ibrutinib changes the metabolic profile CLL cells. Even after short exposure to the drug the cells were less capable of utilizing FFA, and after longer exposure, the cells could no longer utilize FFA. Whether ibrutinib induced reduction in FFA metabolism decreases the proliferation capacity of CLL cells remains to be determined. Disclosures Burger: Pharmacyclics: Research Funding. O'Brien:Janssen: Consultancy, Honoraria; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding. Jain:Pfizer: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Novimmune: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Incyte: Research Funding; ADC Therapeutics: Consultancy, Honoraria, Research Funding; Seattle Genetics: Research Funding; Genentech: Research Funding; BMS: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novartis: Consultancy, Honoraria. Wierda:Novartis: Research Funding; Abbvie: Research Funding; Acerta: Research Funding; Gilead: Research Funding; Genentech: Research Funding.

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