B cell-derived anti-beta 2 glycoprotein I antibody contributes to hyperhomocysteinaemia-aggravated abdominal aortic aneurysm

Abstract Aims Overactivated B cells secrete pathological antibodies, which in turn accelerate the formation of abdominal aortic aneurysms (AAAs). Hyperhomocysteinaemia (HHcy) aggravates AAA in mice; however, the underlying mechanisms remain largely elusive. In this study, we further investigated whether homocysteine (Hcy)-activated B cells produce antigen-specific antibodies that ultimately contribute to AAA formation. Methods and results ELISA assays showed that HHcy induced the secretion of anti-beta 2 glycoprotein I (anti-β2GPI) antibody from B cells both in vitro and in vivo. Mechanistically, Hcy increased the accumulation of various lipid metabolites in B cells tested by liquid chromatography-tandem mass spectrometry, which contributed to elevated anti-β2GPI IgG secretion. By using the toll-like receptor 4 (TLR4)-specific inhibitor TAK-242 or TLR4-deficient macrophages, we found that culture supernatants from Hcy-activated B cells and HHcy plasma IgG polarized inflammatory macrophages in a TLR4-dependent manner. In addition, HHcy markedly increased the incidence of elastase- and CaPO4-induced AAA in male BALB/c mice, which was prevented in μMT mice. To further determine the importance of IgG in HHcy-aggravated AAA formation, we purified plasma IgG from HHcy or control mice and then transferred the IgG into μMT mice, which were subsequently subjected to elastase- or CaPO4-induced AAA. Compared with μMT mice that received plasma IgG from control mice, μMT mice that received HHcy plasma IgG developed significantly exacerbated elastase- or CaPO4-induced AAA accompanied by increased elastin degradation, MMP2/9 expression, and anti-β2GPI IgG deposition in vascular lesions, as shown by immunofluorescence histochemical staining. Conclusion Our findings reveal a novel mechanism by which Hcy-induced B cell-derived pathogenic anti-β2GPI IgG might, at least in part, contribute to HHcy-aggravated chronic vascular inflammation and AAA formation.

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B cell-derived anti-beta 2 glycoprotein I antibody mediates hyperhomocysteinemia-aggravated abdominal aortic aneurysm

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2019.11.097 ◽

2020 ◽

Vol 140 ◽

pp. 41

Author(s):

Fangyu Shao ◽

Yutong Miao ◽

Juan Feng ◽

Xian Wang

Keyword(s):

Abdominal Aortic Aneurysm ◽

Aortic Aneurysm ◽

B Cell ◽

Glycoprotein I ◽

Abdominal Aortic ◽

Beta 2

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The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy

Blood ◽

10.1182/blood-2010-09-305649 ◽

2011 ◽

Vol 117 (6) ◽

pp. 1928-1937 ◽

Cited By ~ 75

Author(s):

Asish K. Ghosh ◽

Charla Secreto ◽

Justin Boysen ◽

Traci Sassoon ◽

Tait D. Shanafelt ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Tyrosine Kinase ◽

B Cell ◽

Receptor Tyrosine Kinase ◽

Intracellular Signaling ◽

Specific Inhibitor ◽

Lymphocytic Leukemia ◽

Dependent Manner ◽

Docking Site

Abstract Recently, we detected that chronic lymphocytic leukemia (CLL) B-cell–derived microvesicles in CLL plasma carry a constitutively phosphorylated novel receptor tyrosine kinase (RTK), Axl, indicating that Axl was acquired from the leukemic B cells. To examine Axl status in CLL, we determined the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B cells by Western blot analysis. We detected differential levels of P-Axl in CLL B cells, and further analysis showed that expression of P-Axl was correlated with the other constitutively phosphorylated kinases, including Lyn, phosphoinositide-3 kinase, SyK/ζ-associated protein of 70 kDa, phospholipase C γ2 in CLL B cells. We found that these intracellular signaling molecules were complexed with P-Axl in primary CLL B cells. When Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, bosutinib (SKI-606), or a specific-inhibitor of Axl (R428), robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner. Therefore, we have identified a novel RTK in CLL B cells which appears to work as a docking site for multiple non-RTKs and drives leukemic cell survival signals. These findings highlight a unique target for CLL treatment.

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Aid-Initiated DNA Lesions Are Differentially Processed in Distinct B Cell Differentiation Stages in a Locus-Dependent Manner.

Blood ◽

10.1182/blood.v120.21.2376.2376 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2376-2376

Author(s):

Zhangguo Chen ◽

Sawanee Viboolsittiseri ◽

Maxwell Eder ◽

Shunzong Yuan ◽

Jing H. Wang

Keyword(s):

B Cells ◽

B Cell ◽

Conflicts Of Interest ◽

Genetic Alterations ◽

Dna Lesions ◽

Dependent Manner ◽

B Cell Differentiation ◽

Activation Induced Deaminase ◽

Differentiation Stage ◽

Activated B Cells

Abstract Abstract 2376 Activation induced deaminase (AID) initiates U:G mismatches that are subsequently converted into point mutations or DNA double-stranded breaks. AID-mediated DNA alterations in switch (S) regions at the Igh locus frequently occur in both antigen-stimulated germinal center (GC) B cells and cytokine-activated B cells. To investigate whether AID-initiated U:G lesions are differentially processed in a differentiation stage-specific manner at non-Ig loci to maintain genome stability, we established a knock-in model by inserting an Sg2b region into the first intron of proto-oncogene c-myc. We found that the inserted Sg2b region mutated at an extremely low level and did not enhance genomic instability of c-myc locus in antigen-stimulated GC B cells. In contrast, the inserted Sg2b region mutated more frequently and increased c-myc locus abnormalities in cytokine-activated B cells. Furthermore, uracil glycosylase deficiency led to increased mutation frequency at the c-myc locus. These results reveal that AID-initiated lesions are differentially processed via error-free or error-prone repair in a differentiation stage-specific and locus-dependent manner. Our data might provide mechanistic explanation for differential frequency of AID-mediated genetic alterations in distinct subtypes of common B cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

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Antibody Production Remains Intact Despite Loss of Bone Marrow B cells in Murine Norovirus Infected Stat1−/− Mice

Comparative Medicine ◽

10.30802/aalas-cm-21-000054 ◽

2021 ◽

Author(s):

Daniel E Eldridge ◽

Charlie C Hsu

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Cell ◽

Cell Loss ◽

Dependent Manner ◽

Intestinal Epithelial ◽

Murine Norovirus ◽

Laboratory Mice ◽

T And B Cells ◽

Humoral Immune

Murine norovirus (MNV), which can be used as a model system to study human noroviruses, can infect macrophages/monocytes, neutrophils, dendritic, intestinal epithelial, T and B cells, and is highly prevalent in laboratory mice. We previouslyshowed that MNV infection significantly reduces bone marrow B cell populations in a Stat1-dependent manner. We show here that while MNV-infected Stat1−/− mice have significant losses of bone marrow B cells, splenic B cells capable of mounting an antibody response to novel antigens retain the ability to expand. We also investigated whether increased granulopoiesis after MNV infection was causing B cell loss. We found that administration of anti-G-CSF antibody inhibits the pronounced bone marrow granulopoiesis induced by MNV infection of Stat1−/− mice, but this inhibition did not rescue bone marrow B cell losses. Therefore, MNV-infected Stat1−/− mice can still mount a robust humoral immune response despite decreased bone marrow B cells. This suggests that further investigation will be needed to identify other indirect factors or mechanisms that are responsible for the bone marrow B cell losses seen after MNV infection. In addition, this work contributes to our understanding of the potential physiologic effects of Stat1-related disruptions in research mouse colonies that may be endemically infected with MNV.

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The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.

Journal of Experimental Medicine ◽

10.1084/jem.165.6.1675 ◽

1987 ◽

Vol 165 (6) ◽

pp. 1675-1687 ◽

Cited By ~ 54

Author(s):

A G Rolink ◽

T Radaszkiewicz ◽

F Melchers

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

The Other ◽

Cell Populations ◽

Foreign Antigen ◽

Alloreactive T Cells ◽

Polyclonal Activator ◽

Activated B Cells

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

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Phospholipid flippases enable precursor B cells to flee engulfment by macrophages

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814323115 ◽

2018 ◽

Vol 115 (48) ◽

pp. 12212-12217 ◽

Cited By ~ 9

Author(s):

Katsumori Segawa ◽

Yuichi Yanagihashi ◽

Kyoko Yamada ◽

Chigure Suzuki ◽

Yasuo Uchiyama ◽

...

Keyword(s):

Plasma Membrane ◽

B Cells ◽

B Cell ◽

Developmental Stages ◽

Peritoneal Macrophages ◽

Dependent Manner ◽

Wild Type ◽

Immature B Cells ◽

T Lymphoma ◽

Precursor B Cells

ATP11A and ATP11C, members of the P4-ATPases, are flippases that translocate phosphatidylserine (PtdSer) from the outer to inner leaflet of the plasma membrane. Using the W3 T lymphoma cell line, we found that Ca2+ ionophore-induced phospholipid scrambling caused prolonged PtdSer exposure in cells lacking both the ATP11A and ATP11C genes. ATP11C-null (ATP11C−/y) mutant mice exhibit severe B-cell deficiency. In wild-type mice, ATP11C was expressed at all B-cell developmental stages, while ATP11A was not expressed after pro−B-cell stages, indicating that ATP11C−/y early B-cell progenitors lacked plasma membrane flippases. The receptor kinases MerTK and Axl are known to be essential for the PtdSer-mediated engulfment of apoptotic cells by macrophages. MerTK−/− and Axl−/− double deficiency fully rescued the lymphopenia in the ATP11C−/y bone marrow. Many of the rescued ATP11C−/y pre-B and immature B cells exposed PtdSer, and these cells were engulfed alive by wild-type peritoneal macrophages, in a PtdSer-dependent manner. These results indicate that ATP11A and ATP11C in precursor B cells are essential for rapidly internalizing PtdSer from the cell surface to prevent the cells’ engulfment by macrophages.

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B Cell Immune Profiles in Essential Thrombocythemia with Calr Mutations: Clinical and Molecular Correlates

Blood ◽

10.1182/blood.v126.23.1610.1610 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1610-1610

Author(s):

Yu-Cheng Chang ◽

Ken-Hong Lim ◽

Huan-Chau Lin ◽

Yi-Hao Chiang ◽

Ling Huang ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Essential Thrombocythemia ◽

Jak2 V617f ◽

Healthy Adults ◽

Tlr4 Expression ◽

Reactive Thrombocytosis ◽

Membrane Bound ◽

Calr Mutations ◽

Activated B Cells

Abstract Introduction: Essential thrombocythemia (ET) is a BCL-ABL1-negative myeloproliferative neoplasm (MPN), and is characterized by increased number of mature megakaryocytes (MKs) in the bone marrow and sustained thrombocytosis in the peripheral blood. We have reported that activated B cells are increased in patients with essential thrombocythemia, and can facilitate platelet production mediated by cytokines, such as interleukin-1beta (IL-1β) and interleukin-6 (IL-6) regardless JAK2 V617F mutational status (Thromb Haemost. 2014, 112: 537). Recently, Calreticulin (CALR) mutations were discovered in JAK2/MPL-unmutated essential thrombocythemia (ET) and primary myelofibrosis. Although CALR mutations may be associated with activated JAK-STAT signaling pathway, its exact molecular pathogenesis remains elusive in MPN. Interestingly, in vitro study has shown that CALR is capable of driving B cells activation through the toll-like receptor 4 (TLR4) pathway (J Immunol.2010; 185: 4561). Here we sought to evaluate the association between CALR mutations and B cell immune profiles in ET patients. Methods: Fifty-four patients diagnosed with ET based on the 2008 WHO classification were enrolled into this study. CALR mutations were screened by high-resolution melting analysis and nucleotide sequencing. JAK2 V617F and MPL mutations were screened by allele-specific PCR and nucleotide sequencing, respectively. B cell populations, granulocytes/monocytes membrane-bound B cell-activating factor (mBAFF) and CALR levels, B cells TLR4 expression and intracellular levels of IL-1β/IL-6 and the expression of CD69, CD80, and CD86 were quantified by flow cytometry. Serum BAFF and plasma CALR concentrations were measured by ELISA. Forty-eight healthy adults and 17 patients with reactive thrombocytosis were used for comparison. The association between clinical, laboratory and molecular characteristics were studied. Statistical significance was defined as a two-sided p value <0.05 and SPSS version 22.0 (IBM, New York, USA) was used for all analyses. Results: In this series, 19 (35.2%) patients harbored 8 types of CALR exon 9 mutations including 4 (7.4%) patients with concomitant JAK2 V617F mutations. Compared to JAK2 V617F mutation, CALR mutations correlated with younger age at diagnosis (p=0.04), higher platelet count (p=0.004), lower hemoglobin level (p=0.013) and lower leukocyte count (p=0.013). Among all ET patients, CALR mutations correlated with significantly lower serum BAFF level (median 1.6 ng/mL, p =0.049) and higher fraction of B cells with TLR4 expression (median 11.3%, p=0.021). Compared to healthy adults, patients with ET had statistically significant higher serum BAFF concentrations and higher mBAFF levels on both granulocytes and monocytes, and higher fraction of B cells with TLR4 expression and higher fractions of B cells with intracellular IL-1β and IL-6 expression irrespective of their genotypes. ET patients with both JAK2 and CALR mutations had statistically higher number of CD69-positive and CD86-positive activated B cells when compared with healthy adults. Among the three mutational groups of ET patients, there were no significant differences in granulocytes/monocytes mBAFF, in the fraction of B cells with intracellular IL-1β or IL-6 expression, and the numbers of CD80-positive and CD86-positive activated B cells. Granulocyte membrane-bound CALR levels were highest in patients with reactive thrombocytosis. Plasma CALR concentrations were highest in patients with reactive thrombocytosis (mean +/- SE: 9.04 +/- 0.59) and lowest in CALR -mutated ET patients (5.35 +/- 0.90, p <0.001). Conclusions: Activation of B cells is universally present in ET. Both granulocyte membrane-bound CALR levels and plasma CALR concentrations were lower in CALR-mutated ET patients suggesting that CALR may not play a major role in the activation of B cells in these patients. Disclosures No relevant conflicts of interest to declare.

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Abstract 484: Multimerization of BAFF Regulates B Cell Function and Growth of Aortic Aneurysms

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.484 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Vlad Serbulea ◽

Philipp Jakobs ◽

Srabani Sahu ◽

Prasad Srikakulapu ◽

Coleen A McNamara ◽

...

Keyword(s):

Oxygen Consumption ◽

B Cells ◽

Angiotensin Ii ◽

B Cell ◽

Oxygen Consumption Rate ◽

Consumption Rate ◽

Plasma Cells ◽

Aortic Aneurysms ◽

Metabolic Reprogramming ◽

Metabolic Phenotype

B cell activating factor (BAFF) regulates differentiation and survival of B cells by binding to the surface receptors BAFF receptor (BR3), transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA). During differentiation, intracellular metabolic reprogramming is crucial, such as, naïve B cells are metabolically quiescent, whereas, antibody producing plasma cells are metabolically active. We have reported that depletion of B cells protects mice from abdominal aortic aneurysm (AA), however it is not clear how B cells promote AA growth. BAFF exists as a 3mer (binds only to BR3) or as a 60mer (binds to BR3, TACI and BCMA). Therefore, we hypothesize that BAFF multimerization regulates the immune and metabolic phenotype of B cells by binding to BAFF receptors and modulate AA growth. Immunohistology was performed on AA tissues collected from patients undergoing open AA repair. Experimental AA was induced by elastase perfusion of abdominal aorta or angiotensin II infusion (1000 ng/kg/min) method in 8 weeks old male C57BL/6 or apolipoprotein E knockout mice, respectively. Western blotting, flow cytometry and Seahorse extracellular flux assays were used to determine immune and metabolic changes in B cells in response to recombinant BAFF 3mer and 60mer. BR3+ B cells were detected in the milieu of BAFF in human AAs. Mouse AAs demonstrated significant infiltration (>50/section) of CD138+ plasma B cells, but few (4-10/section) CD20+ B cells. In vitro, BAFF 3mer induced canonical NF-kB, whereas, 60mer induced both canonical and non-canonical NF-kB signaling. Moreover, the 3mer significantly decreased mitochondrial density, oxygen consumption rate, and surface expression of IgD and IgM indicating a metabolically quiescent state of B cells. However, these parameters were significantly increased by the 60mer similar to plasma cells. Anti-BR3 IgG1, but not a control IgG1 antibody decreased BAFF 60mer-induced oxygen consumption rate by 50%. In a pilot study (n=10/group), anti-BR3 IgG1, but not the control IgG1 aggravated angiotensin II-induced AA growth. Altogether, our results suggest that BAFF 3mer and 60mer oppositely regulate immune and metabolic phenotype of B cells and inhibition of BAFF-BR3 signaling is detrimental for AA growth.

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Abstract 464: Chemokine Receptor CCR6 Expression on B Cells Augments Local IgM Production and Atheroprotection

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.464 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Cited By ~ 1

Author(s):

Prasad Srikakulapu ◽

Chantel McSkimming ◽

Coleen McNamara

Keyword(s):

B Cells ◽

B Cell ◽

Adoptive Transfer ◽

Chemokine Receptors ◽

Immune Cell ◽

Dependent Manner ◽

Perivascular Adipose Tissue ◽

Cell Recruitment ◽

Cell Numbers ◽

Atherosclerosis Development

Background: CCR6 mediates immune cell recruitment to inflammatory sites and has cell type-specific effects on diet-induced atherosclerosis in mice. Recent studies implicate the local immune responses in the adventitia/perivascular adipose tissue (PVAT) in atherosclerosis development. We have previously demonstrated that adoptive transfer of CD43 - splenocytes (B cells) into B cell deficient μMT -/- ApoE -/- mice results in reduced diet-induced atherosclerosis in a CCR6-dependent manner. Notably, there were significantly greater numbers of B cells in the aorta including PVAT of μMT -/- ApoE -/- mice which received splenic B cells from CCR6 +/+ mice compared to CCR6 -/- mice, despite no difference in B cell numbers in blood, spleen and peritoneal cavity, suggesting that CCR6 expression on B cells is important in B cell aortic homing. Production of IgM antibodies is thought to be a major mechanism whereby B cells limit atherosclerosis development. Yet whether B cells produce IgM locally in the PVAT and whether this is regulated by chemokine receptors such as CCR6 is unknown. Methods and Results: FACS experiments demonstrated high numbers of B cells available in the PVAT than aorta of young ApoE -/- (49121±11190 and 80±11; p<0.001, n=7) mice. ELISPOT experiments demonstrated significantly fewer IgM secreting cells were in the PVAT of ApoE -/- CCR6 -/- mice compared to ApoE -/- CCR6 +/+ mice (100±25 vs 850±150, p<0.05, n=5), despite no differences in IgM secreting cell numbers in spleen and bone marrow. Adoptive transfer of CD43 - splenic B cells from ApoE -/- CCR6 -/- and ApoE -/- CCR6 +/+ mice into secretory IgM deficient ApoE -/- sIgM -/- mice demonstrated significantly reduced atherosclerosis in mice that received B cells from ApoE -/- CCR6 +/+ mice compared to those that received B cells from ApoE -/- CCR6 -/- mice. Moreover, the B cells from ApoE -/- CCR6 +/+ mice attenuated atherosclerosis only when they were capable of secreting IgM. FACS data from human blood demonstrated that circulating B and T cells but not monocytes express CCR6, suggesting potential human relevance to our murine findings. Conclusion: Results provide evidence that CCR6 expression on B cells mediates B cell recruitment into aorta and/or PVAT to provide atheroprotection via IgM secretion.

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