bluuebunny/crossref_metadata_2025_split huggingface dataset:
Elucidating the role of ATM in BER and MMR during B cell CSR
Authors: Sible Emily , Fiorica Giuseppe , Rahman Sadia , Attaway Mary , Vuong Bao Q. 
Published: January 2023

Abstract
Secondary immunoglobulin isotypes are produced by class switch recombination (CSR), which requires AID-dependent DNA deamination of intronic switch (S) regions within the immunoglobulin heavy chain gene. Non-canonical repair of deaminated DNA by mismatch repair (MMR) or base excision repair (BER) creates staggered DNA breaks that promote recombination between S regions. ATM-dependent phosphorylation of AID at serine-38 (pS38-AID) promotes its interaction with a BER protein, suggesting that ATM regulates CSR through BER. However, the mechanism by which ATM participates in BER remains unknown. ATM−/− mice were bred to knockout mice of the MMR gene MSH2. Surprisingly, the predicted Mendelian frequencies of ATM−/−MSH2−/− adult mice were not obtained and preliminary data indicate a late embryonic lethal phenotype. To bypass the observed lethality, ATM was conditionally deleted on an MSH2−/− background using a floxed ATM allele [ATMF] and B cell-specific Cre recombinase expression. These ATMd/dMSH2−/− mice and B cells display a CSR phenotype comparable to ATM−/− B cells, which suggest that ATM may function genetically upstream of MSH2 and implicate a role for ATM in both MMR and BER.
H3K9 dimethyltransferase G9a is an important epigenetic modulator of B cell differentiation
Authors: George-Alexander Lou-Ella , Kania Anna , Hicks Sakeenah , Mi Tian , Scharer Christopher D. , Boss Jeremy 
Published: January 2023

Abstract
B cell differentiation is a tightly regulated process coordinated by the timed expression of various transcription factors and chromatin accessibility changes mediated by histone modifying enzymes. The histone methyltransferase (HMT) G9a, dimethylates histone H3 lysine 9 (H3K9) at promoters and inhibits gene expression via recruitment of proteins that impair chromatin accessibility. HMTs are expressed ubiquitously but display distinct enzymatic activities and patterns of chromosomal localization. During B cell differentiation, G9a was found to co-localize with Blimp-1 which is required to silence genes associated with a B cell phenotype and cellular proliferation. However, the B cell processes that are modulated by G9a mediated dimethylation remain to be elucidated. To assess the role of G9a in B-cell differentiation, we crossed G9afl/fl mice onto the CD19Cre/+ background (G9aKO mice). Stimulation of CD19Cre/+ (CreCtrl) and G9aKO mice with the T cell independent antigen LPS resulted in a significant increase of activated B cells and plasmablast in G9aKO mice. Further characterization of this phenotype, identified a skewing within the mature B cell population towards marginal zone (MZ) B cells in G9aKO mice. Regions with chromatin accessibility and expression changes identified by ATAC-Seq and RNA-Seq elucidated the B cell processes that are modulated in G9a deficient mice. B cell processes that are subject to direct modulation by G9a will be determined using CUT&RUN. Together, our data shows the importance of G9a in epigenetic modulation required for B cell function.
Molecular mechanisms by which interferon regulatory factor 5 (IRF5) transduces signaling downstream of BCR and BCR/TLR9 pathways
Authors: Rice Matthew R , Brune Zarina , Guo Qin , Barnes Betsy J 
Published: January 2023

Abstract
The transcription factor IRF5 modulates human B cell activation, proliferation, and plasmablast differentiation via an intrinsic mechanism. Targeted knockdown of IRF5 in human primary naïve B cells revealed a significant reduction in CD19+CD86+ B cells, which demarcate early activated B cells, after BCR/TLR9 stimulation, resulting in reduced clonal expansion and plasmablast differentiation. The mechanism(s) by which IRF5 modulates early B cell activation is not known. We previously found that BCR stimulation alone was insufficient to induce IRF5 nuclear translocation (activation), while BCR/TLR9 stimulation induced robust IRF5 activation. Thus, the focus of this study is to determine the mechanism(s) by which IRF5 functions downstream of BCR and dual BCR/TLR9 signaling at the early time points of activation (5 and 15 min). Experiments were performed in human B cells and B cells from wild-type and Irf5−/− mice. Signaling and protein interactions were examined by Western blot, immunoprecipitation, flow cytometry, and biochemical assay. We identified Bruton’s tyrosine kinase (Btk) as a novel interacting partner of IRF5 in both unstimulated and stimulated human and murine B cells. Of note, active phosphorylated Btk was bound to IRF5 only in stimulated cells. Preliminary data also suggest a defect downstream of Btk activation in Irf5−/− B cells. Together, these data indicate a conserved role for IRF5 in the early stages of B cell activation via BCR/TLR9 signaling.
TLR adaptors MyD88 and TRIF are critical for extrafollicular B cell responses to influenza
Authors: Lam Jonathan , Baumgarth Nicole 
Published: January 2023

Abstract
Antibodies produced during primary influenza infection can be protective and form rapidly. This occurs independently of germinal centers (GCs) in reactions known as extrafollicular B cell (EF) responses, where antibody-secreting cells (ASCs) blast and produce antibodies in the medulla of the mediastinal lymph node. While the EF response to influenza is known to be mostly T-dependent, it is unknown why B cells are shunted away from a GC fate and what factors lead to generation of EF ASCs. We show that absence of both Toll-like receptor (TLR) adaptors MyD88 and TRIF (double knockout, DKO) cause severe, B cell-intrinsic reductions in EF responses to influenza. Observing a reduction of the B cell differentiation factor IRF4 in DKO plasmablasts in vivo, we used in vitro culture systems to assess BCR signaling and T cell help by stimulating B cells with anti-IgM(Fab)2 and CD40L and/or BAFF, respectively. DKO B cells showed reduced survival in culture, which was rescued by providing either CD40L or BAFF. Strikingly, DKO B cells barely proliferated in response to any dose of anti-IgM, with or without CD40L/BAFF. High-dose BCR stimulation further reduced the viability of DKO B cells, which correlated with reduced IRF4 induction and altered NFkB signaling. Specifically, we show by image flow cytometry reduced nuclear localization of NFkB c-Rel, a positive regulator of IRF4, following BCR stimulation of DKO compared to wild type B cells. We propose that EF responses during influenza infection require not only antigen and T cell help, but also MyD88 and/or TRIF signaling to drive activation of c-Rel. This enhances antigen-specific B cell survival, proliferation, and the strong upregulation of IRF4 required for plasmablast differentiation.
Toll-like receptor signaling directly modulates B cell antibody responses
Authors: Nelson Alexander J , WU Yee Ling 
Published: January 2023

Abstract
Toll-like receptor (TLR) signaling alone or in concert with other cellular or molecular signals helps shape responses by different cell types. Murine B lymphocytes express cell surface-bound TLRs 1, 2, 4, 6 and endosomal TLRs 3, 7, 9. This study aims to delineate the effects of these TLRs on B cell responses including proliferation, activation and antibody class-switch recombination (CSR).
We stimulated mature, naïve B cells in vitro with individual TLR agonists alone or together with T cell help (via ligation of CD40) and the TH2 cytokine interleukin-4 (IL-4). We measured proliferation by CellTrace Violet assay, cell activation by surface expression of B cell activation markers, and analyzed antibody class switching by surface expression and secreted levels of antibody isotypes by flow cytometry.
We found that in the presence of a minimal level of anti-CD40, stimulation with TLRs 1/2, 2/6, 4, and 9 agonists induced cell proliferation and up-regulation of activation markers including CD69 and CD80. While both types of TLR agonists down-regulated class switching to IgG1, stimulation of the surface-bound TLRs (1/2, 2/6 and 4) increased class switching to IgG3 and IgG2b. In contrast, stimulation of endosomal TLR9 inhibited switching to all isotypes. The addition of IL-4 induced robust cell proliferation and class switching to IgG1, and TLR 1/2, 2/6, 4, and 9 agonists further enhanced these IL-4-mediated B cell responses.
Altogether, our data demonstrate differential effects of surface bound and endosomal TLRs on B cell responses. Activation of surface-bound TLRs, but not endosomal TLR9, triggers CSR to TH1-associated antibody isotypes, while both groups of TLRs synergize the effect of IL-4 on driving CSR to IgG1.
TLR7/8 and TLR9 ligands induce phenotypic and transcriptional changes in human transitional B cells
Authors: Posner Jourdan K , Chang Sandra P 
Published: January 2023

Abstract
The maturation of human transitional B cells into mature, naïve follicular or marginal zone B cells is a key step in B cell development. Transitional B cells are a prominent B cell population in newborns and very young children who receive many vaccines during early childhood. Pattern recognition receptor (PRR) ligands are attractive candidate vaccine adjuvants due to their targeted mechanisms of action. However, the effects of PRR ligands on transitional B cell fate are not fully understood. We have demonstrated that human cord blood transitional B cells predominantly mature into follicular-like B cells when stimulated in vitro with R848 (TLR7/8 ligand) or CpG (TLR9 ligand) in the presence of IL-4. Additionally, we have profiled the transcriptome of human transitional, follicular and marginal zone-like B cells and identified genes uniquely associated with each B cell subset. Several of the genes identified during our transcriptome analysis were expressed in our in vitro-stimulated transitional B cells. These gene expression changes following PRR ligand stimulation indicate that differing stimuli can skew transitional B cell fate. Our characterization of PRR ligand-induced phenotypic and transcriptional changes associated with mature follicular and marginal zone B cell development may influence the rational approach for vaccine adjuvant development, especially for newborns and very young children.
Transient Receptor Potential Vanilloid 4 (TRPV4) Protects the Lung from Bacterial Pneumonia via MAPK Molecular Switching
Authors: Scheraga Rachel Greenberg , Abraham Susamma , Grove Lisa M , Southern Brian D , Crish James , Hasday Jeffrey D , McDonald Christine , Olman Mitchell A 
Published: January 2023

Abstract
Mechanical cell-matrix interactions can drive the innate immune responses to infection, however the molecular underpinnings of these responses remain elusive. We have discovered that the biophysical properties of the matrix in the range of injured fibrotic lung (≥ 25 kPa) conditions the macrophage response to LPS and infection respectively through the mechanosensitive cation channel, TRPV4 in vitro and in vivo. Studies suggest that LPS-induced macrophage activation is controlled in part by the MAPK pathway (i.e. p38, ERK, JNK). Thus, we investigated if TRPV4 plays a role in the macrophage activation response after LPS through alteration of the MAPK pathway. TRPV4 KO mice exhibited reduced lung bacterial clearance by macrophages (6-fold, p=0.012) after intratracheal P. aeruginosa administration and increased lung injury as measured by inflammatory cell infiltration (≥80±3%, p&lt;0.05), vascular permeability (BAL total protein ≥63±6%, p&lt;0.05), and pro-inflammatory cytokine secretion in BALF (IL-6, CCL2, CXCL1 ≥71±4%, p&lt;0.05). LPS-induced p38 activation was decreased (69%, p&lt;0.05) while JNK activation was increased (2-fold, p&lt;0.05) in a stiffness-dependent manner with no change in ERK activation in TRPV4 KO BMDMs. Inhibition of p38 (SB203580, BIRB796) decreased phagocytosis whereas inhibition of JNK (SP600125) decreased cytokine secretion (IL-6, CCL2, and CXCL1) after LPS (2 fold, p&lt;0.05). DUSP1/MKP1 (MAPK phosphatase) protein was reduced by 3-fold in TRPV4 KO and inhibition of DUSP1 decreased phagocytosis and selectively increased activation of JNK. These data are the first to demonstrate new roles for macrophage TRPV4 in regulating innate immunity in a mechanosensitive manner, through MAPK activation switching.
Sts-1 and Sts-2 regulate the anti-fungal immune response
Authors: Frank David , Carpino Nick 
Published: January 2023

Abstract
Mice lacking expression of the phosphatases Sts-1 and Sts-2 (Sts−/−) are significantly resistant to disseminated candidiasis caused by the fungal pathogen Candida albicans. Resistance is associated with diminished levels of many inflammatory molecules, a reduction in kidney leukocyte infiltrates by 48 hours post infection, and an absence of inflammatory lesions. Unlike wild type mice, Sts−/− mice clear the infection from the kidney following inoculation with supra-lethal doses. To better understand parameters underlying the Sts−/− phenotype of enhanced resistance, we examined the kinetics of fungal clearance at early time points. We observed enhanced fungal clearance within Sts−/− kidneys beginning at 12–18 hrs. post infection. This corresponds to the timeframe when innate leukocytes such as Ccr2+ inflammatory monocytes (IMs) begin entering the tissue to counter the infection. To examine the role of IMs in promoting enhanced fungal clearance evident in Sts−/− animals, we infected mice lacking Ccr2 expression. Loss of Ccr2 abrogated both the fungal clearance and the survival advantage of Sts−/− mice. Analysis of innate anti-fungal effector responses revealed enhanced production of reactive oxygen species by phagocytes lacking Sts expression, with no apparent differences in the responses of other effector pathways. Downstream of fungal receptor Dectin-1, the Syk kinase was hyper-activated in Sts−/− cells. Our results identify Syk as a target of Sts activity in phagocytes and highlight a novel mechanism regulating the anti-fungal immune response. Further understanding of this regulatory pathway could aid in the development of therapeutics to protect against invasive candidiasis.
Production of leukotriene signaling mediators is limited by ORMDL3/serine palmitoyltransferase/5-lipoxygenase crosstalk
Authors: Draber Petr , Paulenda Tomas , Utekal Pavol , Halova Ivana , Kuchar Ladislav , Kuda Ondrej , Vavrova Petra , Schuster Bjoern , Potuckova Lucie , Cernohouzova Sara , Draberova Lubica , Bambouskova Monika , Bugajev Viktor 
Published: January 2023

Abstract
Leukotrienes and sphingolipids are critical lipid mediators participating in cellular signal transduction and development of various diseases. Metabolic pathways initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to leukotrienes and serine palmitoyltransferase (SPT) de novo synthesis of sphingolipids. Previous studies showed that endoplasmic reticulum membrane protein ORMDL3 inhibits the activity of SPT and sphingolipid synthesis. However, the role of ORMDL3 in synthesis of leukotrienes is not known. In this study, we used peritoneal-derived mast cells (PDMCs) isolated from mice with ORMDL3 knockout (KO) or control mice and examined their properties. We found that PDMCs with ORMDL3 KO exhibited increased calcium response and ß-glucuronidase release when activated with antigen. These events were accompanied by increased phosphorylation of IκB-α and TNF-α production. Lipid analysis showed that ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids, but also increased production of leukotriene inflammatory mediators, such as LTB4 and LTC4. These data were supported by the finding that ORMDL3 physically interacts with 5-LO. Further studies showed that 5-LO interacts with the SPT long-chain (LC)1 and SPTLC2 subunits and decreases the ceramide levels. In line with these findings, 5-LO knockdown increased the ceramide levels, and silencing of SPTLC1 decreased transition of arachidonic acid to leukotrienes. These results demonstrate physical and functional crosstalk between leukotriene and sphingolipid metabolism pathways leading to production of lipid signaling mediators.
The Role of Phosphodiesterase (Pde) 3B in the Inflammatory Response of Macrophages to LPS
Authors: Liu Yusen , Meng Xiaomei , Wang Xiantao , Snedden Ali , Kirk Sean G. , Cash Charles J. , Murphy Parker R. , Hafner Markus , Nelin Leif D. , Chen Bernadette 
Published: January 2023

Abstract
Pde3B hydrolyzes both cAMP and cGMP, important second messengers in a variety of cellular processes. Pde3B has been shown to regulate NLRP3 inflammasome activity in adipose tissue. To understand whether Pde3B plays a role in the macrophage response to bacterial infection, we derived macrophages from the bone marrow of wildtype and Pde3B knockout mice. Macrophages were then stimulated with LPS for 6 h or left untreated, and total RNA was prepared for RNA-seq analyses. Our data indicate that Pde3B expression was inhibited by LPS in macrophages. Knockout of Pde3B resulted in significant changes in the expression of a large group of genes: 427 genes were up-regulated by at least 2-fold while 428 genes were down-regulated by more than 50% in LPS stimulated macrophages as the result of Pde3B deficiency. Gene profiling indicates that a number of biological processes are affected by Pde3B knockout, including cellular ion/cation homeostasis, immune system processes, and chemotaxis. Interestingly, Pde3B deficiency disturbed the expression of several chemokines: Cxcl-1, -2, and -3 expression were decreased by 96, 60, and 75%, respectively. In contrast, Ccl-4, -7, -12, -22, Cxcl-9 and -l11 expression were enhanced 1.8, 1.5, 1.9, 2, 3, and 3-fold, respectively. Differential expression of dual specificity phosphatase (Dusp)-1, -2, -3, -4, -5, -6, -10, -11, and -16 were also seen between LPS-stimulated wildtype and Pde3B-deficient macrophages. Since the Dusps are important regulators of MAP kinases that control the expression of chemokines, alterations in the expression of Dusps provides a plausible explanation for the changes in chemokine expression. Our studies suggest that Pde3B may regulate chemokine expression via modulating MAP kinases.
Epigenetic regulation of C-Kitlo FcɛR1a+ mast cells by cannabidiol during staphylococcal enterotoxin B-induced liver injury
Authors: Culpepper Courtney D , Miranda Kathryn , Wilson Kiesha , Nagarkatti Prakash S. , Nagarkatti Mitzi 
Published: January 2023

Abstract
Although recent studies have established cannabidiol (CBD) as a potent anti-inflammatory agent, the mechanisms governing these processes remain unclear. Our previous research demonstrated that CBD treatment induced expansion of CD11b+Gr-1+MDSC in the peritoneum, which suppressed T cell functions leading to attenuation of staphylococcal enterotoxin B (SEB)-mediated liver inflammation in mice. Using the same model, we characterize the effects of CBD administration in the liver. Briefly, mice were injected intraperitoneally (I.P.) with 50mg/kg of CBD for 4 days (d), followed by SEB challenge on d 3. Flow cytometry analysis revealed a significant reduction in Vβ8+ CD8+ T cells. In agreement with our earlier findings, mice treated with CBD displayed robust expansion of Gr-1+ MDSC in the liver, which was primarily comprised of Ly6CHI MHCIILO leukocytes. As mast cells have been found to play a role in CBD-mediated induction of MDSC via increased peroxisome proliferator-activated receptor gamma (PPARγ) signaling, we examined changes to liver mast cell populations. We discovered two distinct subsets of mast cells, C-KithiFcɛR1a+ and C-Kitlo FcɛR1a+present in the liver. CBD treatment promoted expansion of the C-Kitlo FcɛR1a+ subset. Analysis by miRNA microarray displayed enrichment of miRNAs targeting mast cell growth factor, KIT ligand (KITLG), and significant downregulation of miR-217-5p, which is conserved for the co-activator of PPARγ (PPARγC1A). Increased gene expression of PPARγ was confirmed by single-cell RNA sequencing (sc-RNAseq). Taken together, our results identify CBD induced epigenetic reduction of C-Kit expression and KITLG signaling in mast cells to favor PPARγ signaling as a novel anti-inflammatory mechanism.
Differentiation of antibody-secreting cells in the thymus of human neonates
Authors: Cordero Hector , King Rodney G , Dogra Pranay , Dufeu Chloe , See Sarah B. , Chong Alexander M. , Uhlemann Anne-Catrin , Ho Siu-Hong , Farber Donna L , Kearney John F , Zorn Emmanuel 
Published: January 2023

Abstract
Thymic resident B cells were reported for the first time more than 40 years ago. Yet, their phenotype and significance remain unclear. In this study, we investigated the heterogeneity of thymic B cells in neonates as an indication of their possible function. Using thymuses from 21 neonates, we observed two consistent subsets, CD19+CD21+CD35+ and CD19+CD21−CD35−, accounting for virtually all thymic B cells. ELISPOT assays revealed the presence of IgG, IgA, IgM and even IgE antibody-secreting cells within the CD21−CD35− subset but not the CD21+CD35+ subset. To characterize the heterogeneity of CD21−CD35− subset, we used a single-cell RNA-sequencing and identified 4 separate clusters, 3 of which included cells with a phenotype and transcriptome consistent with that of B cell activation and proliferation. The more distant cluster corresponded to plasma cells (PCs) expressing IGHG, IGHA, and IGHE, implying prior class-switch recombination. To assess their specificity, we generated recombinant monoclonal antibodies (rAbs) from 362 PCs using a paired IgH and lgL chain expression cloning strategy. For comparison, rAbs were also generated from 296 undifferentiated CD21+CD35+ thymic B cells. The PC contingent was highly enriched in clones reactive to apoptotic cells when compared to CD21+CD35+ naïve B cells, an essential characteristic of natural antibodies. Remarkably, we identified individual PC clones reactive to some bacterial species also recognized by natural antibodies. Taken together, we demonstrate the differentiation of plasma cells reactive to apoptotic cells and common bacteria within the thymus, suggesting that thymus is a source of natural antibodies, contributing to newborn humoral immunity.
Differential survival requirements for Lck- and CD3e-dependent signaling defines two populations of ATMKO thymic T cell lymphomas
Authors: Hathcock karen S , An Joseph , Xu Max , Hodes Richard J 
Published: January 2023

Abstract
The product of theataxia-telangiectasia mutated gene (ATM) is a serine-threonine kinase thatcommunicates between molecules that detect dangerous DNA double strand breaksand effector mechanisms that maintain genomic integrity. ATM deficiency in bothhumans and mice is associated with genomic instability and increased malignancies. ATMKO mice exclusively develop thymic T cell lymphomas that resemble thymically-derivedT-ALL that occur in human ATM-deficient ataxia telangiectasia patients. Flowcytometry identified two subsets of ATMKO T cell lymphomas based on surfaceexpression of CD3ɛ, TCRβ and pre-Tα:Cells that express surface CD3ɛ, TCRβ and pre-Tα(surface receptor high; sRhi) and cells that do not express detectable surface CD3ɛ, TCRβ orpre-Tα(sRlo). When assayed for CD25 and CD44 expression sRhi lymphomas resemble DN3or DN4 cells while sRlo lymphomas resemble more immature DN1 or DN2 cells. Pre-TCRsignaling in normal DN3 and DN4 thymocytes provides an essential survivalsignal that is proposed to be constitutive and ligand-independent. Therefore, weasked if surface sRhi and sRlo cells are dependent on pre-TCR signaling byincubating cells with either Lck inhibitor, CAS 213743-31-8, or CD3ɛshRNA to inhibit pre-TCR signaling. Both treatments significantly inhibitedsurvival of sRhi lymphomas, while survival of sRlo cells was largely unaffected. These results suggest that ATMKO T celllymphomas arise from cells transformed at different stages of DN development andthat sRhigh cells, like normal DN3 and DN4 thymocytes, retain apre-TCR-dependent survival requirement. These results also suggest that pre-TCRsignaling may be a useful target for clinical intervention in treatment ofhuman T-ALL that express a pre-TCR.