bluuebunny/crossref_metadata_2025_split huggingface dataset:
Desmosterol, an intermediate of cholesterol biosynthesis, modulates B cell activation and functions
Authors: Gauronskas Phillip Joseph , Waseem Tayab , McDonald Jeffrey , Fernandez-Hernando Carlos , Galkina Elena 
Published: January 2023

Abstract

Introduction
Atherosclerosis is a disease of large/medium size vessels attributed to a hyperlipidemic environment and a low-grade chronic inflammation. Normally, macrophages uptake modified low-density lipoprotein (mLDL), resulting in excess intracellular desmosterol, an immediate precursor to cholesterol, which promotes anti-inflammatory signaling. In contrast, mLDL uptake by macrophages in atherosclerosis produces foam cells that play an atherogenic role. While it has been previously shown that B cells can uptake mLDL, it remains unclear how mLDL uptake modulates B cell phenotype.

Methods
Murine B cells were treated with 1 mM of desmosterol for 12/24 hrs, cholesterol efflux genes were analyzed using qPCR. CFSE-labeled splenocytes were incubated with LPS/BAFF, treated with 1 mM of desmosterol for 72 hrs, and analyzed via FACS. B cells isolated from transgenic mice ooverexpressing DHCR24, a dehydrocholesterol reductase, were analyzed for desmosterol content by lipidomics, Ca2+ flux by FACS, and DNP-Ficoll induced Igs production in vivo by ELISA.

Results
Desmosterol uptake results in an increased expression of cholesterol efflux genes and attenuated B cell proliferation. Overexpression of DHCR24 leads to a decreased intracellular desmosterol, hyper-response of B cells shown by increased Ca2+. DNP-Ficoll immunization of B cell-deficient mice populated with either DHCR24- or WT B cells induce more anti-IGM specific Abs in the DHCR24-B cell recipients.

Conclusions
These results suggest desmosterol is synthesized and sensed by B cells, and its accumulation regulates B cell activation thresholds in vitro and in vivo. These novel data offer new insights for B cell biology and atherosclerosis pathology.
A histone code for inducible transcription in immune responses
Authors: de Paz Alexia Martinez , Beguelin Wendy , Sahin Merve , Leslie Christina S. , Melnick Ari M. , Josefowicz Steven Z. 
Published: January 2023

Abstract
Complex organisms have been subjected to intense selective pressure by host-pathogen coevolution to adapt robust immune responses to pathogen sensing. These responses occur by inducing specific genes within the context of large genomes condensed into chromatin. Despite general description of the chromatin changes that occur across inducible genes, little is known of how specific chromatin features, including histone post-translational modifications (PTM), functionally contribute to transcriptional output of responsive genes.
We have described two “signaling-to-chromatin” pathways leading to phosphorylation of histone H3 at residues S28 and S31 that are “read out” by chromatin regulatory factors to augment transcription. H3S28 phosphorylation drives early chromatin activation preceding transcription. This modification delineates expansive chromatin architectural domains containing responsive genes. We are pursuing the hypothesis that H3S28 phosphorylation increases chromatin interaction dynamics within the compartment to favor specific enhancer-promoter interactions and concentrate transcription factors. H3.3S31 is co-transcriptionally phosphorylated along the bodies of induced genes, and coordinately stimulates co-activators and ejects repressors to augment transcription.
We hypothesize that these stimulation-dependent histone phosphorylations act synergistically as a dedicated histone code selectively employed at stimulation responsive genes in macrophages and B cells, with potential for involvement in diverse cell types.
Our data suggest that co-option of these signaling pathways may contribute to lymphomagenesis, and their disruption could represent novel therapeutic strategies.
Assessment of function of neutrophil-derived properdin in a novel functional assay
Authors: Menon Smrithi Sugumaran , Galwankar Neeti , Moore Sara R. , Ferreira Viviana P. 
Published: January 2023

Abstract
The alternative pathway (AP) of complement is an essential immune effector mechanism. Properdin (P), the only positive regulator of the AP, is required for stabilization of AP enzymatic convertases. P also binds to certain surfaces, in vitro, activating complement by recruiting a de novo C3bBb convertase. P is present in serum as dimers (P2), trimers (P3) and tetramers (P4) in a 1:2:1 ratio, with tetramers the most active, followed by trimers and dimers. Activated neutrophils release properdin from secondary granules into the cellular microenvironment. However, the distribution of neutrophil-secreted P oligomers (which would correlate with P function), and whether this distribution modulates local inflammatory processes, are unknown. We developed a novel ELISA-based assay that detects functional differences between P oligomers, based on the immobilization of P and assessment of its ability to generate C3 convertases leading to C3b deposition. Pure P2, P3, and P4 were functionally distinct in this assay and serum-derived P behaved similar to pooled P2-P4, as expected. Surprisingly, P from PMA-activated neutrophil supernatants had significantly lower activity than serum P (p&lt;0.0001), and varied between healthy donors. To understand whether serum is required for enhancing P function (i.e. by allowing P polymerization), neutrophil supernatants were pre-incubated with P-depleted serum, followed by measurement of P function by ELISA. No increase in neutrophil-derived P function was observed. Additional studies are underway to understand the mechanisms involved in the lower function of neutrophil-derived P, with the aim of contributing to understanding molecular mechanisms of AP regulation in inflammatory microenvironments.
Transcriptional regulation of IL10 gene in human macrophages
Authors: Kwon Geunho , Kang Kyuho 
Published: January 2023

Abstract
IL-10 is an anti-inflammatory cytokine with a crucial role in immune homeostasis. Dysregulated production of IL-10 has been linked to certain autoimmune and inflammatory diseases. IL-10 is strongly induced by TLR ligands and suppressed by IFN-γ as part of the synergistic activation of inflammatory genes. However, the molecular mechanisms that regulate expression of the IL10 gene, especially in human macrophages, are incompletely understood. By using epigenomic analysis of ATAC-seq and ChIP-seq analysis we have identified human macrophage-specific enhancers in the IL10 gene locus. In these putative enhancers, we observed that chromatin accessibility and histone acetylation are increased by LPS stimulation. We also found that p300 was frequently recruited to these macrophage-specific cis-regulatory elements (CREs) in LPS-stimulated macrophages. One of these potential LPS-induced enhancers, CRE+6, is located 6kb upstream of the IL10 transcriptional start site and contains transcription factor binding motifs and autoimmune disease-associated SNPs. CRE+6 showed constitutive binding of the macrophage-lineage-determining factor PU.1 and LPS-inducible binding of AP-1 and STAT3. LPS stimulation increased eRNA transcription from CRE+6. The functional relevance of CRE+6 eRNA was confirmed by eRNA-targeted knockdown experiments. Strikingly, the mechanism by which IFN-γ suppresses IL10 expression appeared to be decommissioning of enhancers, as IFN-γ strongly suppressed TLR-induced positive enhancer marks and eRNA transcription. Our study implicates that crucial enhancer elements cooperate with the core promoter in LPS- and IFN-γ-mediated regulation of IL-10 gene transcription in human macrophages.
MicroRNAs of the miR-17~92 family inhibit macrophage activation and inflammation
Authors: Zhang Xiang , Hu Xiaoyu 
Published: January 2023

Abstract
Production of pro- and anti-inflammatory cytokines by macrophages is tightly controlled by multiple layers of mechanisms including those involving miRNA-mediated regulation of post-transcriptional events. By genetically deleting all three individual miRNA clusters in the miR-17~92 family, we found that this family of miRNAs inhibit macrophage activation by promoting expression of a key anti-inflammatory cytokine IL-10 and thus suppress production of pro-inflammatory mediators such as TNF. Consistent with the heightened inflammatory responses in vitro, myeloid-specific ablation of miR-17~92 family miRNAs in vivo led to exacerbated obesity under the condition of a high-fat diet and poor survival in the DSS-induced colitis model. Moreover, miRNA-deficient adipose tissue macrophages and intestinal macrophages exhibited enhanced levels of TNF and reduced levels of IL-10. Mechanistically, miR-17~92 family miRNAs sustained IL-10 production in macrophages by promoting transcription of the Fos gene encoding a key transcription factor driving Il10 expression. The positive effects of miR-17~92 family miRNAs on Fos expression are secondary to negative regulation of Fos by transcription factor YY1 that is a direct target of miR-17~92 family miRNAs. Taken together, these results identified miR-17~92 family miRNAs as crucial regulators of the balance between pro- and anti-inflammatory cytokines and illustrated key roles for three clusters of the miR-17~92 family in cooperatively regulating macrophage activation and inflammatory responses.
CARD19 interacts with MICOS complex proteins and protects against mitochondrial dysfunction in macrophages
Authors: Rios Kariana E , Beauregard Chelsi , Zhou Ming , Conrads Thomas P , Schaefer Brian C 
Published: January 2023

Abstract
CARD19 is a mitochondrial protein of unknown function; gene expression databases indicate that CARD19 is highly expressed in myeloid cells. We have observed that Card19 −/− mice injected with LPS display elevated levels of some pro-inflammatory cytokines relative to Card19 +/+ mice. Because CARD19 is a mitochondrial protein and mitochondrial dysfunction is frequently associated with immune dysregulation and chronic inflammation, we sought to identify the function of CARD19 in macrophages. We demonstrated via super resolution microscopy that endogenous CARD19 colocalizes with mitochondrial markers in bone marrow derived macrophages (BMDMs) and has a punctate distribution. Through immunoprecipitation and mass spectrometry analyses, we determined that CARD19 interacts with MIC19 and other components of the mitochondrial contact site and cristae organizing system (MICOS) complex. We further demonstrated that CARD19 colocalizes with MIC19 in mitochondrial sub-domains via super resolution microscopy. We then measured outcomes of mitochondrial stress in BMDMs. Consistent with previous reports of MICOS deficiencies, we found that Card19 −/− BMDMs have a modestly decreased oxygen consumption rate (OCR) measured by a Seahorse extracellular flux analyzer relative to Card19 +/+ BMDMs. Additionally, Card19 −/− BMDMs display elevated levels of mitochondrial reaction oxygen species (mROS) and a population of mitochondria with a decreased mitochondrial membrane potential, as measured by flow cytometry. Based on these data, we propose that CARD19 may be a previously unknown regulator of MICOS function which potentially links this complex to other mitochondrial and/or non-mitochondrial CARD proteins in macrophages.
Sex-dimorphic trajectories in aging macrophage populations
Authors: Benayoun Bérénice A , Lu Ryan , Sampathkumar Nirmal Kumar 
Published: January 2023

Abstract
The existence of human supercentenarians reveals a surprising predictor for exceptional longevity: being female. Not only are 33 out of 34 living supercentenarians women, women are also more resistant to most diseases responsible for age-related morbidity in the US. However, because most molecular aging studies generally opt to use only one sex, sex-driven differences in aging remain poorly understood. A key compartment that can actively respond to sex-specific inputs throughout life is the immune system. Indeed, the majority of age-related diseases share common inflammatory mechanisms, a phenomenon described as “inflamm-aging”. Macrophages play an important role in the inflammatory response throughout life, and are considered major mediators of this phenomenon. Thus, to unbiasedly dissect sex differences in immune aging, we generated ‘omics’ data from 4 and 20 months old female and male mice. Intriguingly, we found that transcriptional aging in primary macrophage populations varies strongly between sexes, with up to 20-fold more aging changes in female vs. male cells. Pathways specifically downregulated in females with aging included lysosome, inflammation and phagolysosome. We confirmed experimentally that metabolic preferences of macrophages are indeed directly modulated in this context (e.g. glycolytic preference for male-derived cells). Our results support the notion that there are functional differences in aging trajectories in the immune system of female vs. male mice. Our research could provide new insights into the molecular underpinnings of sex-dimorphism in aging and disease.
Templated mutagenesis in B cell non-Ig genes
Authors: Navarrete Karla , Dale Gordon A. , Woodruff Matthew C. , Scharer Christopher D. , Boss Jeremy M. , Sanz Ignacio , Jacob Joshy 
Published: January 2023

Abstract
The immunoglobulin genes of germinal center B cells (GCB) undergo somatic hypermutation in order to generate antibody diversity. Additionally, several non-immunoglobulin (non-Ig) genes are known to accumulate mutations in the germinal center. There are two methods by which mutations are introduced at the Ig and non-Ig loci. The first is through the accumulation of pseudorandom point mutations as a result of activation-induced cytidine deaminase (AID)-dependent double stranded breaks (DSB). The second is through the process of gene conversion, which involves the use of a template sequence to repair a DSB resulting in the template being copied at the site of repair. Findings from our lab suggest that some mutations in the non-Ig genes of human GCB arise through a gene conversion-like mechanism, referred to as templated mutagenesis. We sorted 4 B cell populations, including light zone and dark zone GCB from a young human tonsil and sequenced ~500bp sections from non-Ig genes including FAS, RHOH, and BTG1 via Illumina MiSeq. Previously, our lab has developed a computational pipeline called TRACE, that is able to match mutation clusters within an input sequence to partially homologous donor templates elsewhere in the genome. TRACE-identified donor templates are statistically likely to have been used in a templated mutagenesis event which led to the mutation of the gene. Through this analysis, we found that unlike gene conversion observed in chickens, human templated mutagenesis occurs between sequences on different chromosomes. Furthermore, donor template sequences tend to originate in introns and intergenic regions, not exons. These findings have opened an avenue into the study of non-Ig templated mutagenesis in human B cells.
RIP Kinase death pathways regulate B cell homeostasis and splenic architecture
Authors: McLennan Alexandra L. , Parthasarathy Raksha , Hägglöf Thomas , Mattke Aiden , Dudley Elizabeth , Leadbetter Elizabeth A. 
Published: January 2023

Abstract
Receptor-interacting protein kinase (RIPK) 1, RIPK3, and Caspase-8, are essential adapter proteins in death receptor-induced apoptosis and programmed necrosis. Apoptosis and necroptosis are crucial for normal cell development, homeostasis, and immune responses. RIP kinases and Caspase-8 form parts of multiprotein complexes like the Death-Inducing Signaling Complex (DISC) and the necrosome, thus mediating signaling events in these cell death pathways. We compared RipK3−/−, RipK1−/−RipK3−/−Caspase-8−/−, RipK3−/−Caspase-8−/−, and RIPK1 kinase-dead (RIPK1kd) mice to define the role of RIP kinase inflammatory pathways and Caspase-8 in immune responses and lymphocyte development.
Preliminary studies show that lack of RIPK1 and Caspase-8, but not RIPK3, leads to the accumulation of Marginal Zone (MZ) B cells. Evidence from mixed-bone marrow chimera experiments reveals an intrinsic requirement for RIPK1 and caspase 8 in normal B cell differentiation post-T2 B cell status. Immunofluorescence showed splenic architecture disorganization in the T cell zone and MZB populations in RIPK-deficient mice. Surprisingly, humoral immune responses to T-dependent and T-independent antigens were largely normal, but immunization-induced splenic GC formation was diminished in mice lacking both Caspase-8 and RIPK3. Elucidation of cell development/death in the context of RIPK1 (whose ubiquitination status influences cellular homeostasis) could lead to better priming of B cells for chemotherapy-induced apoptosis. Previous studies also suggest human RIP kinases are potential therapeutic targets for treating a wide range of autoimmune, inflammatory, and neurodegenerative diseases.
Myocardial associated B cells are a dynamic subgroup of circulating B cells, in equilibrium with splenic and circulating B cells, but with organ specific features
Authors: Rocha-Resende Cibele , Yang Wei , Adamo Luigi 
Published: January 2023

Abstract

Study objective
Recent evidence shows that subgroups of circulating B cells accumulate intravascularly in the naïve murine heart. However, the timing of their appearance and their organ specificity remain unclear.

Methods and Results
We performed a systematic analysis of B cells isolated from the myocardium and other organs, from mid development to early adulthood. We found that B cells are present in the developing heart already at E13.5. Myocardial associated B cells were mostly intravascular and in close association with the endothelium. Their phenotype changed rapidly. Through flow cytometry and proteogenomics analysis we found that heart associated B cells were initially almost exclusively CD11b+ and CD11b−CD21−CD23− but became mostly CD11b−CD21+CD23+ cells at 5 weeks of age. Importantly, comparative analysis of B cells isolated from perfused hearts, livers, lungs, spleen and peripheral blood at different time points showed that the B cells isolated from the heart were in equilibrium with both splenic/circulating B cells and B cells isolated from other organs, but they had specific features. This finding was confirmed through simultaneous 10X single cells sequencing of hashtag labelled B cells.

Conclusions
Taken together these observations highlight that myocardial associated B cells are part of a tightly regulated population of circulating B lymphocytes that exists in multiple organs and is in dynamic equilibrium with the spleen and peripheral blood B cells. Further work will be needed to identify the molecular mechanisms that lead to the accumulation of specific subsets of circulating B cells in peripheral organs and to investigate their functional significance.
The class II peptide editor, H2-M, affects the development and repertoire of B-1 cells
Authors: Ghosh Debopam , Pham Tho D , He Xiao , O’mara Mary E , Kantor Aaron B , Nguyen Khoa , Sengupta Deepanwita , Eisenlohr Laurence C , Jensen Peter E , Herzenberg Leonore A , Boyd Scott D , Ghosn Eliver E.B. , Mellins Elizabeth D 
Published: January 2023

Abstract
Non-classical major histocompatibility complex class-II (MHCII) protein H2-M edits peptides bound to conventional MHCII (pMHCII) in favor of stable pMHCII complexes. Although the impact of H2-M deficiency on pMHCII expression and T cell activation is well-studied, information on how this protein affects the development and phenotypic profile of APCs is lacking. We found that the absence of H2-M in C57BL/6 (B6) mice, resulted in a loss of surface expression and peptide-cargo of MHCII molecules in B cells across lymphoid organs. Lower pMHCII expression reduced MHCII association with BCR, affecting the integrated BCR signaling. Importantly, in H2-M deficient B6 mice, compared to the wildtype mice, frequency and abundance of B-1 cells, but not conventional B-2 cells, was reduced in the spleen and peritoneum. This H2-M mediated effect on the B-1 cell population was only evident in the B6 background (I-Ab), but not in BALB/c (I-Ad/I-Ed), indicating an MHCII haplotype-dependent phenomenon. A decrease in B-1 cell number also was evident in immature B-1 cells, emphasizing that H2-M deficiency affects B-1 cell development. In H2-M KO mice compared to WT B6, B-1 cells display a significantly lower self-renewal capacity and a higher rate of apoptosis. Furthermore, H2-M deficiency alters the B-1 BCR repertoire, selecting for B-1 cells specific for highly abundant epitopes, but not for low-frequency epitopes. Collectively, these data identify the impact of H2-M/MHCII interaction as a regulator of BCR signaling that influences the selection, maturation, and maintenance of mature B-1 cell clones in the periphery.
Alteration in TNFα mediates impaired antigen-specific humoral responses in sepsis survivors
Authors: Rana Minakshi , La Bella Andrea , Lederman Rivka , Ochani Mahendar , Volpe Bruce T. , Sherry Barbara , Diamond Betty 
Published: January 2023

Abstract
Sepsis survivors show impaired responsiveness to antigen (Ag), which is linked to increased susceptibility to infection. Using the cecal ligation and puncture (CLP) model of sepsis, we showed that CLP surviving mice have a reduced antibody response to the T-dependent Ag, NP-CGG, compared to mice that underwent a sham procedure, but an intact response to the T-independent Ag, NP-Ficoll. CLP mice have reduced TNFα levels due to sustained vagus nerve activity. We hypothesized that reduced TNFα production might lead to disruption in follicular dendritic cell (FDC) function and impaired germinal center (GC) responses. FDCs play a key role in GC responses and TNFα is critical for FDC clustering and maturation. Immunofluorescence staining in spleens showed a reduced number of FDC clusters and reduced binding of immune complexes on FDCs in CLP mice compared to sham mice. NP-specific GC B cells sorted from CLP mice immunized with NP-CGG exhibit reduced TNFα, AICDA, and BCL6 mRNA expression compared to sham mice. To confirm the role of the vagus nerve in impaired FDC clusters, CLP mice were subjected to bilateral subdiaphragmatic vagotomy 2 weeks post-CLP. These mice exhibit increased anti-NP IgG levels in response to immunization with NP-CGG compared to non-vagotomized CLP mice. FDC are radioresistant cells; therefore, we lethally irradiated sham and CLP mice and reconstituted them with bone marrow cells from naive mice that never experienced sepsis. CLP mice still show an impaired Ag-specific response confirming involvement of FDCs and vagus nerve signals. In summary, our data suggest that altered vagus nerve activity, low TNFα, and disruption in FDC function contributes to reduced Ag-specific humoral responses in sepsis survivors.