bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crispr/Cas9 Gene Editing Targeted to an Intron of a Novel Isoform of the β-Subunit of Thyroid Stimulating Hormone in Peripheral Leukocytes
Authors: Klein John R , Pawar Hitesh N 
Published: January 2023

Abstract
Hashimoto’s disease and Graves’ disease are two forms of autoimmune thyroiditis (AIT). Although various treatments protocols are available for each disease, the specific events involved in the induction of AIT remain largely unknown. We identified a novel splice variant of the β-subunit of thyroid stimulating hormone (TSHβ) in humans and mice, referred to as TSHβv, that is produced almost exclusively by bone marrow cells and peripheral leukocytes. In mice, pituitary-derived TSHβ uses exon 4 and exon 5 to code for the TSHβ polypeptide, whereas TSHβv uses exon 5 and the 3′ end of intron 4. We recently demonstrated that during viral and bacterial infection, peripheral leukocytes traffic to the thyroid and produce large amounts of TSHβv intrathyroidally. We have speculated that although this may have beneficial physiological effects for the host, it also may be a factor in the initiation and/or perpetuation of AIT. To explore this, we made Crispr/Cas9 knockout mice in which the production of immune system TSHβv but not pituitary TSHβ is disrupted by insertion of a stop codon in the terminal portion of intron 4. Of 24 founder mice, 3 had gene editing in intron 4 as determined by PCR and sequence analyses. Those animals were back-crossed with wildtype mice to get F1 heterozygous colonies and clean genomic DNA with deletions at the stop codon insertion site. F1 offspring from two of the founder mice lacked the gene-edited site in intron 4. However, 6 offspring from the third founder animal had the expected editing in intron 4. Those animals are being bred to obtain homozygous mice for experimental use. To our knowledge, this is one of the few examples of gene disruption targeted to an intron with potential functional deletion of a novel isoform.
Epigenetic programming underpins B cell dysfunction in human SLE
Authors: Scharer Christopher , Blalock Emily , Mi Tian , Barwick Benjamin , Jenks Scott , Neary Bridget , Patterson Dillon , Deguchi Tsuneo , Lee Eun-Hyung , Wei Chungwen , Sanz Ignacio , Boss Jeremy M 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naïve cells. Although these cells have distinct markers and phenotypes, their epigenetic architecture and how it contributes to SLE remains poorly understood. To establish if epigenetic mechanisms contributed to pathogenic B cell programming in SLE, we systematically determined the DNA methylation, chromatin accessibility and transcriptome landscape of human B cell subsets from SLE and healthy subjects. To comprehensively cover the B cell lineage, we isolated resting naïve, activated subsets, class-switched memory, plasmabasts, and a newly defined effector B cell subset. These data define a differentiation hierarchy between the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Intriguingly, an SLE epigenetic signature was already established in resting naïve cells and persisted in more differentiated subsets. DNA methylation profiling revealed a set of CpG surrounding IFN response genes whose methylation status were highly predictive of disease activity. The AP-1 and EGR transcription factor networks were highly enriched in the SLE accessible chromatin and dysregulated genes. Together, these transcription factors acted in synergy with TBET to shape the epigenome of expanded SLE effector B cell subsets. These results provide insights into the epigenetic programming of human B cell subsets, identify loci that correlate with the expansion of distinct pathogenic B cell subsets, and suggest that early alterations in the epigenome may predispose B cells towards a disease fate.
In vivo depletion of microRNA-183-96-182 cluster alleviates autoimmunity in B6-lpr lupus mice
Authors: Wang Zhuang , Heid Bettina , Lu Ran , Khan Deena , Ahmed S Ansar , Dai Rujuan 
Published: January 2023

Abstract
We have previously reported a significant upregulation of microRNA-183-96-182 cluster (miR-183C) in splenocytes of several murine lupus models. In this study, we found that the upregulation of miR-183C is inversely related to the expression of Foxo1 and Foxo3a, which play important roles in the regulation of T lymphocyte homeostasis and immune responses. Importantly, we found that inhibition of miR-183C with specific antagomirs significantly reduces lupus-related inflammatory cytokines expression such as IFNg, IL-6, and IL-17 in in vitro activated splenocytes from both MRL and MRL-lpr mice. We further showed that inhibition of miR-183C increases expression of Foxo1 and that inhibition of Foxo1 with siRNA increases IFNg and IL-6 in activated splenocytes from MRL mice. These data suggest that the miR-183C may regulate inflammatory response by targeting Foxo1.
To further characterize the role of miR-183C in lupus pathogenesis, we developed B6-lpr mice with conditional depletion of miR-183C in T and B lymphocyte (miR-183C−/− B6-lpr) by serial crossbreeding of B6-lpr with miR-183Cfl/fl and hCD2-iCre mice. To our surprise, there was no significant difference in the production of IFNg and IL-6 in in vitro activated splenocytes from knock out (miR-183C−/− B6-lpr) mice when compared to that from control (miR-183Cfl/fl B6-lpr) mice. Impressively, we found that miR-183C−/− B6-lpr had reduced serum anti-dsDNA levels, spleen and lymphoid node weight when compared to controls. The histopathological analysis also revealed reduced inflammation in kidney in miR-183C−/− B6-lpr mice. Together, our results suggest a potential role of miR-183C in lupus pathogenesis and that miR-183C might be a new potential target for lupus therapy.
Distinctions in gene-expression in PBMC from male and female SLE patients
Authors: Olferiev Mikhail , Fernandez David , Greenman Dina , Peng Mary , Kirou Kyriakos , Crow Mary K 
Published: January 2023

Abstract
The female predisposition for multiple autoimmune diseases is well known. The mechanisms might be related to the female reproductive system, sex hormones, incomplete X-chromosomal inactivation etc. We investigated gene expression in PBMC of quiescent SLE patients and corresponding healthy donors of both genders (10 matching samples per group) using the RNA-seq approach.
Principal component analysis differentiated the 4 donor groups, with PC1 reflecting disease and PC2 reflecting gender. Male SLE patients received the highest score on PC1 and were placed closer to females than healthy males on PC2. We identified 796 and 99 differentially expressed genes in male and female SLE patients compared to HD (p&lt;0.05, logFC=1.5). Fewer genes (20 vs. 27) differed between genders in SLE vs. HD (p&lt;0.05, logFC=1.5). Weighted Gene Co-expression Network Analysis (WGCNA) identified 24 clusters of co-expressed genes. The most intriguing were clusters that differed in male and female HD and were upregulated in SLE of both genders. Those clusters include genes related to EGF receptor ligands (AREG, EREG, HBEGF), transcription factors ATF3, NR4A1, NR4A2, NR4A3, cytokines and chemokines (CCL3, CXCL2, CXCL8, CCL3L3, IL1A, IL1B).
Despite being clinically similar, male SLE patients showed a higher level of affected genes and had a lower divergence from females based on gender-related genes. Male HD had the lowest basal level of pro-inflammatory genes and EGF receptor ligands. EGFR is expressed in pituitary and involved in the reproductive hypothalamic-pituitary axis, possibly providing relative protection of males. We propose that gender-specific differences in gene expression reveal immunopathogenic mechanisms in autoimmune disease.
A non-depleting PC61 antibody allows residual CD25 function that maintains regulatory T cell-mediated immune homeostasis
Authors: Hayes Erika , Hagan Cassidy , Campbell Daniel J. 
Published: January 2023

Abstract
IL-2 is a critical regulator of immune homeostasis through its role in the maintenance and function of regulatory T cells (TR) and its stimulatory capacity on effector cells. Most TR constitutively express the high affinity IL-2 receptor component CD25, which increases their sensitivity to IL-2 and allows them to outcompete NK cells and memory T cells for access to IL-2 in vivo. Historically, the blocking CD25 antibody clone PC61 has been used to examine the role of CD25 in IL-2 signaling in mice. However, interpretation of results has been difficult due to uncertainty of whether the observed in vivo effects are mediated by CD25 blockade or TR depletion. We used an engineered isoform of PC61 (PC61N297Q) that cannot deplete CD25-expressing cells to specifically explore how functional inhibition of CD25 impacts TR mediated immune homeostasis. In vivo treatment of B6 mice for one week with IL-2 neutralizing antibodies (αIL-2) reduced TR frequency and number by half in lymphoid tissues, resulting in immune dysregulation that included enhanced DC activation. By contrast, treatment with PC61N297Q did not decrease TR abundance compared to controls. Although PC61N297Q reduced TR sensitivity to IL-2 by about 100-fold compared to control TR, this was not sufficient to erase their selective advantage over effector cells for IL-2 access in vivo, as normal levels of pSTAT5 were maintained in TR stained directly ex vivo from mice treated with PC61N297Q. Residual IL-2 signaling in PC61N297Q treated TR was CD25 dependent, since it could be blocked with an antibody that directly binds IL-2 at the CD25 binding site. Thus, we conclude that reducing the CD25 function on PC61N297Q treated TR is not sufficient to alter immune homeostasis.
CD2-iCre mice reveal a role for ABCB7 in B cell development and bone marrow homeostasis
Authors: Lehrke Michael Jonathan , McCue Shaylene , Rajcula Matthew , Shapiro Michael , Shapiro Virginia Smith 
Published: January 2023

Abstract
ABCB7 is a mitochondrial transporter that exports iron-sulfur (Fe-S) cluster intermediates from the mitochondria to the cytoplasm. Using CD2-iCre ABCB7 cKO mice, we found ABCB7 is required for early B cell development. These mice have a severe block in pro-B cell development at the Fr. C to Fr. C′ transition, with reduced expression of intracellular μ and pre-BCR. CD19-cre and CD23-cre ABCB7 cKO mice have normal B cell development and peripheral B cell populations, indicating ABCB7 is required only during early B cell development. We observed reduced expression of Runx1, Ikaros, Foxo1, E47 (E2A), and Pax5 in CD2-iCre ABCB7 cKO pro-B cells. Interestingly, we also observed decreased BACH2 expression, which indicates elevated heme synthesis is occurring in the absence of ABCB7. We hypothesize that in the absence of ABCB7, developing B cells enhance heme synthesis to dispose of accumulating mitochondrial iron. Using a hen egg lysozyme BCR transgene, we were able to rescue B cell development in CD2-iCre ABCB7 cKO mice. Intriguingly, at the age of 5–8 weeks old, CD2-iCre ABCB7 cKO mice become lethargic and hunched. CBCs reveal an increase in peripheral blood neutrophils. Both the spleen and bone marrow become highly infiltrated by CD11b+Ly6Glow/+Ly6C− cells that are immature and mature neutrophils. As other knockout mice with pro-B cell blocks do not have neutrophilia, we considered if increased iron or heme is responsible for our observation. As heme can stimulate TLR4, we propose that excess heme from developing ABCB7-deficent B cells activates macrophages. In support of this, hemin induced GM-CSF production in BMDM cultures. We are currently exploring the mechanism by which excess heme production can stimulate the production of neutrophils.
MicroRNA-142 Is an Essential Negative Regulator of B Cell Maturation and Malignant Transformation
Authors: Graham Natalie Marie , Wang Wei-Le , Magilnick Nathaniel , Lee Jaewoong , Wang Huafeng , Zhang Bin , Marcucci Guido , Muschen Markus , Reyes Estefany , Boldin Mark P 
Published: January 2023

Abstract
microRNA-142 (miR-142) is expressed predominantly in cells of hematopoietic origin and plays a vital role in the regulation of innate and adaptive immunity. We have previously shown that miR-142 ablation in mice results in a marked expansion of the immature and mature B cell compartments. To better understand the role of miR-142 in B cell ontogenesis, we examined the early B cell development in miR-142 null mice using Hardy fraction analysis. Our findings indicate that miR-142 deletion significantly upregulates the frequency of pro-B cells, large pre-B cells, and immature B cells (Fractions B, C, and E), while decreasing the relative number of the earliest B cell progenitors (pre-pro-B cells; Fraction A). Our data links accumulation of pro-B and large pre-B cells in miR-142−/− bone marrow to an increase in their survival capacity. Moreover, we established miR-142 as a negative regulator of interleukein-7 receptor (IL7R), a cytokine receptor which plays a critical role in B cell maturation from pro-B to pre-B cell stage. In agreement with this notion, miR-142-deficient pro-B cells displayed elevated IL7R levels on their surface and proliferated more vigorously than WT cells in response to IL-7 stimulation in vitro. To test the effect of miR-142 dysregulation on malignant B cell transformation, we bred miR-142−/− mice with Eμ-myc transgenic mice, which spontaneously develop a mix of B cell lymphoblastic leukemia and lymphoma. Interestingly, miR-142 haploinsufficiency dramatically accelerated development of the disease in Eμ-myc transgenic mice. Collectively, our results establish miR-142 as an essential negative regulator of primary B cell development, whose dysregulation may contribute to malignant B cell transformation.
TCR-ligand interactions are required for murine epidermal Vγ3Vδ1 T cell development
Authors: Witherden Deborah , Garijo Olivia D , Kelly Ryan , Komori H. Kiyomi , Havran Wendy L 
Published: January 2023

Abstract
Dendritic epidermal T cells (DETC), which bear the Vγ3Vδ1 T cell receptor (TCR), are the exclusive T cell population resident in the murine epidermis where they act as sentinels for neighboring keratinocytes. γδ T cells differentiate from lymphoid precursors largely within the thymic microenvironment in a highly organized manner. Cellular selection processes are also believed to be involved and indeed several lines of evidence suggest that proper development and homing of the Vγ3Vδ1+ subset does require positive selection events in the fetal thymus. To evaluate the role of TCR interactions with cognate ligand in Vγ3Vδ1 T cell development and maturation, we used a soluble DETC TCR tetramer to characterize ligand expression in the fetal thymus and skin. We show that temporal expression of ligand in the thymus closely mimics the window of DETC intrathymic development. Ligand expression is independent of Skint1, a molecule shown to be important for thymic maturation of DETC and their subsequent residence in the epidermis. Furthermore, using the soluble DETC TCR tetramer as a ligand blocking reagent, we show that in fetal thymic reaggregation cultures, TCR-ligand interactions are essential for DETC development and maturation. Current experiments are investigating the nature of the ligand-expressing thymic epithelial cell population. Understanding TCR-ligand interactions involved in DETC development may provide important insight into identifying the enigmatic DETC antigen.
Atrophied thymus creates tTreg repertoire holes altering an antigen-specific population in the periphery
Authors: Thomas Rachel Ruth , Oh Jiyoung , Wang Weikan , Su Dongming 
Published: January 2023

Abstract
We have shown that there is an increased ratio of total thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells generated by the atrophied thymus related to aging. This observation, coupled with the accumulation of peripheral Treg (pTreg) cells with age, poses the difficult question of why Tregs in the periphery of the elderly are unable to suppress amplified self-reactivity-induced inflammaging. We utilized a chimeric mouse model with immune system reconstitution in which lethally irradiated rat insulin promotor-driven (RIP) mOVA host mice received mixed OT-II TCR transgenic and wild-type bone marrow, each expressing distinct congenic identifiers (CD45.1 vs CD45.2). In this system, OVA serves as a mock self-antigen, which is expressed mainly in the pancreas of the host mice. Further, our mOVA host mice carried a FoxN1-floxed gene, for induction of conditional FoxN1 knock-out, resulting in thymic atrophy analogous to age-related thymic atrophy. We observed that chimeric mice with induced thymic atrophy exhibited significantly decreased OVA-specific (OT-II) Tregs, but not total (pan) Tregs, in the spleen and pancreas. The specific Treg cells of mice with thymic atrophy were also more instable, showing relatively decreased FoxP3 expression, and had greater plasticity, showing an increased Th1-like (IFNγ+FoxP3+) Treg phenotype. Overall, our preliminary results suggest that thymic atrophy creates changes in the antigen-specific repertoire during tTreg generation resulting in potential “holes” that may contribute to inflammaging in the elderly and negatively impact Treg cell-mediated regulation in the aged immune system. (Supported by NIH/NIAID 1R01AI121147)
Neurokinin A renders mast cells responsive to autocrine IL-10
Authors: Sumpter Tina L , Li Yuting , Rao Mekhala , Gohel Dhruv , Falo Louis D , Larregina Adriana T 
Published: January 2023

Abstract
Interleukin (IL)-10 is produced by skin-resident mast cells (MCs). However, little is known about mechanisms regulating IL-10 production by MCs. In this study, we investigated the capacity for tissue-derived factors, such as neuropeptides, to regulate IL-10 release from MCs. Neurokinin A (NKA), a tachykinin family member, is highly expressed in the skin, but few studies have addressed its impact on immune cells. In this study, we specifically hypothesized that NKA would affect IL-10 levels in MCs. We investigated the effects of NKA in vitro using bone marrow (BM)MCs, loaded with IgE then activated with cross-linking antigen. Neurokinin A affected three levels of IL-10 regulation in IgE-activated BMMCs: 1) NKA augmented IL-10R expression, 2) NKA increased IL-10 transcription 3) NKA decreased release of an IL-10-degrading protease, without affecting degranulation. Neurokinin A potently reduced IL-4, IL-6 and IL-13 transcription and STAT5 phosphorylation. Neutralization of IL-10 reversed the effects of NKA on transcription and on STAT5 phosphorylation. In the skin, intradermal administration of NKA into MC-sufficient mice upregulated expression of IL-10 RNA, while NKA had no effect on IL-10 RNA when administered into mice lacking cutaneous MCs. We used the murine passive cutaneous anaphylaxis (PCA) model to investigate the relationship between NKA and IgE-initiated MC activation in the skin. Neurokinin A reduced early and late PCA in a manner requiring MC-derived IL-10. Collectively, we have identified a novel mechanism through which the neuropeptide, NKA, acts on MCs to regulate IL-10, broadening our understanding of the role of neuropeptides in MC function.
Ceramide-CD300f interaction inhibits Mrgprb2-mediated mast cell activation and pseudo-allergic drug reactions in mice
Authors: Takamori Ayako , Ando Tomoaki , Kaitani Ayako , Maehara Akie , Izawa Kumi , Okumura Ko , Kitaura Jiro 
Published: January 2023

Abstract
We have previously identified ceramide as a ligand for an inhibitory receptor CD300f (also called LMIR3) and demonstrated that ceramide-CD300f interaction inhibits IgE- and mast cell-dependent allergic responses. According to a recent report, basic secretagogues including inflammatory peptides and drugs associated with allergic-type reactions (c. g., compound 48/80, Ciprofloxacin, or Icatibant) activated connective tissue-type mast cells via Mrgprb2 in mice or via MRGPRX2 in human. To investigate a role of CD300f in pseudo-allergic reactions in mice, compound 48/80, Ciprofloxacin, or Icatibant together with Evans blue dye was intradermally injected into wild-type or CD300f-deficient mice. The results showed that CD300f deficiency enhanced vascular permeability in skin treated with basic secretagogues. Interestingly, ceramide-CD300f interaction inhibited compound 48/80-stimulated degranulation of peritoneal mast cells. Moreover, pretreatment with anti-ceramide antibody or ceramide vesicles enhanced or inhibited vascular permeability in compound 48/80-stimulated skin of wild-type mice. On the other hand, the same pretreatment failed to influence vascular permeability in compound 48/80-stimulated skin of CD300f-deficient mice. Thus, ceramide-CD300f interaction inhibits IgE-independent Mrgprb2-mediated pseudo-allergic reactions as well as IgE-dependent allergic reactions.
A review of the evidence for the existence of and relationship between hypermobile Ehlers-Danlos syndrome (hEDS), postural orthostatic tachycardia syndrome (POTS), and mast cell activation syndrome (MCAS)
Authors: Kohn Alison , Chang Christopher 
Published: January 2023

Abstract
The association of hypermobile Ehlers-Danlos syndrome (hEDS), mast cell activation syndrome (MCAS) and postural orthostatic tachycardia syndrome (POTS) is unclear. We conducted an extensive review of the literature using 2 different search strategies. A narrower strategy included 88 searches of various combinations of terms for each of the three conditions, yielding 19 unique papers. A broader search included 136 searches of various combinations of terms but included all forms of EDS and yielded 40 unique papers. Of these, only 4 and 9 papers from the narrower and broader search strategies were original research articles. None of these papers resulted from a combination of the search terms for the three conditions. All three clinical entities are controversial in either existence or pathogenesis. MCAS is a poorly defined clinical entity and many studies do not adhere to the proposed criteria when establishing the diagnosis. Patients previously diagnosed with EDS hypermobility type may not meet the new, stricter criteria for hEDS, but may for a less severe hypermobility spectrum disorder (HSD). The pathophysiology of POTS is still unclear. An evidence-based, common pathophysiologic mechanism between any of the two, much less all three conditions, has yet to be described. Our review of the literature shows that current evidence is lacking on the existence of MCAS or hEDS as separate or significant clinical entities. Studies proposing a relationship between the three clinical entities are either biased or based on outdated criteria. The reason behind the purported association of these entities stems from an overlapping pool of vague, subjective symptoms, which is inadequate evidence to conclude that any such relationship exists.