bluuebunny/crossref_metadata_2025_split huggingface dataset:
Destination active chromatin: RAG2 regions that pave the way to V(D)J recombination
Authors: Simpson Destiny A , Rodgers William , Rodgers Karla K 
Published: February 2023

Abstract
V(D)J recombination is required to establish the diversity of antigen receptors in the adaptive immune system. Recombination activating gene 2 (RAG2), a protein subunit of the nuclear V(D)J recombinase, rearranges gene segments to generate functional genes that encode antigen receptors. Based on our previous results, we hypothesized that a negatively charged region (the acidic hinge) in RAG2 functions to regulate localization of the V(D)J recombinase with chromatin. To test this, we generated the acidic hinge mutant 1 (A1; D/E 370–383 A), acidic hinge mutant 2 (A2; D/E 404–410 A), and D400H (a known mutation that causes immunodeficiency) to determine impact on chromatin association and V(D)J recombination activity. Dynamics of RAG2 mobility with the chromatin were determined with fluorescence recovery after photobleaching (FRAP) experiments with stably expressed GFP-tagged wild type (WT) or mutant RAG2 in RAG2−/− pro-B cells. Changes in V(D)J recombination activity and in RAG2 localization was determined for cells expressing the WT versus mutant RAG2 proteins. FRAP experiments indicated that the acidic hinge mutants had more tightly bound interactions with chromatin, as compared to WT RAG2. A1 resulted in increased V(D)J recombination, whereas, A2 and D400H led to decreased V(D)J recombination. Interestingly, these results correlated with an increased and a decreased nuclear localization of A1 and A2, respectively, relative to WT RAG2. These results indicate that the acidic region has a role in the cellular localization of the V(D)J recombinase, affecting its recombination efficiency. However, D400H localized similarly to WT RAG2, indicating the residue has a more direct role in the nuclear function of the V(D)J recombinase.
Individual zinc finger domains of Ikaros have specific roles in regulating murine myeloid hematopoietic development
Authors: Vestbostad Johanne , Podgorica Mirjam , Strege Matthew , Pinheiro Douglas , Munthe Ludvig , Schjerven Hilde 
Published: February 2023

Abstract
The Ikzf1 gene (encoding Ikaros) is essential for B cell development, but also plays important roles in the development of other hematopoietic lineages. Ikaros is a zinc finger (ZnF) transcription factor, with ZnF1 through ZnF4 involved in DNA binding, and ZnF5-ZnF6 required for dimerization. Previous work in our group has shown that germline deletion of the first or fourth ZnFs (ΔF1 and ΔF4 mice) resulted in different defects in lymphoid development. Here, we present our results from investigation of the roles of ZnF1 and ZnF4 in myeloid cell development.
Flow cytometry analysis of bone marrow (BM), peritoneal cavity, lung and spleen cells show that ΔF1 and ΔF4 mice have significantly altered levels of eosinophils, basophils, monocytes, and mast cells. Most importantly, the observed changes differ between the two mutants.
Interestingly, we observed a systemic increase in eosinophils in all tissues investigated in one of our ZnF-mutant mice. When culturing BM cells ex-vivo, we see that cells from the mutant mice proliferate at a higher rate than that of wt, and respond more actively to cytokine stimulation. Furthermore, ex vivo differentiation assay displayed a ZnF-dependent defect in mast cell/basophil development, and skewing towards eosinophil development, supporting the finding that ZnF1 and ZnF4 of Ikaros differentially regulate granulocyte development.
In addition to studying immune cell subsets and ex-vivo stimulation assays, we aim to sort granulocyte cell populations from the ΔF1 and ΔF4 mice and perform RNA-seq to investigate differential gene regulation. We conclude that Ikzf1 plays a role in the regulation of eosinophils, mast cells and basophils, and will present results to date.
Single-cell trajectory analysis reveals a melanoma-driven distinct hematopoietic response in murine spleen
Authors: Kara Nihan , Samusik Nikolay , Shi Xiaoshan , Lomas Chip , Widmann Stephanie , Tyznik Aaron J 
Published: February 2023

Abstract
Hematopoietic stem and progenitor cells (HSPCs) are a rare population of precursor cells residing in bone marrow that replenish blood cells throughout adult life. Growing evidence suggests that tumor progression can interfere with normal hematopoiesis, skew the host system to undergo myeloid biased changes and cause extramedullary hematopoiesis (EMH) in organs such as the spleen. In this study, we investigated tumor-driven phenotypic and molecular alterations in HSPCs during EMH using single-cell multiomics. We used a B16-F10 melanoma mouse model which showed splenomegaly and alterations in the composition of the HSPCs in the spleen consistent with EMH. We then isolated four HSPC populations from the spleen and bone marrow of melanoma-burdened mice using the BD FACSMelody™ Cell Sorter and used the BD Rhapsody™ Single-Cell Analysis System for the downstream single-cell multiomics analysis. We utilized oligo-conjugated antibodies for NGS-based sample multiplexing (BD® Single-Cell Multiplexing Kit) and protein detection (BD® AbSeq Assay), along with whole transcriptome analysis for a comprehensive analysis of mRNA expression. We observed tumor-driven distinct hematopoietic response and alterations in developmental trajectories of HSPCs during EMH using the X-shift algorithm paired with single-cell force-directed layout visualization. Our findings highlight melanoma-driven perturbations in hematopoiesis by utilizing single-cell multiomics in a murine model.
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Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice
Authors: Costa Thiago Alves da , Peterson Jacob , Lang Julie , Shulman Jeremy , Freed Brian M , Boackle Susan A , Lauzurica Pilar , Torres Raul M , Pelanda Roberta 
Published: February 2023

Abstract
Central B cell tolerance, the process responsible for mitigating the development of autoreactive B cells, has been extensively studied in mice, while little is known of how human B cells are tolerized in the bone marrow due to the inability to phenotypically distinguish autoreactive and non-autoreactive immature B cells and the difficulty in accessing fresh human bone marrow biopsies. Using a human immune system mouse model where all human Igk+ B cells are autoreactive and therefore tolerized in the bone marrow, we show that human autoreactive immature B cells are phenotypically different from non-autoreactive due to the differential expression of CD69, CD81, CXCR4 and other surface glycoproteins, while also exhibiting lower activation of ERK. Upon treatment of human immune system mice and autoreactive mice with a CXCR4 antagonist we show that signaling through this receptor is necessary to prevent both human and mouse autoreactive B cell clones to egress the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.
MiRNA-17-92 cluster is required for the embryonic development of Langerhans cells and dendritic epidermal T cells
Authors: Zhou Li , Landschoot Lauren , Yu Qian , Yao Yi , Mi Qing-Sheng 
Published: February 2023

Abstract
Mouse epidermis contains two major immune cells, antigen presenting Langerhans cells (LCs) and dendritic epidermal T cells (DETCs), which regulate skin immunity and involve in disease pathogenesis. Recent lineage-tracing studies have uncovered that mouse DETCs and LCs are derived from embryonic yolk-sac-derived hematopoietic precursors and self-maintain after birth. However, detailed regulating mechanisms related to their ontogeny and homeostasis remain unclear. MicroRNAs (miRNAs) are important post transcriptional regulators of protein-coding genes. Using a CSF1R-cre-mediated Dicer deletion mouse model, we found the critical involvement of miRNAs in the ontogeny of both LCs and DETCs. To further investigate the role of individual miRNAs in the ontogeny of LCs and DETCs, we evaluated the expression of miRNAs in the precursors of LCs and DETCs at different embryonic developmental stages and identified that miR17-92 cluster was highly expressed and dynamically regulated. We next generated a Csf1rCre-mediated miR17-92 deletion mouse model (miR17-92 KO) and found that conditionally deletion of miR17-92 led to significantly reduced number and interrupted phenotypes of epidermis LC and DETC precursors at E18.5 and P0 of miR17-92 KO embryos. Defective LC precursors were also identified at E14.5 and E16.5 embryonic skin, while diminished number and blocked maturation of Vg3+ DETC precursors were identified in the thymus of the same embryonic stages in miR17-92 KO mice. Overall, our results indicate that miR17-92 serves as a key epigenetic regulator in the ontogeny of LCs and DETCs. Detailed molecular mechanisms underlining the miR17-92 mediated LC and DETC developmental regulation are currently under investigation.
The role of Klf4 in thymic epithelial cells
Authors: Depoërs Lucyle , Brochu Sylvie , Houques Chloé , Dumont-Lagacé Maude , Perreault Claude 
Published: February 2023

Abstract
Thymic epithelial cells (TECs) are essential for the development and the selection of naïve T cells that will be able to discriminate the non-self from self-peptides. The thymus is the organ that starts degenerating the earliest in the organism, leading to a decrease in naïve T cell production. Starting from 1 year old in humans, thymic involution accelerates at puberty, partially caused by an increase in circulating sex steroids (SS). SS induce thymic involution, mediated indirectly by nuclear receptors in TECs. Little is known about how and why the age-related thymic involution is induced. We identified Klf4 as a potential regulator of TEC function and differentiation. Klf4 is a transcription factor that induces differentiation and inhibits proliferation in epithelial cells from various tissues. We showed that Klf4 expression is negatively correlated with TEC proliferation in different contexts. Interestingly, Klf4 is known as a mediator of nuclear receptors response and is a sex hormone target in several tissues. We discovered that Klf4 was more expressed in male TECs than in female TECs. Importantly, TECs are less proliferative in males than in females. Moreover, Klf4 expression was reduced when males were castrated. These results suggest that Klf4 has an important role in TEC homeostasis and is a mediator of SS response. We are currently testing the role of Klf4 in TECs by phenotypic and transcriptomic analysis on transgenic mice in which Klf4 is specifically deleted in TECs. Using testosterone treatment, we are studying the effect of SS on TEC and thymic output with or without Klf4 expression. Finally, we will assess the direct KLF4 target genes in TECs using ChIP-Seq.
Topological and transcriptional control of TCRβ antigen receptor diversification
Authors: Nganga Vincent K , Allyn Brittney , Hayer Katharina , Collins Patrick , Oyeniran Clement , Bassing Craig , Oltz Eugene 
Published: February 2023

Abstract
Antigen receptor (AgR) loci undergo distinct developmentally regulated changes that include a transcriptional burst and locus compaction to facilitate recombination between V and (D)J gene segments during the primary diversification process. In order to understand how this contributes to diverse AgR repertoires, we generated mice lacking T cell receptor beta (TCRβ) cis-regulatory and CTCF binding elements (CBEs) with potential to serve as locus loop anchors. We then systematically compared epigenetics, transcriptomes, and 3D genome topologies of wild-type and mutant thymocytes populations coupled with their TCRβ repertoires. We find that in DN thymocytes, contacts to the recombination center (RC) are limited to other transcriptionally active chromatin comprising of all Vβ segments, while excluding neighboring heterochromatic regions, supporting our prediction that compaction is driven by homotypic chromatin forces. We identify CBEs and cis regulatory elements that either a) serve as boundary elements, b) control select Vβ accessibility and interaction with the RC, or c) control proximal versus distal skewing of the repertoire. Collectively, our results reveal mechanisms by which CBEs cement TCRβ topological structures and allow efficient recombination of the distal Vβs during AgR diversification.
Natural killer cells as key regulators of cutaneous wound healing
Authors: Jordon Alexander R.D , Crane Meredith J , Jamieson Amanda M 
Published: February 2023

Abstract
Annually in the U.S, the cost to treat the more than 6 million sufferers of chronic, non-healing wounds exceeds 50 billion U.S.D. Despite extensive characterization of the functionally diverse cellular and molecular constituents found in the wound bed, wound healing therapies are lacking. The pro- and anti-inflammatory capabilities of the innate immune system make it critical in the wound healing process. Natural killer (NK) cells are innate lymphocytes which exhibit similar pro- and anti-inflammatory capabilities. Despite the breadth of their functions, ranging from anti-microbial to maintaining a healthy pregnancy, there is a paucity of information regarding NK cells in the wound healing process. Using mouse wound models, we have identified 2 distinct populations of NK cells abundantly present in seven-day old sterile, cutaneous wounds. In addition, we have identified NK cells in the wound effluent of patients who underwent corrective surgery for idiopathic scoliosis. Previous work in different models suggests that these 2 NK cell populations exhibit proliferative effector functions related to their potential to facilitate organogenesis and T cell activation. Interestingly, these are both processes implicated in late stage wound healing. Using our mouse model of cutaneous wound healing we have shown that NK cell depletion delays closure. Furthermore, we show that NK cell depleted wounds demonstrate an aberrant growth factor response. With this work we aim to determine the impacts wound natural killer cell phenotype and function have on cutaneous wound healing outcomes and to determine the role of natural killer cells in wound growth factor expression.
Interleukin-22 by altering type O-glycan profile of mucin plays a protective role in animal model of experimental colitis
Authors: Singh Ankita , Beaupre Michael , Lin Xun , Gaudino Stephen , Kumar Pawan 
Published: February 2023

Abstract
IL-22 is known to play both protective and inflammatory role in the intestine. Thus, the effect of IL-22 in intestine is complex, and cell type-specific function of this cytokine requires further investigation.
To identify target cell type of IL-22, we have used entire intestinal epithelial cell (IL-22Ra1ΔIEC), Paneth (IL-22Ra1ΔDefa6) and Lgr5+ intestinal stem cell (IL-22Ra1ΔLgr5)-specific IL-22Ra1 knockout mice models. Experimental colitis was developed by providing dextran sulphate sodium (DSS) in drinking water for first 8 days, and change to normal water for next two days. On examining DSS induced colitis development, significant increase in disease severity as revealed by reduced colon length, greater weight loss and histopathological changes in tissues were observed in IL-22ra1ΔIEC as compared to IL-22ra1fl/fl mice. No difference in disease severity was observed in IL-22ra1ΔDefa6 and IL-22ra1ΔLgr5 mice compared to their respective littermate control IL-22Ra1fl/fl mice. Quantitative PCR data show increased expression of inflammatory cytokines in distal colon of IL-22ra1ΔIEC compared to IL-22ra1fl/fl mice. Alcian blue staining revealed no difference in goblet cell number, however, expression of selective glycosyltransferase, required for mucin type-O glycosylation core extension, was significantly reduced in distal colon of IL-22Ra1ΔIEC compared to IL-22Ra1fl/fl mice on day 10 DSS/water treatment. Mucin type-O glycan analysis by MALDI-TOF revealed an increase in core 1 and decrease in core 4 structure in IL-22Ra1ΔIEC mice compared to IL-22Ra1fl/fl mice at steady state.
Thus, IL-22 through glycosyltransferase activity may maintain intestinal barrier integrity and alleviate severity of experimental colitis.
SerpinB7 deficiency contributes to development of psoriasis via Calcium-mediated keratinocyte differentiation dysfunction
Authors: Zheng Huaping 
Published: February 2023

Abstract
Defective execution of protease and protease inhibitor, which meditates abnormal signaling cascade, is merging as key contributors to autoimmune disease. SerpinB7, is identified as a skin-specific endogenous protease inhibitor, the role and underlying mechanism in psoriasis is poorly understood. Here we found that SerpinB7 is highly expressed in psoriatic keratinocytes of patient and imiquimod-induced psoriatic lesion in mice. SerpinB7−/− mice showed abnormal epidermal barrier integrity and skin architecture in homeostasis, and aggravated psoriatic lesion with inhibiting terminal differentiation and increasing inflammatory cells infiltration compared to WT after IMQ treatment. Mechanistically, SerpinB7 deficiency resulted in excessive proliferation and impaired differentiation, as well as increased chemokines and antimicrobial peptide expression in human epidermal keratinocyte and mouse primary keratinocyte. Transcriptome analysis demonstrate that SerpinB7 has a significant correlation with keratinocyte differentiation, consistent with WGCNA analysis from GTEx. Notably, we demonstrated that SerpinB7 deficiency prevented the increase of intracellular Ca2+ influx and the generation of mitochondria-derived reactive oxygen species (ROS), which were partly eliminated by antioxidant NAC and intracellular Ca2+ chelator BAPTA-AM. More importantly, iTraq and iTraq-tail proteomics demostrated SerpinB7 deficiency attenuated proteolytic networks that control calcium flux in keratinocytes. Our findings first described the critical role of SerpinB7 in the regulation of keratinocytes differentiation and psoriatic microenvironment, implying a new candidate for therapeutic targets in psoriasis.
Microvesicle-associated immunomodulators are critical regulators of immune dysfunction following burn injury
Authors: Willis Micah L , Mahung Cressida , Coleman Leon , Wallet Shannon , Cairns Bruce , Maile Robert 
Published: February 2023

Abstract
~450,000 people in the USA suffer from burn injury each year, with ~3,500 deaths. Burn injury is characterized by early (0–72h post injury) systemic cell death and widespread release of cytokines and chemokines, followed by immunosuppression with increased risk of opportunistic infection. We and others have described DAMP (damage associated molecular patterns)/TLR (Toll-like Receptor) ligation as driving this response. Microvesicles (MV) are extracellular vesicles released during physiological or pathological processes. We have previously reported that following burn injury there is an increased frequency of heterogenous MV, with a population of MV that contained co-localized HMGB1 DAMP and IL-1b. We hypothesize that burn induced MV are an unidentified reservoir of immunomodulators and drive immune dysfunction. To test this, we utilized burn patients from the NC Jaycee Burn Center, and our established model of burn injury in which wildtype C57BL/6 mice were exposed to a cutaneous burn. MV isolated from the plasma of patients 24h after injury were either lysed or left intact. Concentrations of membrane-associated and free cytokines/chemokines were quantified by Luminex assay. We observed that some immunomodulators such as IL-27, IL-10, IL-16 and MCP-1 were exclusively found in MV. We then performed adoptive transfer studies whereby MV from burn or unburned mice isolated 24h after injury were transferred into unburned mice by tail vein injection. We observed that adoptive transfer of burn-induced MV caused significantly increased plasma levels of IL-10, IL-6, IL-8 and MCP-1 24h after transfer compared to MV isolated from uninjured controls. These data indicate that injury-induced MV play a critical role in immune dysfunction.
Changes in peripheral intraepidermal nerve fiber densities in CD137 ligand – mediated neuropathic pain following sciatic nerve crush
Authors: Jordan Ethan E , Thakkar Bianca , Cao Ling 
Published: February 2023

Abstract
Previously, we have shown that the co-stimulatory molecule, CD137L plays a significant role in the development of neuropathic pain-like behaviors following sciatic nerve crush (SNC). Intraepidermal nerve fiber (IENF) density has shown high specificity and reliability in testing for small fiber sensory neuropathy. Here, we investigated the effects of CD137L depletion on the changes of IENFs following SNC. Both wild type (WT) C57BL/6 and B6_CD137LKO mice underwent SNC and sham surgeries. The ipsilateral and contralateral hind paws from individual mice were collected on days 0 (naïve), 3, 7, 14, 28, and 56 following surgery (2 males and 2 females per group per time point). All tissues were processed for fluorescent immunohistochemistry (IHC), which was performed with antibodies against PGP9.5 (pan-neuronal marker for total nerve fibers) and CGRP (peptidergic nociceptive fibers) followed by respective fluorescence-labeled secondary antibodies and fluorescence-labeled IB4 (non-peptidergic nociceptive fibers). The density of fibers was determined based on the respective IENF numbers (PGP9.5+, CGRP+ or IB4+) and the length of corresponding skin tissue. At the baseline, CD137L KO and WT mice had comparable levels of IENF densities for all types of fibers examined. Although surgery (SNC or sham) induced a transient increase in IB4 IENF density in CD137L KO mice at day 28, neither surgery induced significant changes in IENF densities over time. We did not observe significant differences between SNC vs. sham groups in either WT or CD137L KO mice. Therefore CD137L-mediated neuropathic pain behaviors cannot be attributed to reduction of hind paw IENF densities.
Immune and microRNA differentials post-simulated deep spaceflight
Authors: Paul Amber M , Cheng-Campbell Margareth , Blaber Elizabeth A , Anand Sulekha , Bhattacharya Sharmila , Zwart Sara R , Crucian Brian E , Smith Scott M , Meller Robert , Grabham Peter , Beheshti Afshin 
Published: February 2023

Abstract
Spaceflight can cause immune system dysfunction in astronauts with little understanding of immune outcomes in deep space. This study assessed immune responses in mice following simulated deep spaceflight, along with retrospective data from astronauts on the International Space Station (ISS). We simulated microgravity using the hindlimb unloaded mouse model alone or in combination with the newly developed simulated galactic cosmic rays or solar particle events irradiation at NASA’s Space Radiation Laboratory. Immune differentials revealed unique immune diversity following each experimental platform, suggesting consequences for deep spaceflight. Furthermore, circulating plasma microRNA sequencing analysis revealed immune system dysregulation. An ISS astronaut cohort showed elevated inflammation during low-Earth orbit missions, which supports our simulated ground experiments. Herein, distinct circulating immune biomarkers are defined by irradiation type, coupled to simulated microgravity, which can be promising targets for countermeasure developments and programmatic queries.