bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of β-Adrenergic Receptor Kinases in the Internalization of C-C Chemokine Receptor Seven (CCR7) in Mammalian T Cells
Authors: Bill Colin A , Sanchez Anahi , Amparan Jailene , Vines Charlotte M 
Published: January 2023

Abstract
Ligand mediated GPCR internalization/signaling is regulated by G protein-coupled receptor kinase (GRK) induced receptor phosphorylation and recruitment of β arrestins. The GPCR chemokine receptor, CCR7, has two ligands, CCL19 and CCL21 that mediate chemotactic migration of CCR7 expressing immune cells, including naïve T cells. Distinct internalization/signaling pathways are activated in T cells depending upon which ligand is bound to the receptor. It remains unclear to what extent the β-Adrenergic Receptor Kinase family, GRK2 and GRK3 regulate CCR7 internalization.
Incubation of primary mouse T cells or human T cell lines, Hut 78 and CEM with 200 nM CCL19, but not CCL21 led to rapid CCR7 internalization. To determine the roles of GRK2 and GRK3 in CCR7 internalization, we co-transfected HEK cells with plasmids expressing either GRK2 or GRK3 along with arrestin-3, which we have previously shown is required for CCR7 internalization. Immunofluorescence microscopy showed that CCR7 co-localized with arrestin-3 in cells co-transfected with GRK2 after 30 minutes incubation with CCL19, but not CCL21. Limited co-localization of CCR7/arrestin-3 was observed in cells co-transfected with GRK3. Further CCL19/CCR7 incubation studies showed that GRK2-dependent CCR7 internalization was dependent upon the expression level of GRK2 with high protein levels being inhibitory. To further assess the role of GRK2, we used a GRK2 kinase dead mutant (GRK2-K220R) that does not facilitate the internalization of CCR7, GRK2-K220R did, however, result in some clustering of CCR7/arrestin-3. Overall, these data suggest that GRK2 plays an important role in CCR7 internalization in mammalian T cells; however, other GRKs are likely involved in this process.
Improvements in Ultra-Sentive Single Molecule Assays to Extend Quantitative Measurement of Cytokines in Plasma and Serum at Attogram per mL Concentrations
Authors: Lambert Jeremy , Duffy David , Rissin David , Kan Cheuk , Tobos Carmen , Wiener Alexander , Meyer Ray 
Published: January 2023

Abstract
The concentration of cytokines in blood are often at pictogram per mL and sub-picogram per mL. Recent advances in the sensitivity of immunoassays, such as single molecule arrays (Simoa), have allowed the detection of many of these cytokines that were previously undetectable in blood. While the limits of detection (LOD) of these new technologies are in the femtogram per mL range, the concentrations of cytokines in many healthy individuals can still not be detected. In fact, the quantitative measurement of several important cytokines are &lt;50% in healthy individuals. We will report on advances to improve the sensitivity of the Simoa technology into the attogram per mL range that improve the quantitative measurement of a broad range of cytokines.
Catalog of ex vivo whole blood stimulation conditions to generate species-specific cytokine controls
Authors: Repic Allison M , Rowse Amber L , Bruder Eric , Frantz Aric M 
Published: January 2023

Abstract
Commercially available reagents for the detection of cytokines in pre-clinical species often rely on cross-reactivity from other species. Species-specific cytokine control materials are ideal for use in determining the suitability of the cross-reactive reagents within the intended pre-clinical species. To generate species specific cytokine positive control materials in a non-invasive manner, a systematic catalog of ex vivo whole blood mitogen stimulation and collection conditions was developed. Since naïve matrix (plasma or serum) is unlikely to have cytokines detectable above the lower limit of quantitation (LLOQ), ex vivo stimulation of whole blood using a variety of mitogens was performed in order to produce measureable cytokine release. Plasma harvested at specific time points from these stimulations was then spiked into naïve (species matched) plasma or serum to achieve analyte concentrations within the analytical measurement range of the assay. This ‘spike-in’ sample serves as a model of the intended test system, and allows for evaluation of the ability to detect species-specific cytokines within both plasma and serum matrices. A range of detection was determined for a number of analytes using a range of concentrations of spike-in using commercially available multiplex assays. By producing this catalog of stimulation conditions including multiple mitogens and durations of challenge for optimized collection per analyte, we are able to quickly generate positive control materials for a wide range of analytes across multiple species and thereby have simplified and streamlined the approach to generating these much-needed materials in the preclinical testing laboratory.
Interleukin-22 alleviates Angiotensin II-induced cardiomyocyte apoptosis by preventing the intrinsic mitochondrial pathway
Authors: Xia Lin , Che Yang , Su Zhaoliang 
Published: January 2023

Abstract

Background
Cardiac apoptosis plays an important role in the development of heart failure, and Angiotensin II (Ang II) plays a central role in the pathogenesis of RAAS-triggered cardiac apoptosis. Interleukin (IL)-22 is an inflammatory cytokine, which is involved in cardiovascular diseases. Recent evidence has demonstrated that there is a close relationship between IL-22 and human hypertension. However, the effects of IL-22 on Ang II-induced cardiomyocyte apoptosis remain unknown.

Methods
MCM cells and primary cardiomyocytes were exposed to Ang II with or without IL-22. The activation of the apoptosis signaling pathways, reactive oxygen species (ROS), supernatant TNF-α, Fas/FsaL, mitochondrial membrane potential (MMP), cytochrome c (Cyc) and the activity of superoxide diamutase (SOD) were detected.

Results
In MCM cells and primary cardiomyocytes, flow cytometry revealed that IL-22 significantly alleviated Ang II-induced apoptosis, and IL-22 receptors (IL-22R1 and IL-10R1) levels were obviously increased. Western blotting revealed that increased cleaved Caspase 3/8 and Bax protein levels were clearly reduced after IL-22 treatment. The Fas/FsaL and TNF-α levels were identical in the cells with or without IL-22 treatment. But ROS levels were markedly reduced after IL-22 treatment via upregulation of SOD activity, and Ang II-induced decrease of MMP was improved by IL-22, reducing the release of Cyc and the levels of cleaved Caspase 9.

Conclusions
IL-22 improved Ang II-induced cardiomyocyte apoptosis via inhibiting the intrinsic apoptosis pathway.
Th17-derived cytokines synergistically enhance IL-17C production by the colonic epithelium
Authors: Swedik Stephanie M , Madola Abson , Levine Alan 
Published: January 2023

Abstract
Tightly regulated communication between the gastrointestinal (GI) epithelium and immune cells in the underlying lamina propria is critical for immune homeostasis. Interleukin (IL)-17C, produced by epithelial cells after exposure to inflammatory stimuli, facilitates cell-to-cell communication by promoting pro-inflammatory responses in Th17 cells. Since Th17-derived cytokines induce the expression of IL-17C, we hypothesize that IL-17C participates in an inflammatory amplification loop with Th17-derived cytokines, contributing to GI disease. We report that a Th17-derived cytokine TNFα induces IL-17C mRNA and protein expression in human colonic epithelial cell lines via an NFκB dependent pathway. Co-stimulation of TNFα-treated cells with Th17-derived IL-17A or IL-22 induces a respective 2,000- and 1,000-fold increase in IL-17C mRNA and protein. All three cytokines stimulate an 11,000-fold increase of IL-17C. While neither IL-17A nor IL-22 alone induce IL-17C synthesis, their presence at culture initiation is necessary to achieve synergy, suggesting that they stimulate the cells independently from TNFα. Inhibition of NFκB activation reduces IL-17C production by 99.99%, confirming that TNFα is the primary stimulant. Inhibition of AP-1 reduces IL-17C mRNA synthesis induced by pairwise or triple cytokine exposure to levels equivalent to that of TNFα alone, suggesting a role for this transcription factor in the synergistic response. IL-17A-mediated RNA stability was shown not to contribute to the mechanism of synergy. Taken together, cooperation between the NFκB and AP-1 signaling pathways in colonic epithelial cells may mediate the synergy observed in this novel pro-inflammatory feed-forward loop in the GI tract.
Detoxified dental healing abutments trigger an inflammatory response in-vitro
Authors: Valverde Araceli Maria , Narvekar Aniruddh Nitin , Naqvi Afsar Raza , Nares Salvador 
Published: January 2023

Abstract
Healing abutment (HA) is a titanium screw used in implant dentistry to replace missing teeth. Although designed for single procedure, HA are frequently reused. Pathogen Associated Molecular Patterns (PAMPs), present on HA can elicit immune responses in vivo and may lead to implant failure. The aim of this study is to test four commonly employed protocols for HA detoxification. Forty HA collected from subjects with prior implant placement were randomly distributed and detoxified using four protocols (A–D, N=10/group). Group A: Autoclave sterilized, Group B: Ultrasonic bath + autoclave, Group C: Debrided by prophy jet and glycine powder + autoclave, Group D: Debrided using a non-scratch scrub sponge + autoclave, Control: Unused. Residual protein quantification (N=3/group) and macroscopic debris evaluation (N=2/group) was assessed by Protein BCA Assay Kit and Phloxine B staining, respectively. Human primary monocyte-derived macrophages (Mϕ) were challenged with HA (N=5/group) and supernatants were collected at 4, 24, 48 and 120 h to analyze ten cytokines by multiplex bead assay. Control group did not show any detectable protein, debris or cytokine production. Among the treatments, protocol C and D were highly effective in decontamination as observed by reduced protein and debris quantification. Protocol C showed reduced cytokine (TNF-α, IL-8, IL-6, IL-10 and CXCL-10) secretion both at early and late time points compared to other decontamination protocols (A, B or D). The most effective detoxification method for HA is Protocol C. Compared to unused HA, none of the protocols completely decontaminated HA. Potent immunogenicity of decontaminated HA suggests that the practice to reuse HA should be reconsidered.
TNFR2 signaling enhances ILC2 survival, function and induction of airway hyperreactivity
Authors: Hurrell Benjamin P , Galle-Treger Lauriane , Jahani Pedram Shafiei , Howard Emily , Helou Doumet Georges , Banie Homayon , Soroosh Pejman , Akbari Omid 
Published: January 2023

Abstract
Group 2 innate lymphoid cells (ILC2s) can initiate pathologic inflammation in allergic asthma by secreting copious amounts of type-2 cytokines, promoting lung eosinophilia and airway hyperreactivity (AHR), a cardinal feature of asthma. We discovered that the TNF/TNFR2 axis is a central immune checkpoint in murine and human ILC2s. TNFa is a pleiotropic proinflammatory cytokine which is elevated in the airways of patients with severe asthma, signaling through two main receptors with opposing functions: TNFR1 and TNFR2. We found that murine ILC2s selectively express and induce TNFR2 upon IL-33 activation, whereas they fail to express – or induce – TNFR1. Strikingly, blocking the TNF/TNFR2 axis inhibits ILC2 survival, cytokine production, ILC2-dependent AHR and airway eosinophilia. The mechanism of action of TNFR2 in ILC2s is through utilizing non-canonical NFkB pathway as a NFkB inducing kinase (NIK) inhibitor blocks the costimulatory effects of TNFa both in vitro and in vivo. Similarly, human ILC2s selectively express TNFR2 and using the model of humanized mice that our laboratory recently developed, we show that TNFR2 engagement in human ILC2s enhances survival and activation, ultimately promoting AHR through a NIK-dependent pathway. These findings highlight the role of the TNF/TNFR2 axis in pulmonary ILC2s, suggesting that targeting TNFR2 or relevant signaling represents a novel strategy for treating patients with ILC2-dependent asthma.
High fat diet facilitate duffy antigen receptor for chemokine (DARC) expression in mononuclear cells by IL-22 dependent manner
Authors: Lee Hyung Keun , Kim Eun Young , Chang Eun Ju 
Published: January 2023

Abstract
Though interleukin-22 (IL-22) is recently suggested to play a role in immune-inflammatory system, however, the specific role of IL-22 for accumulation of adipose tissue in the context of obesity and its metabolic consequences has not been clearly documented. The purpose of this study is determine the role of IL-22 on adipose tissue accumulation and inflammation through mouse and human model. We here identified a previously unknown subset of monocyte-derived Duffy antigen receptors for chemokines (DARC)+ macrophages in epididymal fat adipose tissue and found that they are preferentially recruited into the crown-like structures of adipose tissue in the mouse upon high fat diet-induced obesity. Importantly, DARC+ macrophages highly express the IL-22 receptor (IL-22Ra1). Exposure to recombinant IL-22 shifts macrophages to an alternative M2 polarization pathway and augments DARC expression via a STAT5b signaling axis. STAT5b directly binds to the DARC promoter and a STAT5 inhibitor abrogates the IL-22-mediated induction of DARC. These M2-like DARC+ subpopulations of monocytes/macrophages were elevated in obese db/db mice compared to WT lean mice. Furthermore, subsets of CD14+ and/or CD16+ monocytes/macrophages within human peripheral blood mononuclear cell populations express DARC and the prevalence of these subsets is enhanced by IL-22 stimuli. This suggested that IL-22 is a critical cytokine that promotes the infiltration of adipose tissue macrophages, which regulate inflammatory processes. Taken together, our present findings provide important insights into the molecular mechanism by which IL-22 signal modulates DARC expression in M2-like macrophages.
Artificial thymic organoids reveal an early block in T cell differentiation in patients carrying mutations in IKZF1
Authors: Bosticardo Marita , Pala Francesca , Kuehn Hye Sun , Montel-Hagen Amelie , Crooks Gay M , Rosenzweig Sergio D , Notarangelo Luigi D 
Published: January 2023

Abstract
IKZF1 is an essential transcription factor expressed throughout hematopoiesis and involved in both lymphocyte and myeloid differentiation. Heterozygous germline mutations in IKZF1 give rise to distinct clinical phenotypes. IKZF1 haploinsufficiency causes common variable immunodeficiency (CVID) associated with B cell immune deficiency, B-ALL susceptibility and autoimmune manifestations, without clinical T cell defects. Patients carrying dominant negative (DN) IKZF1 mutations have combined immunodeficiency (CID), with an increased proportion of naïve T cells and impaired T cell function. Mouse models of IKZF1 mutations have a distinct phenotype from patients and some models present a more severe defect in T cell development. To address these controversies and gain novel insights into the effects of distinct IKZF1 mutations on human T cell development, we used the artificial thymic organoid (ATO) system, based on the 3D aggregation and culture of a delta-like Notch ligand 4-expressing stromal cell line (MS5-Dll4) with CD34+ cells, to analyze T cell development from CD34+ cells obtained from one patient with IKZF1 haploinsufficiency and one patient with DN IKZF1 mutation. Both patients showed a similar early block in T-cell differentiation at pre-T cell stage. However, the patient with IKZF1 haploinsufficiency showed a more pronounced leakiness, with a residual production of CD3+TCRαβ+ cells, which could account for the milder T-cell phenotype. Interestingly, the DN patient presented an increased accumulation of CD4−CD8β− CD8αα+ cells. These results show an unexpected role for IKZF1 in humans in early stages of T-cell differentiation and indicate IKZF1 as a necessary factor for the induction of CD8β expression in T cells.
The phosphatidinositol-transfer protein Nir3 is a modulator of T cell development
Authors: Helou Ynes , Au-Yeung Byron B , Weiss Arthur 
Published: January 2023

Abstract
Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) functions as a critical source of second messengers implicated in T cell activation. In T cells, hydrolysis of PIP2 by the lipid phosphatase PLCγ1 generates the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), resulting in PKC and Ras/MAPK activation as well as calcium increases that contribute to T cell responses. A secondary consequence of PLCγ1 activation is a transient reduction in PIP2 levels. The PI transfer proteins Nir2 and Nir3 have been shown to replenish PIP2 levels at the PM through delivery of the precursor PI from the endoplasmic reticulum (ER) via non-vesicular transport. In particular, Nir3 has been shown to replenish PI levels during basal signaling and in response to weak signaling. Given the importance of weak signaling in thymocyte positive selection, as well as the elevated levels of Nir3 mRNA during the double positive (DP) stage of thymocytes development (Immgen), we hypothesize that Nir3 is a regulator of PI homeostasis during thymocyte development. Using CRISPR-Cas9 technology, we have generated a Nir3 knockout mouse. DP thymocytes from Nir3 knockout mice have reduced basal calcium levels, as well as reduced TCR-induced calcium flux. Furthermore, introduction of the OT1 transgenic TCR into Nir3 knockout mice resulted in elevated numbers of DP thymocytes and reduced numbers of CD8SP thymocytes, suggesting a block in positive selection. Analysis of TCR signaling in Nir3 knockout OT1 thymocytes revealed attenuated calcium flux as well as defective induction of pERK, pAkt, and pS6. These findings are the first to characterize the function of PI transfer proteins and their importance specifically in T cell development.
Molecular cues dictating positive and negative selection in the thymus
Authors: McIntyre Laura Lynn , Steier Zoe , Lutes Lydia , Streets Aaron , Yosef Nir , Robey Ellen A 
Published: January 2023

Abstract
T cells develop in the thymus where CD4+CD8+ double positive (DP) thymocytes undergo positive and negative selection to generate mature CD4+ and CD8+ single positive (SP) T cells. Positive selection allows for the generation of a diverse repertoire of T cells capable of recognizing peptide:MHC (pMHC) complexes, while negative selection ensures that the mature T cell repertoire is self-tolerant. The cell types involved in thymic selection are well understood and recent work has begun to elucidate temporal and spatial aspects involved in selection. However, the intracellular cues regulating development and CD4 vs. CD8 lineage commitment are incompletely understood. Our work focuses on utilizing a single cell RNA sequencing (scRNA-seq) approach to elucidate the molecular cues guiding thymic selection of CD4+ and CD8+ T cells.
Visualizing thymic regulatory T cell development with single-cell RNA sequencing
Authors: Owen David L. , LaRue Rebecca S , Farrar Michael A 
Published: January 2023

Abstract
CD4SP thymocytes that receive moderate to strong TCR signals upregulate either FOXP3 or CD25, generating distinct Foxp3lo or CD25+ Treg progenitor (TRP) populations, respectively. Stimulation of these TRP populations with IL2 induces co-expression of CD25 and FOXP3, completing thymic Treg differentiation. The alterations in gene expression that drive these respective Treg differentiation programs, as well as the identification of agonist selected cells within conventional CD4SP thymocytes remain unclear. To address these questions, we performed single-cell RNA-Seq with paired TCR sequencing of conventional and Treg lineage CD4SP thymocytes. This data set contained cells of the entire range of maturity, from recent DP-CD4SP converts to mature CD4 T cells competent for thymic export. In conventional CD4SPs we were able to identify a subset of cells undergoing agonist selection that differentiates into TRP cells along with cells undergoing negative selection. The transcriptomes of the subsequent CD25+ and FOXP3lo TRP subsets were clearly distinct. For example, we found that the source of IL2 required for conversion of both TRP subsets into mature Tregs was uniquely found within the most immature CD25+ TRP undergoing agonist selection. Finally, TCR analysis demonstrated that some TCRs were enriched in CD4SP receiving agonist stimulation that gave rise to Tregs, or undergoing negative selection, while others were excluded from this pathway. We are currently carrying out spatial transcriptome studies to identify the specific localization of distinct Treg intermediates within the thymus. Interrogation of these single cell and spatial transcriptomic data sets will generate a comprehensive model of thymic Treg development.
Alteration in Three-Dimensional Microenvironment of Themis-deficient Murine Thymus Using Image Cytometry Approach
Authors: Aziz Nazihah Husna Abdul , Tan Yingrou , Brzostek Joanna , Ng Lai Guan , Gascoigne Nicholas R J 
Published: January 2023

Abstract
Themis is a T-cell intrinsic protein involved in selection during thymocyte development in the thymus. Thus, Themis-deficient mice have gravely reduced numbers of T-cells. Despite extensive research on thymocyte signalling, the exact role of Themis remains a mystery. As the thymic 3-dimensional (3D) microenvironment is a critical element in supporting thymocyte development and vice versa, we were interested to investigate the effect of Themis deficiency on thymic components and processes. In this study, we paired 3D imaging with conventional flow cytometry to understand thymic development in normal and Themis-deficient condition. Current conventional 2-dimensional (2D) histology and 3D reconstruction from 2D sections are unsatisfactory for studying thymus morphology. We thus established a novel approach for whole thymus 3D imaging to visualize developing thymocytes and supporting stroma, such as epithelial cells, myeloid cells and vasculature. 3D imaging coupled with quantitative analysis of images using image cytometry allows for objective comparison of the thymic microenvironment between sample groups. Here, we show an altered medullary and vasculature organization in Themis−/− thymus, suggesting a suboptimal 3D niche supporting thymocyte development. Correspondingly, mature single-positive thymocytes which are dependent on the medullary environment are altered phenotypically. In addition, we detected higher levels of apoptosis in Themis−/− cortex and an increase in clonally deleted double-positive thymocytes. Our findings demonstrate how Themis-deficiency affects developing thymocytes and its supporting stromal components.