bluuebunny/crossref_metadata_2025_split huggingface dataset:
Keratinocyte-derived CCL20 orchestrates epidermal localization of IL-17-producing γδ T cells and ILCs to mediate psoriasis-like skin inflammation
Authors: Singh Tej Pratap , Lu Xinping , Singh Satya P , Zhang Hongwei , Doucet Michele , Kominsky Scott L , Farber Joshua M 
Published: January 2023

Abstract
CCR6 is important for imiquimod (IMQ)-induced psoriatic dermatitis in mice, which depends on motile CCR6+ γδ T cells that produce IL-17 and IL-22. CCR6 ligands include CCL20 and β-defensins. We used new strains of Ccl20 tdTomato knock-in mice, Ccl20fl/flmice, and Ccr6fl/fl GFP knock-in mice to determine sites of production and action of CCL20 and its role in the localization of γδ T cells and ILCs during imiquimod (IMQ)-induced inflammation. IMQ treatment of Ccl20+/tdT mice led to expression of Ccl20 in keratinocytes (KCs), including in the infundibulum and isthmus of the hair follicles. Ccl20−/− mice showed reduced inflammation and expression of Il17 and Il22 in the treated skin. Deletion of Ccl20 specifically in the KCs by treating Ccl20fl/flK14-CreERT mice with tamoxifen before applying IMQ significantly decreased the accumulation of IL-17-producing gd T cells and ILCs in the epidermis. Depleting KC-derived CCL20 at day 4 when inflammation was well established also reduced the number of epidermal γδ T cells and ILCs. In both cases the percent of IL-17-producing γδ T cells and ILCs did not change, demonstrating that migration to the epidermis and the ability to produce IL-17 are regulated independently. Multi-photon microscopy of IMQ-treated Ccr6+/gfp and Ccr6gfp/gfp(CCR6-deficient) micerevealed a CCR6-independent step whereby dermal CCR6+ cells accumulate at the hair follicles, followed by a CCR6-dependent step whereby the cells move into the superficial epidermis.
Engagement of CD45 differentially alters early signaling events in human T cells costimulated through TCR+CD28 compared with TCR+ICAM-1
Authors: Bhatta Anuja , Chan Marcia , Benedict Stephen H 
Published: January 2023

Abstract
Our lab has been developing the concept that co-stimulation of human naïve CD4+ T cells through different costimulatory molecules can tune differentiation to different phenotypes. Much of our work has compared costimulation through TCR/CD3+CD28 with costimulation through TCR/CD3+ICAM-1. In the absence of exogenously added cytokines, we observed differences in phenotypic outcomes among Th1, Th2 and Treg cells. An open question is where in the signaling pathways induced by these two costimulation regimens do differences occur that contribute to outcome of differentiation. The cell surface glycoprotein, CD45, exerts intracellular tyrosine phosphatase activity immediately after recognition of cognate Ag by the TCR. CD45 plays a crucial role to initiate T cell signaling by dephosphorylating a negatively regulatory tyrosine residue in the C-terminal region of Src family kinases such as Lck. Here, we investigate the early signaling process by comparing events that follow engagement of CD45 alone or in association with the two co-stimulatory molecules: ICAM-1 or CD28. Using protein blotting, we have confirmed that engagement of CD45 alone induces changes in protein phosphorylation. We have observed that CD45 engagement affected differently the composition of signaling complexes formed during costimulation through CD3+CD28 compared with CD3+ICAM-1. We hypothesize that during TCR recognition of cognate antigen, engagement of CD45 differentially guides signaling events in T cells depending on the mode of costimulation. A goal is to learn whether different outcomes of naïve T cell differentiation can begin at the earliest steps of T cell activation and ultimately how these signals progress during the activation process.
Molecular background of Orai1 accumulation in the immunological synapse
Authors: Vörös Orsolya , Panyi György , Hajdu Péter 
Published: January 2023

Abstract
CRAC channels have a vital role in the activation of T lymphocytes, through their calcium influx. CRAC is composed of the ER-membrane resident STIM1 (stromal interaction molecule 1) and the plasma membrane Orai1 pore-forming subunit. Both are accumulated at the interface of the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). CRAC channelopathies due to mutations in either Orai1 or STIM1 are characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia. The down-regulation of actin-binding protein expression (e.g. HS1), which may be responsible for the immobilization of ion channels, hampers Ca2+-signaling and IS formation of T lymphocytes. We hypothesized that actin-binding proteins modulate the trafficking of the CRAC channels to the IS, which is crucial for the Ca2+-signaling. By means of GST-affinity assay we could show that HS1, which is an actin-regulatory adaptor protein, binds to the N-terminus of the Orai1. N-terminal truncated Orai1 channels (ΔN1: 1–74, ΔN2: 1–88) were created and expressed in Jurkat cells. Then Orai1 channel expressing Jurkat cells were conjugated to CD3/CD28 beads as an APC and Orai1 polarization was determined: the Orai1-ΔN1 mutant similar to the wild-type Orai1 redistributed to the IS but the Orai1-ΔN2 did not. Furthermore, the HS1 protein did not show IS-accumulation in cells expressing the Orai1-ΔN2 subunits unlike for those transfected with the Orai1-ΔN1 or the wild-type constructs. Overall, these data indicate that Orai1-HS1 interaction could contribute to the anchoring of the Orai1 in the IS and can affect the Ca2+-signaling upon T cell activation.
Role of PAFR-MHC-II-CD38 interactions in infections
Authors: Khan Aslam A 
Published: January 2023

Abstract
Infectious diseases are the most prevalent cause of deaths around the world. Chronic contagious ailments will account for seven out of ten deaths in the world by 2020. The most significant problem in the treatment of the infectious disease is the ever-increasing rate of resistance to drugs mounted by pathogens. Our preliminary results suggest that during antigen presentation the major histocompatibility complex II (MHC-II) engages two associate protein molecules CD38 and Platelet Activating Factor Receptor (PAFR). CD38 and PAFR elicit two counteractive signaling pathways in APCs resulting in the release of either proinflammatory cytokine such as IL-12 or anti-inflammatory cytokines such as IL-10 respectively. Pathogens show remarkable adaptability to the drugs by either metabolizing the drug molecules or by changing the surface profile to neutralize binding of drugs to the pathogens. Therefore, there is a requirement to find new treatment modalities that do not target the pathogens but aid the immune system to eliminate the pathogens. Our preliminary data further suggest that the use of associate molecule during antigen presentation decides the fate of pathogens such as Salmonella Typhimurium or Staphylococcus aureus in macrophages. For example, if MHC-II engages CD38 to signal inside the macrophages leading to reduced pathogen survival in macrophages and mice, while MHC-II-signaling via PAFR aid the pathogen survival in macrophages and mice. Our studies also suggest that combinatorial use of PAFR inhibitor (WEB2086) and antibiotics ampicillin reduced the growth of S. Typhimurium or S. aureus in macrophages and mice.
microRNA-21 controls inflammatory and metabolic program of macrophage and neutrophils during sepsis
Authors: Melo Paulo Henrique , Alvarez Annie Rocio Piñeiros , Serezani Carlos Henrique 
Published: January 2023

Abstract
Sepsis is associated with a hyper inflammatory state and switch of metabolic profile of immune cells leading to organ damage and lethality. MiR-21 is one of the most abundant microRNAs in phagocytes and plays important roles in both increasing and dampening inflammation. Here, we aim to investigate the role of miR-21 in inflammatory and metabolic state of phagocytes during acute sepsis. Sepsis induced by CLP (Cecal ligation and puncture) enhanced miR-21 expression in neutrophils and macrophages from peritoneal cavity and lung. MiR-21 deficient myeloid cells mice (miR21Δmyel) show increased sepsis survival, which was correlated with increased bacterial clearance and decreased production of IL6 and IL1β, and reduced of heart and liver damage markers (CK-MB and AST, respectively). Next, we sought to test whether decreased inflammation in miR21Δmyel is dependent on the aerobic glycolysis. Both peritoneal and BALF cells from miR21Δmyel septic mice showed decreased expression of genes encoding glycolytic molecules. Also, decreased HK1 was associated with lower lactate release than septic miR21fl/fl mice. Corroborating the in vivo data, LPS-challenged BMDM and peritoneal neutrophils from miR21Δmyel also exhibited lower expression of glycolytic genes and inflammatory cytokines than miR21fl/fl cells. Both, macrophages and neutrophils were submitted to glycolytic stress assay after LPS stimulation in order to confirm the functional impact of miR-21 over glycolysis. Similarity in both cells the lack of miR-21 leads to decrease of ECAR (extracellular acidification rate). In conclusion, higher expression of miR-21 after sepsis and LPS stimulation leads to assembly of inflammatory and glycolytic metabolic program in myeloid cells.
CD73-dependent adenosine dampens IL-1β-induced CXCL8 production in gingival fibroblasts via HO-1 and pAMPK
Authors: Ramos Erivan Schnaider , Pedram Michael , Lee Renee E. , Exstrom Drake , Yilmaz Ozlem , Coutinho-Silva Robson , Ojcius David M. , Morandini Ana Carolina 
Published: January 2023

Abstract
During inflammation, stressed or infected cells can release adenosine tri-phosphate (ATP), which can be hydrolyzed to adenosine by ectonucleotidases such as CD39 and CD73. The role of CD73 in the modulation of cytokine release by human gingival fibroblasts (HGF) remains underexplored. Here, we investigated whether CD73-mediated hydrolysis of extracellular ATP (eATP) could affect IL-1β-induced CXCL8 secretion. The mRNA expression of adenosine receptors, CD39, and CD73 of periodontitis samples was retrieved from a public database. HGF mRNA levels were measured after 3, 6 or 24 h of IL-1β stimulation. IL-1β-induced CXCL8 protein levels were measured after pretreatment with 100 μM eATP in the presence of absence of CD73 inhibitor. The effect of eATP degradation to adenosine on CXCL8 levels was investigated using agonist and antagonist of adenosine receptors. The CD39, CD73 and adenosine receptor mRNA were differentially modulated by IL-1β. ATP pretreatment impaired IL-1β-induced CXCL8 secretion and required activation of HO-1 and pAMPK. The inhibition of CD73 or adenosine receptors abrogated the ATP effect on CXCL8 secretion. CD73-generated adenosine dampens IL-1β-induced CXCL8 in HGF via HO-1 and pAMPK. These results imply that CD73 is a negative regulator of the inflammatory microenvironment, suggesting this ectoenzyme should be considered as a target for therapeutic strategies to control periodontal disease.
Resistin-like molecule alpha (RELMα) dampens lung inflammation and promotes wound healing in helminth infection and a 3D lung repair model
Authors: Kim Sang Yong , Li Jiang , Burr Abigail C. , Batugedara Hashini , Nordgren Tara , Zang Xingxing , Nair Meera G 
Published: January 2023

Abstract
RELMα is a small secreted and immunoregulatory protein, also known as hypoxia-induced mitogenic factor (HIMF) and found in inflammatory zone (FIZZ). RELMα, produced by macrophages and epithelial cells in the lung and intestine, has recently been shown by our lab and others to induce wound healing during Nippostrongylus brasiliensis (Nb) infection. However, the mechanisms by which RELMα activates wound healing pathways and what cell-types are activated by RELMα are unclear. We generated constitutive RELMα−/−/TdTomato-red (TdT) reporter mice to delete the RELMα gene and track RELMα promoter activity. We found that following Nb infection, RELMα−/− macrophages exhibited reduced expression of genes associated with wound healing such as Arg1, Mmp19 and Pdgfra. To complement the RELMα−/− mouse in vivo studies, a new endotoxin-free RELMα-human Fc fusion protein was constructed and purified. A RELMα capture assay with the fusion protein demonstrated that RELMα binds to macrophage cell line RAW 264.7. Moreover, 3D lung scaffold and wound healing assays showed that RELMα-Fc promoted tissue repair by lung epithelial cells and mesenchymal stem cells. Lastly, RELMα function in vivo was characterized by RELMα-Fc fusion protein treatment of Nb-infected RELMα−/−/TdT mice, which downregulated immune cell recruitment in the lung compared to control Fc. Ongoing studies include identification of the RELMα receptor using the RELMα fusion proteins and testing whether the wound healing properties of RELMα are effective in the intestine following Heligosomoides polygyrus infection.
Plexin B1 expression on human Treg cells is required for regulation of their phenotypic stability by semaphorin 4A
Authors: Chapoval Svetlana P , Hritzo Molly , Qi Xiulan , Tamagnone Luca , Golding Amit , Keegan Achsah Dorsey 
Published: January 2023

Abstract
We previously reported that neuroimmune semaphorin (Sema) 4A regulates the severity of experimental allergic asthma and increases regulatory T-cell (Treg) numbers in vivo, however the mechanisms of Sema4A action remain unknown. It was also reported that Sema4A controls murine Treg cell function and survival acting through Neuropilin 1 (NRP-1) receptor. To clarify Sema4A action on human T cells, we employed T-cell lines, HuT78 and HuT102, as well as human PBMCs and CD4+ T cells in phenotypic and functional assays. We found that HuT78 demonstrated a T-effector-like phenotype (CD4+CD25lowFoxp3-), while HuT102 expressed a Treg-like phenotype (CD4+CD25hi Foxp3+). Neither cell line expressed NRP-1. However, HuT102 cells expressed high levels of the Sema4A-counter receptor, Plexin B1 whereas HuT78 cells demonstrated high cell-surface expression of Sema4A. All human peripheral blood CD4+ T cells, including Treg cells, expressed PlexinB1 and lacked both, NRP-1 and −2. Culture of HuT cells with soluble Sema4A led to an upregulation of CD25 and Foxp3 markers on HuT102 cells. Addition of soluble Sema4A increased the relative numbers of CD4+CD25+Foxp3+ cells in PBMC and CD4+ T cells which did not express NRP-1 but were PlexinB1+, suggesting the role of this receptor in Treg cell stability. The inclusion of anti-PlexinB1 blocking Ab in cultures before rSema4A addition significantly decreased Treg cell numbers as compared to cultures with rSema4A alone. Sema4A was as effective as TGF-b in iTreg induction from CD4+CD25depleted cells but did not affect Treg cell suppressive activity in vitro. These results suggest strategies for the development of new Sema4A-based therapeutic measures to combat allergic inflammatory diseases.
Identification of distinct highly pathogenic allergen-specific T cells in common allergy and asthma
Authors: Seumois Gregory , Suastegui Ciro Ramirez , Shmiedel Benjamin J , Liang Shu , Shulten Veronique , Panwar Bharat , Madrigal Ariel , Sette Alessandro , Peters Bjoern , Vijayanand Pandurangan 
Published: January 2023

Abstract
Despite the proven critical role of the aero-allergen house dust mite (HDM) in the pathogenesis of airway allergic diseases and asthma, the molecular mechanisms leading to allergen-specific T cell activation, as well as the molecular differences driving to disease between different type of HDM-sensitized patients are unknown. The recent development of robust allergen activation assays combined with single-cell RNA-Seq technologies made us able to perform the first large single-cell RNA-Seq study for around 50,000 cells. We collected blood from 12 asthmatic patients with or without HDM sensitization, 6 patients with Allergic Rhinitis HDM-sensitized and 6 healthy subjects. We stimulated PBMCs with a pool HDM peptides for a period of 6 hours and sorted activated cells based on the expression of 2 surface markers: CD154+ (effector T cells) and CD137high/CD154low (regulatory T cells). We performed standard bulk RNA-Seq and single-cell RNA-Seq. Differential gene expression analysis showed important changes in gene expression upon TCR engagement in both populations. Comparison of disease groups identified several known but also new genes with relevant functional significance in allergy and asthma. Clustering analysis of Effector T cells single-cell RNA-Seq revealed an unexpected level of heterogeneity of gene expression among T cells. Major changes were observed between disease groups, revealing important pathological consequences to the understanding of immunity in allergy. Our study was the first attempt to generate a large-scale of single-cell RNA-Seq datasets from activated HDM-specific T cell. We showed presence of new T-cell subsets with unknown function as well as new molecules of therapeutic interest in allergy and asthma.
Alcohol effects on immune responses of classical pro-inflammatory macrophages
Authors: Hu Yawen , Yoshimura Masami 
Published: January 2023

Abstract
Alcohol consumption modulates immune responses. However, the mechanisms of ethanol’s action on the immune system are not fully understood. Previous studies demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform-specific manner, that type 7 AC (AC7) is most enhanced by ethanol, and that AC7 is highly expressed in immune cells. Therefore, we hypothesized that AC7 is a central player in regulating alcohol’s effects on immune responses. To examine this hypothesis, we generated AC7 knockout (KO) cell lines from a mouse microglia cell line, BV-2, and isolated primary bone marrow-derived macrophages (BMDM) from myeloid lineage-specific AC7 KO mice. Cells were activated by lipopolysaccharides (LPS) and interferon gamma (IFN-γ) to classical pro-inflammatory macrophages. The effects of ethanol on nitric oxide (NO) production of macrophages were examined via Griess reagent. LPS and IFN-γ synergistically increased nitric oxide (NO) production in wild type (WT) BV-2 cells in a dose-dependent manner. Ethanol suppressed LPS and IFN-γ co-activated NO production. AC7 KO BV-2 cells did not respond to LPS or IFN-γ activation. LPS increased NO production of both WT and AC7 KO BMDM. NO production of KO BMDM was significantly lower than that of WT BMDM. IFN-γ increased LPS-induced NO production of both WT and AC7 KO BMDM. Ethanol suppressed LPS-induced NO production in a dose-dependent manner in WT BMDM, while this effect was reduced in AC7 KO BMDM. The results suggest that AC7 is a key factor in the regulation of NO production and that the effect of ethanol on NO production depends on AC7. In the future, we will investigate the iNOS expression signaling cascade of classical pro-inflammatory macrophages in the presence of ethanol.
Myeloid CFTR Loss-of-function Causes Persistent Neutrophilic Inflammation in Cystic Fibrosis
Authors: Wang Guoshun , Ng Hang Pong 
Published: January 2023

Abstract
Persistent neutrophilic inflammation is a hallmark manifestation of cystic fibrosis (CF). However, the mechanism underlying this phenomenal clinical symptom remains incompletely understood. Here we report a pivotal role of CFTR in myeloid immune cells in control of neutrophilic inflammation. Myeloid CFTR-Knockout (Mye-Cftr−/−) mice and Wild-type (WT) mice were challenged peritoneally with zymosan at different doses. The lethal-dose challenge resulted in significantly higher mortality in Mye-Cftr−/− mice, indicating an intrinsic defect in host protection against inflammation in CF. The sub-lethal-dose challenge demonstrated an impaired resolution of inflammation in Mye-Cftr−/− mice, reflected by persistent neutrophilic inflammation, and hyper-inflammation with significantly higher levels of pro-inflammatory cytokines, including the neutrophil-recruiting chemokines MIP-2 and KC, which led to excessive neutrophil recruitment in vivo. Pulmonary challenge with zymosan confirmed the peritoneal finding. To determine the major types of cells responsible for the over-recruitment of neutrophils, zymosan-elicited peritoneal neutrophils and macrophages from Mye-Cftr−/− and WT mice were FACS-sorted and cultured ex vivo. The CF neutrophils produced significantly more neutrophil chemokine MIP-2. Moreover, peripheral blood neutrophils and monocytes from Mye-Cftr−/− and WT mice were cultured and stimulated with zymosan in vitro. Similarly, the CF neutrophils produced significantly more MIP-2. These data altogether suggest that CFTR dysfunction in myeloid immune cells leads to excessive neutrophil recruitment, thus serving as a mechanism for the long-observed neutrophilic inflammation in CF.
A role for allosteric disulfides in regulating the function of human killer cell receptor KIR2DL4
Authors: Rajagopalan Sumati , Mastorakos George , Long Eric O 
Published: January 2023

Abstract
KIR2DL4 is an unusual member of the killer cell Ig-like receptor (KIR) family expressed in NK cells. It resides mostly in endosomes and activates NK cells in response to soluble agonists, such as soluble HLA-G and a monoclonal antibody. KIR2DL4 signals from endosomes for a unique proinflammatory and proangiogenic response, which has relevance to placental development due to the restricted expression of its ligand, HLA-G, by fetal trophoblast cells during early pregnancy. To understand the nature and regulation of KIR2DL4–HLA-G interactions, we used an unbiased random mutagenesis approach and screened mutants of KIR2DL4 in a cellular assay for their ability to load soluble HLA-G into KIR2DL4+ endosomes. With this approach, amino acid residues in KIR2DL4 that are important for endosomal localization and HLA-G recognition were identified. Specifically, we have identified a putative allosteric cysteine in the extracellular domain of this receptor, which may be involved in a disulfide switching mechanism to control receptor internalization and ligand binding. Protein disulfide isomerase (PDI) acting at the cell surface may be responsible for this switch as inhibition of PDI interfered with internalization of KIR2DL4 and uptake of HLA-G into endosomes. The configuration of the relevant disulfide bond is -RHstaple, which is a hallmark of many allosteric disulfides. Thus, KIR2DL4 localization and function is regulated by allosteric disulfide bonds at the cell surface. Studies are underway to explore the dynamic regulation of receptor structure by this allosteric disulfide switch to control ligand binding, localization and signaling by this receptor.
Protein Kinase CK2 Drives Plasma Cell Differentiation
Authors: Wei Hairong , Yang Wei , Yan Zhaoqi , Qin Hongwei , Benveniste Etty 
Published: January 2023

Abstract
Casein kinase 2 (CK2) is a serine/threonine protein kinase composed of three subunits: two catalytic subunits, CK2α and CK2α’, and the regulatory subunit CK2β. It is overexpressed and overactive in B-cell acute lymphoblastic leukemia and diffuse large B-cell lymphomas, leading to inappropriate activation of NF-κB, JAK/STAT and PI3K/AKT/mTOR signaling, but little is known about how CK2 functions in normal B-cell differentiation. Here, we demonstrate that CK2 mRNA and protein expression is induced upon B-cell activation. Using the small molecule inhibitor CX-4945, which targets the catalytic activity of CK2, a significant inhibition of plasma cell differentiation was observed in vitro under T-cell-independent (TI) or T-cell-dependent (TD) conditions via stimulation with lipopolysaccharide (LPS) or CD40L and interleukin 4 (IL-4) +IL-5, respectively. At the cellular level, we found that inhibition of CK2 kinase activity in B-cells resulted in defective mTORC1 signaling upon stimulation with LPS. These results were confirmed in purified mature B-cells from mice with tamoxifen-induced global deletion of CK2α. These data demonstrate that CK2α is an important regulator of B-cell differentiation. Ongoing experiments with B-cell specific deletion of CK2α will further examine the impact of CK2α on B-cell differentiation in vivo.