bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of IL-2- and IL-6-dependent signaling in human Tregs by CISH and SOCS3
Authors: Vujanac Milos , Ding Ying , Malek Thomas R 
Published: January 2023

Abstract
SOCS proteins function as negative regulators of cytokine signaling. However, the role in of these proteins in human Tregs remains poorly understood. To address this issue, CRISPR-Cas9 genome editing was used to probe the role of SOCS3 and CISH in ex vivo expanded human Tregs. High efficiency editing of SOCS3 and CISH was confirmed at the DNA and RNA levels and the protein level for CISH. Deletion of SOCS3 resulted in sustained activation of STAT3 upon treatment with IL-6 whereas IL-2-induced STAT5 activation and IL-10- induced STAT3 activation were not affected. These results suggest that in Tregs SOCS3 specifically targets signaling initiated through IL-6R. Proliferative capacity and expression of Treg-specific phenotypic markers were not affected by SOCS3 ablation. RNA-seq revealed a common transcriptional signature between IL-6-treated wild-type and SOCS3-edited cells, showing upregulation of pro- and anti-inflammatory response genes and other genes that may affect Treg stability. Other differentially expressed genes in SOCS3-edited cells belong to pathways that may play a role in how Tregs integrate cytokine signals. In contrast, CISH editing resulted in increased IL-2-dependent activation of STAT5 and STAT3 and led to increased proliferation of Tregs. These effects were accompanied by only minor change in the expression of Treg-specific phenotypic markers. Our data indicate a restricted role of SOCS3 and CISH in regulating the responses of Tregs to IL-6 and IL-2, respectively. These activities likely contribute to maintaining Treg homeostasis under normal and inflammatory conditions.
Supported by: NIH R01AI131648
Characterization of anti-porcine CXCL10 monoclonal antibodies
Authors: Lunney Joan K , Hailstock Taylor , Dai Chaohui , Aquino Jovan , Chick Shannon , Walker Kristen , Manirarora Jean N , Gourapura Renukaradhya J , Sullivan Yvonne B , LaBresh Joanna 
Published: January 2023

Abstract
C-X-C motif chemokine ligand 10 (CXCL10) facilitates chemoattraction of important immune cells to tissues. As part of the USDA-NIFA Swine Immune Toolkit Initiative, our goal is to provide the veterinary community with new commercial immune reagents and standardized assays for future research efforts. For the chemokine target CXCL10, we used yeast expressed, recombinant porcine CXCL10 (rPoCXCL10) to generate a panel of α-CXCL10 monoclonal antibodies (mAbs). Each of the α-PoCXCL10 mAbs was assessed by ELISA using cross-inhibition analyses of biotinylated mAbs, and direct binding to orthologous yeast expressed CXCL10 proteins. As a result, we assigned six distinct epitope groups for the nine generated α-CXCL10 mAbs. Subsequently, we screened AF647-tagged α-CXCL10 mAbs for intracellular staining of pig immune cells using different stimulation conditions. Of the nine mAbs only two detected intracellular CXCL10 expression in PMA/ionomycin or rPoIFNγ-stimulated porcine cells. Further, cell characterization assays verified CXCL10+ cells as CD3-CD4-CD172+, with occasional CD4+ subsets. Overall, we have determined that α-CXCL10-1.4 mAb is the best mAb clone for intracellular staining and recommend it for use in analysis of intracellular signaling to detect interactions that regulate cell migration. A sandwich ELISA was also developed to quantitate CXCL10 protein expression; it verified reactivity with native porcine CXCL10. Overall, these tools will be useful for evaluating immunity against swine infectious diseases and host-responses to vaccines. New reagents identified by the Swine Immune Toolkit Initiative will undoubtedly advance future swine research efforts. Supported by USDA-NIFA AFRI grant # 2019-67015-29815.
Supported by USDA-ARS project # 8042?32000?102 and USDA-NIFA AFRI grant # 2019-67015-29815
Chemokines and anionic phospholipids: new binding partners for microbial killing and apoptotic cell clearance
Authors: Pontejo Sergio , Murphy Philip 
Published: January 2023

Abstract
Chemokines constitute a large family of small cytokines that regulate leukocyte migration in immunity and inflammation, some of which also have direct bacteria-killing properties. While the molecular mechanism by which chemokines kill bacteria remains poorly understood, chemokines mediate cell migration by interacting with cell surface glycosaminoglycans (GAGs) and G protein-coupled receptors (GPCRs). We have discovered that many chemokines interact with high affinity with anionic membrane phospholipids, including phosphatidylserine (PS) and cardiolipin (CL) which are selectively exposed on the membrane of apoptotic cells and apoptotic extracellular vesicles (ApoEV), and bacteria, respectively. This adds to GPCRs and GAGs a third class of cell surface binding site for chemokines and expands the scope of chemokine action to the biological niches occupied by PS and CL. For instance, we found that ApoEV induce phagocyte migration for apoptotic cell clearance by presenting PS-binding chemokines on the vesicle surface to GPCRs expressed by phagocytes. This is a novel PS-dependent mechanism for chemokine delivery by extracellular vesicles potentially relevant to other areas of immunology and indicates that chemokines may play a more prominent role in the removal of dying cells than previously reported. On the other hand, we discovered that only CL-binding chemokines are antimicrobial and that CL-deficient bacteria are resistant to chemokine treatment, indicating that chemokines kill bacteria by targeting CL on the bacterial membrane. In short, we have identified chemokines as the first family of soluble cytokines whose bioactivities can be regulated by direct interaction with membrane phospholipids.
Supported by NIAID/NIH intramural research program
IL-18R signaling impairs hematopoietic recovery after severe, shock-like infection
Authors: Howard Jennifer E , MacNamara Katherine C 
Published: January 2023

Abstract
Severe infection dramatically alters blood production, but the mechanisms impacting hematopoietic stem and progenitor cell (HSC/HSPC) recovery have not been carefully investigated. Using Ixodes ovatus Ehrlichia (IOE), a pathogen that causes severe shock-like illness and bone marrow (BM) aplasia, we explored the kinetics and mechanisms of hematopoietic recovery. Doxycycline treatment is necessary to prevent IOE-induced death and control bacterial burden in mice, but is not sufficient to prevent HSC/HSPC loss. During recovery from acute infection (12dpi) we saw increased cellularity of LT-HSCs and MPPs, accompanied by diminished function measured by decreased colony forming units (CFUs) in methocult assays. Reduced CFUs indicate a deficiency in BM myelopoietic function during recovery phase post- IOE. We previously showed that IL-18R-deficient mice exhibited improved CFUs at 12dpi, suggesting IL-18R signaling is detrimental to recovery from shock-like IOE. Utilizing a mixed BM chimeric mouse model (50:50 WT:IL-18R−/−), we observed that during recovery at 15dpi, IL-18R−/− cellularity predominates the LT-HSC and MPP2 populations, demonstrating an intrinsic role for IL-18R in limiting recovery of these populations. Additionally, BM chimeras at 15dpi showed increased levels of cell death (Annexin V+ and 7AAD+) in WT HSCs compared with IL-18R−/− HSCs. Transplantation of whole BM from mixed BM chimeric mice revealed a pronounced bias for IL-18R−/− cells, indicating that IL-18R−/− cells had a competitive advantage and improved function after IOE infection. Together, these data support the conclusion that IL-18R signaling impairs HSC function during recovery from severe, shock-like infection via decreased HSC viability.
This work was supported by National Institutes of Health funding from NIGMS (R35GM131842) to KCM.
IL-7 and IL-7R regulate fetal macrophage establishment via survival and proliferation
Authors: Leung Gabriel , Valencia Clint , Krum Brian , Beaudin Anna E 
Published: January 2023

Abstract
We have recently reported that IL7R signaling regulates fetal macrophage development, in addition to lymphocyte lineage commitment and development. Specifically, IL7R deletion and pharmacological blockade during a defined fetal developmental window impairs the establishment of fetal macrophage populations in the liver, lung, brain, and epidermis. We next hypothesized that IL-7 is also required for fetal macrophage establishment and that IL-7 signaling activates developmental programs that facilitate macrophage development via proliferation and survival. Immunofluorescent staining of E14.5 murine embryos revealed abundant co-localized IL7R and F4/80 expression in fetal liver, lung, brain, and skin. IL7R+ F4/80-expressing cells were also found in close proximity to IL-7+ cells, suggesting they participate in IL-7 signaling. Moreover, germline deletion of IL-7 also resulted in postnatal depletion of fetal-derived macrophage populations. Antibody blockade of IL-7R receptor during late gestation revealed tissue-specific deficits in survival and proliferation the day before birth. Specifically, liver and lung macrophages showed increased apoptosis, as determined by Annexin V staining, while brain and epidermal macrophages were less proliferative, as determined by Ki67 staining. Here we have shown that IL-7, in addition to IL7R, regulates establishment of tissue-resident macrophages and that fetal blockade of IL7R signaling regulates establishment by impairing survival and proliferation during late gestation. Ongoing work seeks to define IL7/IL7R signaling pathways in developing macrophages, as well as use genetic models to pinpoint the requirement for IL7R in fetal myeloid specification.
Supported by grants from NIH (R01 10057561).
Age Associated B Cells positively associate with atherosclerosis in mice and Humans
Authors: Srikakulapu Prasad , Pattarabanjird Tanyaporn , Bontha Sai Vineela , Upadhye Aditi , Drago Fabrizio , Marshall Melissa , McNamara Coleen 
Published: January 2023

Abstract
Age-Associated B cells (ABCs: CD19+ CD11c+) have recently been reported as a new subset of B cells with unique cell surface and transcriptional signatures. ABCs are mostly comprised of antigen experienced memory B cells that arise in response to microbial infections. ABCs have been reported to be elevated in autoimmune diseases, where in, these cells have characteristics of auto-antibody producing memory B cells. However, the role of ABCs during atherosclerosis has not be studied. Atherosclerosis is a chronic inflammatory disease and the major underlying cause for cardiovascular diseases (CVD). We for the first time report the role of ABCs in both atherosclerotic mice and in human CVD. Chow fed 50- and 100- week old, male, ApoEKO mice were used to study atherosclerosis. Lesion area was measured following Sudan-IV enface staining of aorta. 100-week-old mice developed significantly more disease than the 50-week-old mice. Further, through flow cytometric analysis ABCs were detected in spleen, bone marrow (BM) and blood of these mice. The frequency of ABCs from total B cells was significantly higher in all these tissue compartments in 100-week-old mice compared to 50-week-old mice. Additionally, most ABCs (&gt;80 %) were actively proliferating cells (Ki67+), as compared to normal CD11c− B cells (~35 %). In mice, we observed a significant correlation of atherosclerosis disease levels with the frequency of ABCs in spleen (p=0.004, R2=0.61), BM (p=0.01, R2=0.53), and blood (p=0.0005, R2=0.75). In humans as well, the frequency of circulating ABCs was significantly higher in CVD subjects compared to non-CVD subjects. Results from this study suggest a strong association of ABCs with aggravated atherosclerosis in both murine models and humans with CVD.
Arterial antibodies promote atherosclerotic plaque development in mice
Authors: Taylor Joshua A , Hutchinson Mark A , Gearhart Patricia J , Maul Robert W 
Published: January 2023

Abstract
Atherosclerosis is characterized by the progressive buildup of cholesterol-rich plaques within the arterial wall. Plaques activate the immune system, of which B cells play a major role. Antibodies bind autoantigens released from necrotic cells and form immune complexes to increase inflammation. We isolated aorta-associated B cells from atherosclerotic ApoE-deficient mice on a high-fat diet and performed single cell sequencing of the B cell receptors to obtain antibody pairs. 32 over-represented antibodies were identified and then expressed in Expi293F cells. Epitope determination of these antibodies was performed against a human proteome array, and 15 potential autoantigens bound the monoclonal antibodies with significant affinity and specificity. The 12 most-promising antigens were scrutinized for their location in plaque tissue by immunohistochemistry using the cognate antibodies. Nine of these relevant antigens were then successfully synthesized using the pET bacterial expression system, and purified proteins were used to interrogate antibodies in human and mouse sera by ELISA. Mice were then immunized with the 5 best antigens to confirm their immunogenicity. The relevant antigens and antibodies are now being tested to see if they will protect or promote atherosclerosis development, and if they can be used as biomarkers in human disease.
This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.
Whole-slide multispectral imaging reveals the immune subtypes of melanoma associated with the tumor microenvironment: An automated 7-color mIF assay
Authors: Mehra Navi , Patel Bhavika , Allen Stephanie , Boldt Brittney , Ramirez Noah , Mammadova Najiba , Haggerty Agnes 
Published: January 2023

Abstract
Immunotherapy and precision medicine are rapidly developing approaches to cancer therapy. Biomarkers that detect the tumor and tumor microenvironment allow for the development of strategies that accelerate the advancement of treatments to enhance a patient’s immune system. Akoya’s MOTiF™ PD-1/PD-L1 Auto Melanoma Kit is a validated, multiplex immunoassay enabling detection of the 6 most clinically relevant immuno-oncology and spatial biomarkers: FoxP3, PD-L1, Sox10/S100, PD-1, CD8 and CD68. In this study, the MOTiF™ PD-1/PD-L1 panel was used to analyze the tumor microenvironment and specifically assess immune phenotypes within melanoma samples from 3 patients. This study demonstrates a fully optimized and end-to-end workflow solution for biomarker discovery in melanoma. We demonstrate the utility of Akoya’s MOTiF™ PD-1/PD-L1 Melanoma panel kit in studying the cellular diversity while retaining spatial context. Stained slides were analyzed using the InForm® and PhenoptrReports image analysis platforms to identify phenotypes and better understand spatial relationships between cell phenotypes. The MOTiF™ PD-1/PD-L1 panel kit provides reproducibility across different patient samples. This data provides insight into the innate and adaptive immune landscape for targeted design of new immunotherapies as well as improved efficacy and reduced toxicity in the treatment of melanoma.
Androgen Receptor Deficiency Eliminates Sex Differences in Mast Cells
Authors: Moeser Adam James , Tang Yu Ping , Thelen Kyan , Fardisi Mahsa 
Published: January 2023

Abstract
A sex bias exists in mast cell (MC)-associated inflammatory diseases with females at increased risk. Our previous studies demonstrated that MCs exhibit a sexually dimorphic phenotype with male MCs exhibiting significantly reduced concentration and release of MC granule mediators (histamine, serotonin, and proteases) which was associated with reduced severity of anaphylaxis in males. Further, we showed that perinatal androgens are important in organizing male MC sex differences. The mechanisms by which androgens organize MC sex differences remain poorly understood. Utilizing male mice with reduced androgen receptor (AR) expression induced by Cre-LoxP technology (induced TFM; iTfm), we tested the hypothesis that ARs mediate sex differences in MC histamine storage and release, and severity of anaphylaxis. Isolated peritoneal MCs (pMCs) from iTFm male mice exhibited a feminized MC phenotype characterized by a greater level of histamine compared with control male pMCs, but similar to female controls. Following induction of IgE-mediate anaphylaxis in vivo, iTFM male and female pMCs contained lower (n=5–6; P&lt;0.01) histamine contents compared with control males, suggesting enhanced release of histamine. Preliminary results also indicate that iTFm male mice exhibit greater IgE-mediated histamine releases and hypothermia. Experiments with bone marrow derived MCs (BMMCs) showed that IgE-mediated histamine release from iTFm male and female BMMCs was greater (n=5–6; P&lt;0.01) relative to control male BMMCs. Together, these studies demonstrate that sex differences in MC phenotype and function are mediated via Ars and that iTFm mice are a valid model to explore AR-mediated sex differences in MC function and disease pathogenesis.
Supported by NIH R01 HD072968, NIH R21 AI140413
Fluoxetine-mediated suppression reveals a role for P2X receptors in mast cell function and allergic disease
Authors: Burchett Jason , Haque Tamara , Kee Sydney Ann , Sebti Said , Martin Rebecca , Ryan John J 
Published: January 2023

Abstract
There is a clinical need for new treatment options addressing allergic disease because current approaches are often suboptimal. Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that have been shown to possess anti-inflammatory properties, but the mechanism of action is unclear. Because mast cells are critical effectors of allergic inflammation, we tested SSRI effects on IgE-induced mast cell function. We show that the SSRI fluoxetine suppresses IgE-mediated degranulation and cytokine production in mouse and human mast cells. Fluoxetine was also effective in a model of allergic airway inflammation, where it reduced bronchoresponsiveness and inflammation. IgE crosslinkage elicited rapid ATP release from mast cells, suggesting ATP-P2X receptor signaling may potentiate IgE-driven responses. We subsequently found that fluoxetine inhibits ATP-induced mast cell function. We propose that fluoxetine acts by blocking an ATP-P2X autocrine or paracrine loop that augments mast cell-mediated inflammation. These data support the potential repurposing of fluoxetine in allergic disease.
Supported by NIH/NIAID R01138495 R01164710
TGF-β promotes glycolysis through PFKL in activated macrophages and exacerbates sepsis by disrupting blood coagulation
Authors: Gauthier Thierry 
Published: January 2023

Abstract
The metabolism of macrophages plays a key role in their immune responses, but the mechanisms by which the metabolism is regulated remain largely unknown. We show here that TGF-β induces a unique phenotype of macrophages and regulates the glycolysis of macrophages independently of inflammatory cytokine production. Specifically, TGF-β increased expression and activity of phosphofructokinase-1 liver type (PFKL) in macrophages and thus promoted their glycolysis during cell activation, yet paradoxically suppressed the production of proinflammatory cytokines in the same macrophages. The upregulation of glycolysis was mediated by an mTORc-MYC dependent pathway, whereas the inhibition of cytokines was ascribed to downregulated activation of the major pro-inflammatory transcription factors, namely AP-1, NFkB and STAT1. Importantly, we found that in LPS-induced sepsis model, TGF-β enhancement of macrophage glycolysis led to a decreased survival in mice by increasing blood coagulation. Analysis of cohorts of patients with sepsis and covid-19 revealed that the expression of PFKL, TGF-b receptors TGFBRI and coagulation factor F13A1 in monocytes positively correlated with the progression of the disease. Thus, TGF-β is emerging as a critical cytokine regulating macrophage metabolism and could serve as a therapeutic target in patients with sepsis and Covid-19.
Supporte by NIH/NIDCR intramural funding
Levels of epigenetic regulators TET2 and DNMT1 correlate with virus-stimulated pDC differentiation from IFN-α producing cells to antigen presenting cells
Authors: Codrington Alicia L , Singh Sukhwinder L , Fitzgerald-Bocarsly Patricia L 
Published: January 2023

Abstract
Plasmacytoid dendritic cells (pDC) are potent producers of IFN-α in response to DNA and RNA viruses and subsequently mature into antigen presenting cells. We investigated the role of DNA methyltransferase 1 (DNMT1) and Ten-eleven translocation methyl-cytosine dioxygenase 2 (TET2), which are generally known as gene silencers and activators, respectively, in pDC activation. PBMC from healthy donors were stimulated with Herpes Simplex Virus (HSV) or Influenza-A virus (IAV) for 6- and 24h. PBMC were surface-stained for pDC markers CD123, HLA-DR and CD11C and co-stimulatory markers CD80 and CD86, then intracellularly for DNMT1, TET2 and IFN-α, and acquired by flow cytometry. mRNA expression of DNMT1 in purified pDC was determined by qRT-PCR. At 6h, DNMT1 protein was expressed at equivalent levels in pDC with and without stimulation and in IFN-α+ and IFN-α-pDC, and in unstimulated controls. In contrast, TET2 proteins were elevated in IFN-α+ but not IFN-α-pDC and controls at 6h. At 24h, DNMT1 increased while TET2 was reduced in stimulated pDC, correlating with high IFN-α levels at 6h and low levels at 24h. There was no change in DNMT1 gene expression in virus-stimulated vs. unstimulated controls. CD80 and CD86 were elevated at 24h compared to 6h in stimulated pDC, confirming pDC differentiation from IFN-α producing to antigen presenting cells. At the point of pDC differentiation, TET2 levels were decreased and DNMT1 increased. These results suggest that TET2 is demethylating and causing activation of genes associated with pDC IFN-α production, while DNMT1 methylates and silences genes in this pathway. This results in an initial induction of TET2 and IFN-α production followed by upregulation of co-stimulatory markers, DNMT1 and loss of IFN-α.
Supported by 1. AAI trainee fellowship Careers in Immunology Fellowship Program AAI EIN #: 52-2317193 2. R01Al106125