bluuebunny/crossref_metadata_2025_split huggingface dataset:
Therapeutic NKG2D Ligation Facilitates Improved Wound Healing
Authors: Schenkel Jason M , Kubasiak John C , Dolubzino Hubert , Kohlhapp Frederick J , Tarabichi Saeed , Lee Michael , Guevara-Patino Jose A , Zloza Andrew 
Published: January 2023

Abstract
NKG2D is a C-type lectin-like receptor in the CD94/NKG2 family and is expressed by a variety of leukocyte populations including natural killer (NK) cells, γδ T cells, CD8+ T cells and certain innate lymphoid cell (ILC) subsets. NKG2D can bind proteins in two protein families – MIC and RAET1/ULBP. Expression of NKG2D ligands is low/absent under homeostatic conditions, but these proteins are upregulated in several contexts, including infection, cancer, physiologic stress, and senescence. NKG2D-driven activation of NK and CD8+ T cells has previously been shown to result in augmented control of infections and tumor growth. However, the role of NKG2D in the context of tissue injury and repair has not been established. Here we tested the effects of augmented NKG2D ligation in the context of a skin injury model. Biopsy punches were used to generate six 3- mm wounds on the dorsal surface of C57BL/6 mice. On days 0 and 1, NKG2D stimulating antibodies or isotype controls were injected into the peritoneal cavity and wound healing was assessed daily. Mice treated with NKG2D activating antibodies had more rapid wound repair than controls – wounds were healed by day 5 in NKG2D-treated mice as compared to day 7 in controls. Analogous results were observed in an ex vivo human skin model. Using a biolistic particle delivery system to induce upregulation of RAE (an NKG2D ligand), we observed more rapid wound repair compared to controls. Genetic ablation of NKG2D resulted in a 40% delay in wound healing. Moreover, depletion of NK1.1+ or CD8+ leukocytes blunted the benefit of NKG2D ligation, suggesting that T cells, NK cells, and/or ILCs may play a role in driving tissue repair. Thus, we demonstrate a potential therapeutic function of NKG2D engagement in tissue regeneration.
Bacterial amyloids from biofilms break tolerance in lupus
Authors: Lee Michael Hweemoon , Tursi Sarah A , Green Benjamin L , Caricchio Roberto , Tükel Çagla , Gallucci Stefania 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease, in which infections are considered to play a pathogenic role, but the cellular and molecular culprits remain unknown. Bacterial biofilms are multicellular bacterial communities important in the establishment of chronic infection by pathogens. Bacteria produce amyloids, complex proteins with a conserved beta sheet structure that strengthen the extracellular matrix of their biofilms. We have reported that bacterial and eukaryotic DNA is incorporated into curli fibers, functional bacterial amyloids present in Salmonella and E. coli biofilms. I.P. infections with curli-expressing S. Typhimurium or E. coli Nissle, and also systemic administration of curli/DNA complexes purified from Salmonella biofilms accelerated onset in lupus-prone NZBxW/F1 mice and triggered autoantibodies production in non-predisposed mice, suggesting curli/DNA complexes as novel players in SLE pathogenesis. We present here evidence that curli/DNA complexes can activate multiple B cell subsets in vivo. In addition, curli/DNA complexes are capable of directly inducing in vitro polyclonal activation and proliferation of B cells from wildtype mice and from lupus-prone mice and also from 3H9 mice, which are knocked-in for a rearranged anti-DNA Ig heavy chain and whose B cells are normally tolerized for the self-Antigen. Our results suggest that curli/DNA complexes may induce autoantibody production by directly breaking tolerance in autoreactive B cell.
Enhanced survival of BENTA patient B cells is dependent on MALT1 protease activity
Authors: Arjunaraja Swadhinya , Malinverni Claire , Sukumar Gauthaman , Quanchard Jean , Lott Nathaniel , Dalgard Clifton , Bornancin Frederic , Snow Andrew L 
Published: January 2023

Abstract
BENTA (B cell Expansion with NF-κB and T cell Anergy) disease is a rare, selective B cell lymphoproliferative disorder caused by gain-of-function (GOF) mutations in the lymphocyte scaffolding molecule CARD11. CARD11 is required for downstream activation of NF-κB following antigen receptor engagement, which normally triggers recruitment of BCL10 and MALT1. MALT1 serves both scaffolding and proteolytic functions that initiate and amplify canonical NF-κB signaling. It still remains unclear how continuous CARD11 signaling ultimately drives massive B cell accumulation in BENTA patients. Here we show that in contrast to normal human B cells, BENTA patient B cells exhibited a striking resistance to apoptosis in cell culture, particularly after stimulation. Enhanced survival of patient cells was dependent on MALT1 protease, which is activated constitutively by CARD11 GOF mutants. Treatment with a novel, specific low molecular weight inhibitor of MALT1 protease completely restored apoptosis sensitivity in BENTA patient B cells. RNA-Seq analyses revealed that MALT1 protease inhibition induced a specific subset of pro-apoptotic genes in activated patient B cells, and downregulated multiple genes associated with cell cycle progression and metabolism, in addition to several NF-κB target genes. Although not strictly required for CARD11-dependent NF-κB activation, our results imply that MALT1 protease function governs a pro-survival signaling program in BENTA B cells that likely contributes to the profound B cell lymphocytosis that distinguishes this disease. MALT1 protease thus represents an attractive therapeutic target for reducing B cell burden in these patients, thereby lowering the risk of B cell malignancy later in life.
Development and Characterization of V-PLEX® TH17 Cytokine. Assays
Authors: Yatsula Victoria , Kobayashi Lori , Sharma Deepak , Qualters Elisa , Janaki Lalitha , Weckerle Celine , Harkins Seth B. , Stewart David , Wohlstadter Jacob N. 
Published: January 2023

Abstract
Th17 cytokines are important mediators of the host defense against infection and are increasingly investigated for their role in autoimmune disorders and immune regulation at mucosal surfaces. Here we describe the development, characterization, and analytical validation of a multiplexed immunoassay panel for seven Th17 cytokines, IL-17A, IL-21, IL-22, IL-23, IL-27, IL-31, and MIP-3alpha, on MSD’s V-PLEX platform.
To efficiently and rapidly identify potential antibody pairs, biotinylated capture antibodies and detection antibodies conjugated with SULFO-TAG™ label were screened on MSD’s U-PLEX® platform, which enables the solution phase assembly of capture antibody arrays. Subsequent development used printed arrays of capture antibodies. Antibody concentrations, calibrator curve linearity, dynamic range, specificity, matrix tolerance, and assay robustness were analyzed for each assay during development. Calibration curves demonstrated a three-log dynamic range while achieving a lower limit of quantitation (LLOQ) of less than 1 pg/mL for most assays. Control samples for the assays had CVs of less than 10%. Dilution linearity and spike recovery studies in serum, plasma, urine, and cell culture media were conducted to demonstrate matrix compatibility. Spiked matrix samples were typically found to recover between 80%–120% of the expected value. Cross-reactivity was shown to be less than 0.3% between assays within the panel and less than 0.5% when panned against more than 30 other blood-related biomarkers.
These validated, multiplexed assays provide sensitive measurement of Th17 cytokines in a variety of matrices and can be used as part of a researcher’s pre-clinical and clinical studies.
MicroRNA-142 guards against autoimmunity by controlling Treg cell development and function
Authors: Wang Wei-Le , Ouyang Ching , Cassady Kaniel , Xiong Min , Reyes Estefany , Davis Alicia , Tang Kathie , Zeng Defu , Boldin Mark 
Published: January 2023

Abstract
Regulatory T (Treg) cells are critical in preventing aberrant immune responses. Post-transcriptional gene control by microRNA (miRNA), a large class of small regulatory RNA, has recently emerged as a key genetic element required for Treg cell function. Nevertheless, specific miRNA gene(s) that play a vital role in the regulation of Treg cell activity are still unknown. Here we report that mice with Treg cell-specific ablation of miR-142 (hereafter Foxp3CremiR-142fl/fl mice) developed a fatal systemic autoimmune disorder due to a breakdown in peripheral T cell tolerance. Foxp3CremiR-142fl/fl mice displayed a sharp decrease in abundance and suppressive capacity of Treg cells. Analysis of miR-142-deficient Treg cells revealed excessive IFNγ production and dysregulated IFNγ signaling. We have identified several of the derepressed IFNγ-related genes, including Ifngr2 receptor and Hif1a transcription factor, as direct miR-142 targets. Furthermore, lowering the Hif1a gene dose in Treg cells significantly diminished the hyperactivation of peripheral effector T cells in Foxp3CremiR-142fl/flmice. Thus, miR-142 is an indispensable regulator of Treg cell development and function that mediates its control by attenuating IFNg response.
Downregulation of allograft inflammatory factor-1 prevents B cell infiltration to cardiac tissue during the development of diabetic cardiomyopathy (DCM)
Authors: Sarkar Amrita , Shukla Sanket Kumar , Rafiq Khadija 
Published: January 2023

Abstract
An emerging concept is that inflammation might be linked to alteration of immune system, thus leading to adverse cardiac function, remodeling in DCM. However, the role of inflammatory pathways during development of DCM is poorly understood and controversial, as both positive/negative effects of inflammation have been observed. 8 weeks wild type male mice were injected with streptozotocin (50 mg/kg, 5 days intraperitoneally) and studied 8 weeks after induction of diabetes mellitus having elevated blood glucose levels (&gt;300 mg/dL) and DCM was confirmed through ECG. We found a significant decrease in B cell infiltration into left ventricular heart, but not in other organs, during DCM. Interestingly, downregulation of B cell infiltration correlates with downregulation of a B cell inflammatory molecule expression, Allograft Inflammatory Factor-1 (AIF-1), which has been reported to enhance lymphocyte activation. Our in vitro study showed that B cell migration is regulated by AIF-1 expression. We observed significant migration of B cells to hyperglycemic AIF-1 transfected H9C2cells compared to control cells transfected with an empty vector. In vivo studies showed strong co-localization of AIF-1 and CD19 B-cell marker in nondiabetic heart tissue compared with diabetic heart tissue. Interestingly, AdAIF-1 overexpression promoted B cell migration into diabetic hearts, reduced inflammation and pathological remodeling. These data suggest that AIF-1 downregulation in cardiac myocytes is caused by diabetes that prevents B cell infiltration into hearts, and increase cardiac inflammation that leads to cardiomyocyte death and that overexpression of AIF-1 could be a novel treatment for this condition.
Novel in vitro model of Rich focus formation and mycobacterium infected cell migration at the blood brain barrier
Authors: Walter Fruzsina R. , Gilpin Trey E. , Mergaert Aisha , Sandor Matyas , Fabry Zsuzsanna 
Published: January 2023

Abstract
Tuberculous meningitis is the most lethal form of extrapulmonary tuberculosis with more than 100,000 new cases per year. Mycobacterium tuberculosis (Mtb) invades the central nervous system (CNS) in 1–5 % of all cases with a mechanism not well understood. Today’s knowledge is based on the findings of Rich and McCordock proposing that bacteria disseminate via the blood either within a carrier cell or extracellularly, enter the CNS through the blood-brain barrier (BBB) and form a tuberculoma (Rich focus) which is able to rupture causing meningitis. Previously our laboratory showed that the dissemination of Mycobacterium bovis (BCG) and Mtb from pulmonary granulomas is executed by dendritic cells. Currently no good in vitro model exists which is able to mimic the focus formation and the dissemination of Mtb to the brain. We are the first to assemble a mouse primary cell based in vitro Rich focus model. We show that cellular aggregates consisting of BCG or Mtb infected DCs derived from bone marrow of CD11c eYFP animals and peripheral blood mononuclear cells isolated from P25 T-cell receptor transgenic mice form within 60 minutes in cultures. These in vitro granulomas attach to the brain microvessel endothelial cells and facilitate migration of infected cells across the barrier. Aggregate formation and transmigration interfere with endothelial zonula occludens-1 junctional integrity and induce mitochondrial damage shown by Mitotracker staining. These data shed light on the mechanism how mycobacterium infected cells migrate across the BBB. Understanding the mechanism of Mtb dissemination into the CNS is crucial for developing novel therapeutic target inhibiting TB meningitis.
FMNL1 promotes T cell extravasation and trafficking to sites of inflammation
Authors: Thompson Scott Beckett , Long Robert A. , Chung Jeffrey W. , Estin Miriam L. , Jacobelli Jordan 
Published: January 2023

Abstract
To execute their effector function, activated T cells traffic through the blood stream to sites of inflammation. To enter tissues, T cells extravasate through the vascular endothelial cell wall by a process known as transendothelial migration (TEM). While many of the adhesion molecules and chemokine signaling pathways required for TEM have been previously characterized, little is known about how downstream cytoskeletal effectors mediate the mechanical forces and shape changes needed for T cell TEM. Formin-like 1 (FMNL1) is a terminal cytoskeletal effector directly involved in actin network remodeling. It has been associated with the formation of membrane protrusions that likely function in migration and cell-cell interactions, and is highly expressed by activated T cells. The goal of our study was to examine the role of FMNL1 in activated T cell extravasation and trafficking to sites of inflammation. While activated FMNL1 deficient T cells had normal migration in response to chemokine across 5μm pore transwell membranes, they displayed an impaired ability to migrate through more restrictive 3μm pores. In vitro time-lapse microscopy data suggest that FMNL1 deficient T cells have a reduced ability to complete TEM across brain endothelium. FMNL1 deficient T cells were also impaired in their ability to traffic to the inflamed central nervous system in vivo. Finally, adoptively transferred autoreactive FMNL1 deficient T cells were impaired in their ability to induce Experimental Autoimmune Encephalitis. Together these data suggest that FMNL1 is required for the efficient migration of activated T cells though restrictive endothelial barriers and may be a promising therapeutic target for regulating T cell trafficking in autoimmunity.
Role of Rho kinases in B cell development
Authors: Sambandam Arivazhagan , Caplazi Patrick , Wu xiumin , Jeet Surinder , Liu John , Zhang Juan , Lee Wyne , Pappu Rajita 
Published: January 2023

Abstract
Cytoskeletal rearrangement is a critical component to hematopoietic stem cell development and differentiation into mature leukocyte populations. A number of chemokines and biolipids have been shown to regulate these events via binding to G protein coupled receptors. Many of these receptors couple to the Ga12/13 family of G proteins to regulate actin reorganization via the RhoA small GTPase. The Rho-kinases, Rock1 and Rock2, are activated downstream of RhoA to induce cytoskeletal changes that regulate numerous cellular processes, including contraction, polarity, proliferation, motility, adhesion and viability. A role for RhoA in regulating hematopoiesis and subsequent leukocyte development has been established using genetic studies. However, a similar role for the Rock1 and Rock2 has not been demonstrated. As global deletion of either kinase causes embryonic lethality, we used conditional gene targeting to ablate either the Rock1 or Rock2 allele in adult mice. Rock1flox/flox or Rock2flox/flox mice were crossed to the Rosa26-Cre ERT2 line to globally delete the floxed alleles with tamoxifen. In contrast to deletion of RhoA, conditional deletion of either Rock1 or Rock2 was well-tolerated, with no aberrant effects on hematopoiesis. These results suggest functional redundancy between Rock1 and Rock2 in regulating RhoA activity in hematopoiesis. However, we observed a significant decrease in marginal zone B cell number and frequency in the spleen of mice lacking Rock1. We also observed a follicular B cell defect in the spleen of mice lacking Rock2. These results are consistent with the known roles of GPCRs in B cell migration/positioning, and suggest there are isoform specific functions of these kinases.
Distinct tissue specific functions for bone marrow regulatory T cells
Authors: Camacho Virginia , Wolock Sam , Tenen Danielle , Welner Robert 
Published: January 2023

Abstract
While the influence of T-regs has been explored in numerous contexts, their role within the bone marrow (BM) has not been elucidated. In this study, we establish that BM derived T-regs represent a specialized population with a tissue-specific role. Functional homing assays demonstrate that BM T-regs preferentially traffic back to this site, revealing that specific signaling cues recruit T-regs to this microenvironment. This is supported by RNA-seq data showing that the chemokine and cytokine receptor profiles of BM T-regs differ from T-regs in secondary lymphoid tissues. BM Tregs are also characterized by a higher expression of CD127/IL7Ra conventionally not expressed on peripheral T-regs. Depletion of Tregs via anti-CD25 treatment has widespread effects across several BM populations, especially the stem cell compartment. Loss of T-regs results in increased HSC cycling and proliferation, while fostering myeloid skewed differentiation. Key stem cell features including repopulation capacity and dormancy are also disrupted. Moreover, supportive niche populations are also altered by Treg depletion, as evidenced by increased cycling and proliferation of stromal cells. Non-genotoxic conditioning and transplantation, reveals that the T-reg depleted microenvironment is less supportive of HSCs, thus establishing a novel role for Tregs in maintaining niche function. Remarkably, the effects of Treg depletion are found to be longlasting. In the absence of continued treatment, cohorts sustain a myeloid bias, as well as increased numbers of HSCs and stromal cells. These observations support the conclusion that BM T-regs represent a unique subset that maintains numerous populations and have a specialized function within this tissue.
Maternal Lipid Regulation of the Development of Responsiveness to Allergen in Neonates and Infants.
Authors: Cook-Mills Joan M. , Walker Matthew 
Published: January 2023

Abstract
In animals and humans, offspring of allergic mothers have increased responsiveness to allergen. A major question is what are the maternal factors of allergic mothers that alter offspring responsiveness to allergen? We focused on maternal lipids, because lipids are altered during allergic inflammation and because lipids are transported to the fetal liver and are in the mother’s milk. In our studies, allergic pregnant mice had increased glucosylceramides (GlcCer) in the mother plasma, placenta, and fetal liver as compared to non-allergic mothers. In allergen-challenged pups of allergic mothers, GlcCer was elevated in the pup lung and liver. Maternal GlcCer is directly transported to the placenta, the fetal liver, mother breast tissue and the milk of the pup stomach. Administration of a pharmacolgical inhibitor of GlcCer synthase to allergic mothers reduced GlcCer to the levels of GlcCer in non-allergic mothers and blocked the development of allergic lung inflammation and generation of allergen-specific IgE in allergen-challenged pups of allergic mothers. We also demonstrated that the fetal liver and offspring of allergic mothers have increased numbers of resident dendritic cells (DCs) and that the administration of the GlcCer synthase inhibitor to the allergic mothers blocked the increase in these DCs in the offspring. In summary, allergic mothers had increased GlcCer that is transferred to offspring and mediates changes in offspring DCs and responsiveness to allergen. These data have a significant impact on our understanding of mechanisms of maternal lipid regulation of offspring development of allergies and has the potential to lead to novel interventions that significantly impact risk for allergic disease early in life.
IL-12 induced STAT4 activation plays a role in pro-inflammatory neutrophil functions
Authors: Keeter William Coles , Moriarty Alina , Butcher Matthew J. , Ma Kaiwen W. , Nadler Jerry L. , Galkina Elena 
Published: January 2023

Abstract
Signal transducer and activator of transcription 4 (STAT4) becomes activated via Jak/Tyk kinases in response to IL-12 and other cytokines, leading to expression of genes involved in pro-inflammatory pathways. STAT4 has been well characterized in T lymphocytes and NK cells, yet less is known of its potential role in neutrophils. Neutrophils have recently been shown to play a contributing role in atherogenesis. Our published data demonstrate that global deficiency of STAT4 results in decreased atherogenesis in atherosclerotic ApoE−/− mice. However, the cell specific role of STAT4 in atherosclerosis is unknown. Western blot analysis revealed STAT4 expression and IL-12 induced phospho-STAT4 in isolated neutrophils with no distinguishable difference between blood and bone marrow neutrophils. IL-12 dependent activation of wild-type (WT) neutrophils induced an increase in reactive oxygen species (ROS) production when compared to non-stimulated WT neutrophils in both blood and bone marrow. Similarly, IL-12 stimulated WT neutrophils showed an increase in neutrophil extracellular trap (NET) elastase activity versus unstimulated controls. IL-12 stimulated neutrophils from Stat4−/− mice displayed no changes in NET elastase activity when compared to non-stimulated STAT4 deficient neutrophils suggesting STAT4-dependent NETosis. Furthermore, IL-12 stimulated Stat4−/− neutrophils showed decreases in both ROS production and NET elastase activity when compared to IL-12 stimulated WT neutrophils. Our data suggest a novel pro-inflammatory, IL-12 dependent role of STAT4 in neutrophils. Inhibition of these processes by targeting the IL-12/STAT4 pathway may serve as a possible therapeutic approach to treating atherosclerosis.