bluuebunny/crossref_metadata_2025_split huggingface dataset:
Novel nanofibrous DNA-histone structures mediate reproducible, high-throughput analyses of neutrophil extracellular traps and their effects in vitro
Authors: Louttit Cameron , Weerappuli Priyan , Kojima Taisuke , Maeda Midori , Takayama Shuichi , Moon James J. 
Published: January 2023

Abstract
Termed the “double-edged swords of innate immunity” by Kaplan and Radic, neutrophil extracellular traps (NETs) are vital contributors to innate defenses but also participate in the pathogeneses of manifold diseases including systemic lupus erythematosus, rheumatoid arthritis, thrombosis, and cancer. Despite their vast clinical impact, NETs have proven difficult to study in high quantity and isolation; indeed, over 1.9E9 neutrophils are required for full NET coverage of a 96-well plate. Our group has developed a novel NET-mimicking structure for adaptable and reproducible high-throughput analyses of NETs in vitro. Rooted in a base composition of DNA and histones interacted by slow diffusion across an aqueous two phase system, these structures exhibit the same 10–100 nm diameter fibers as their physiological counterparts and can incorporate additional NET-associated proteins and peptides. This platform therefore enables unprecedented high-throughput probes of the roles of individual NET components in health and disease. In a pilot study exploiting this opportunity, our group expanded upon the previously-reported protection of NETs from nuclease-mediated degradation by the peptide LL-37. The comparison between LL-37-containing structures and control structures without the peptide, a data point unavailable endogenously, revealed a 4.5-fold increase in structural half-life conferred by LL-37 after exposure to DNase I. Similar ongoing experiments are exploring the lupus-linked activation of plasmacytoid dendritic cells by NETs. In this way, the reported platform provides new investigative angles for NET studies while also conferring a reproducibility and statistical power unattainable with cell-based methods.
A direct, bioluminescent caspase-1 assay for monitoring inflammasome activation
Authors: O’Brien Martha A. , Moehring Danielle , Munoz-Planillo Raul , Nunez Gabriel , Callaway Justin , Ting Jenny P , Scurria Mike , Ugo Tim , Bernad Laurent , Cali James , Lazar Dan 
Published: January 2023

Abstract
Caspase-1, an essential component of the inflammasome, is activated in response to inflammatory stimuli, resulting in 1) the processing and release of IL-1b and IL-18, and 2) pyroptosis, an immunogenic form of cell death. To provide a homogeneous method for detecting caspase-1 activity, we developed a bioluminescent assay that combines a substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent added directly to cultured cells. The assay can also measure caspase-1 activity released into the medium. Assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG-132, in the reagent and by use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. Using this assay system, caspase-1 activity has been monitored in THP-1 cells, J774A.1 mouse macrophages, mouse bone marrow-derived macrophages (BMDMs), and human primary monocytes treated with known inflammasome inducers. Caspase-1 activity was not detected in BMDMs derived from Casp 1−/−mice. Measuring caspase-1 activity released into medium enables multiplexing of the sample to monitor additional cell parameters. Multiplexing to monitor caspase-1 activity, cell death, and IL-1b release revealed that unprimed, undifferentiated THP-1 cells stimulated with nigericin or a-hemolysin activate caspase-1 and undergo pyroptosis, but do not release any IL-1b. This suggests that K+ efflux alone can engage the inflammasome and activate caspase-1 in THP-1 cells, providing an interesting system to assess pyroptosis in the absence of IL-1b. This convenient caspase-1 assay will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators.
Real-time imaging of non-adherent cells maintained in contact with adherent cell layers
Authors: Yeh Victor , Chen Cindy , Hung Paul 
Published: January 2023

Abstract
Cell-cell interactions are crucial to the survival and function of cells. Imaging these interactions in real-time is a challenging task with non-adherent cells due to their mobility in suspension, not to mention their communication with other heterotypic cells. Here we present a novel method to trap non-adherent cells in precise locations with a layer of adherent cells in order to study and image their interactions while in contact over time. This platform uses a well plate format to integrate 8 individual microfluidic units. Each unit has a 3 mm × 3 mm × 40 μm (L × W × H) chamber for cell culture, and an array of one hundred 72 × 72 × 15 μm (L × W × H) trap areas within each chamber to immobilize non-adherent cells underneath. The chamber in each unit can be addressed with programmable and on-demand perturbation of up to 2 different reagents to perform live cell imaging assays without disrupting the cell cultures. We have successfully cultured MCF-7 cells using this design, with Jurkat cells subsequently loaded and trapped above the layer of MCF-7. Upon reagent introduction, time-lapse images confirmed that the Jurkat cells remained in place. In conclusion, the microfluidic design allows investigation of cell-cell interactions in a wide range of applications requiring the confinement of non-adherent cells in contact with a different adherent cell layer. The on-demand perturbation feature further increases the capability to control the microenvironment exposed to the cell culture.
Increasing the palette of fluorophores for intravital 2P-imaging of the vascular and immune system in mice
Authors: Saunders Brian T , Li Wenjun , Williams Jesse W. , Kim Ki-Wook , Randolph Gwendalyn J. , Zinselmeyer Bernd H. 
Published: January 2023

Abstract
Disruption in the movement of blood or lymph leads to potentially fatal diseases, most commonly atherosclerosis in the blood and lymphedema/lymphangitis in the lymph. There have been an enormous amount of studies done on blood vasculature in the past few decades, but our understanding of lymphatics in health and disease remains poor. Lymphatics interact systemically and locally with the blood vasculature and we have recently proven that dendritic cells are an important regulator in lymphatic function. Some of these processes can only be proven in a living animal as the interactions and impact of immune cells on vascular flow dynamics and vice versa cannot been mimicked in vitro. Our most recent intravital imaging studies allow the visualization of resident macrophage dynamics within a plaque. Due to the fast movement of cells in the vasculature and the sensitivity of cells to phototoxicity it was challenging to generate a palette of dyes which are low in cytotoxicity able to label the structures and cell populations of interest. It is possible by using fluorophores (Fluorescent proteins and conventional dyes) efficient enough to be excited with low laser power and an emission sufficient enough for being detected. Recently, a femtosecond pulsed Titanium Sapphire (Ti:sap) laser with averaged power ≥ 1W above 1200 nm became available. We have coupled a laser of this type (InSight DS+) together with an conventional Ti:sap-laser (Mai Tai DeepSee) to an customized ultra fast and sensitive scanning Microscope. Using these settings we can image up to five fluorophores in intravital settings. This approach enables us to visualize lymphatics and blood vessels together with myeloid cells and lymphocytes in vivo.
IDO1-targeting intervention by proteasome inhibition in autoimmune diabetes
Authors: Orabona Ciriana , Albini Elisa , Mondanelli Giada , Volpi Claudia , Pallotta Maria Teresa , Puccetti Paolo , Grohmann Ursula 
Published: January 2023

Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1) is a metabolic enzyme involved in the catabolism of tryptophan. Its metabolic activity has become pivotal in the regulation of the immune responses. The functional expression of IDO1 enzyme confers a tolerogenic phenotype to different subsets of dendritic cells (DCs). Our previous data suggested a defective IDO1 expression in a prediabetic phase of nonobese diabetic (NOD) mice, a prototypic model of human type 1 diabetes. Based on the recent finding that IDO1 may be subjected to regulatory proteolysis, which is triggered by IL-6 and mediated by the immunoproteasome, this pathway was explored in splenic plasmacytoid dendritic cells (pDCs) of NOD mice. The in vitro conditioning of murine NOD pDCs with the proteasome inhibitor Bortezomib, increased the functional expression of IDO1 enzyme, conferring those cells an immunoregulatory phenotype that is strongly inhibited for presenting in vivo the diabetogenic antigen IGRP, an effect accompanied by increased IGRP-specific regulatory T cells in pancreatic lymph nodes. The inhibition of IDO1 by 1-methyl tryptophan (MT) completely abolished the immunoregulatory effect of Bortezomib in NOD pDCs, indicating the involvement of the enzyme. Moreover, the pharmacologic treatment of NOD female mice with Bortezomib during prediabetic phase (8–10 weeks old) prevented the onset of hyperglycemia, reducing the diabetes incidence. Overall, our data suggest that the control of proteasomal degradation in pDCs may represent an innovative strategy for inducing/potentiating IDO1-mediated immunoregulation in autoimmune diabetes.
Reinstalling immune regulatory tryptophan catabolism in juvenile diabetes via interleukin 6 receptor blockade
Authors: Mondanelli Giada , Orabona Ciriana , Pallotta Maria Teresa , Albini Elisa , Volpi Claudia , Grohmann Ursula 
Published: January 2023

Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1) is a potent immunoregulatory enzyme that catalyses the degradation of the essential amino acid tryptophan (Trp) along the kynurenines pathway. Significant changes in systemic Trp catabolism have been reported in many diseases, including cancer and autoimmunity. In female nonobese (NOD) mice, IDO1 expression and hence immune tolerance to pancreatic b-cell autoantigens are defective in conventional dendritic cells stimulated with IFN-γ, the main IDO1 inducer. Although the evidences in NOD mice suggest that IDO1 function is impaired, the existence of the IDO1 defect in human T1D (type 1 diabetes) has not been proven yet. Here we monitored the IDO1 expression and activity in peripheral blood mononuclear cells (PBMCs) of children with T1D as compare to age-matched control subject, in response to IFN- γ. Results from kynurenines assay and Western blot analysis demonstrate that the majority of patients with T1D is characterized by defective Trp catabolism. Moreover, our data indicated that this defect is mainly imputable to a SOCS3-mediated, dysregulated IL-6 signaling that would favor IDO1 proteasomal degradation in inflammatory environments, i.e. dominated by IFN-γ. To confirm this, we measured IDO1 expression and activity in PBMCs co-incubated with IFN-γ and Tocilizumab (TCZ), a licensed IL-6 receptor blocker. Results showed that TCZ is able to restore normal levels of IDO1 catalytic activity in response to IFN-γ in approximately 30% of the examined T1D population. Besides further confirming the heterogeneity of the disease, our data indicate the existence of a subset of individuals with T1D who may gain clinical benefit in restoring immunoregulatory mechanisms by treatment with tocilizumab.
Dendritic cell-CD8+ T cell communication is essential for Kinsenoside, a potential immunosuppression drug for autoimmune hepatitis
Authors: Liu Tingting , Xiang Ming , Tan Wanyue , Liu Junjun , Li Hua , Liu Xiulan , Cao Hui , Zhu Hucheng , Tuo Yali , Zhang Yonghui 
Published: January 2023

Abstract
Autoimmune hepatitis (AIH) is a chronic auto-immune inflammation against liver tissue. Here, using T cell-deficient mice, acute AIH mice model and a novel AIH mice model induced by dendritic cells loaded with murine hepatocellular carcinoma cells (DC/Hepa1–6) vaccination, we reveal that Kinsenoside (KD), a major component of Anoectochilus roxburghii, has an powerful therapeutic effect on AIH by limiting the metabolism of immune cells such as DCs and CD8+ T cells, which results in the immunosuppression against autoimmune liver injury. The immunosuppressive and anti-inflammatory effect of KD is triggered by increased the expression of co-inhibitory molecules PD-L1/PD-1 to inhibit DC cross-priming of CD8+ T cell responses but not modulate the hepatotoxicity of CD8+ T cells directly. Furthermore, KD reduces immune cells metabolism and executes immunosuppressive and anti-inflammatory action by binding to the potential target VEGFR2 and blocking the cross-talk of metabolism related PI3K-AKT and inflammation related JAK2-STAT3 signaling pathways.
Programmed cell death-ligand 1 ameliorates psoriatic inflammation by inhibiting TCR-induced IL-17A production of T cells overexpressing PD-1
Authors: Kim Jong Hoon , Choi Young Joon , Lee Byung Ha , Song Mi-Young , Ban Chae Yeon , Kim Jihye , Park Junsik , Kim Song-Ee , Kim Tae-Gyun , Park Su-Hyung , Kim Hyoung-Pyo , Sung Young-Chul , Kim Soo-Chan , Shin Eui-Cheol 
Published: January 2023

Abstract
Psoriasis is one of the most common chronic inflammatory diseases in the skin. Recently, IL-17-producing T cells have been shown to play a critical role in psoriatic inflammation. Programmed cell death-1 (PD-1) is a co-inhibitory receptor expressed on T cells in various chronic inflammatory diseases; however, it remains to be elucidated whether PD-1 is overexpressed on T cells in psoriasis and whether PD-1 agonist alleviates psoriatic inflammation. We examined PD-1 expression on IL-17A-producing T cells from Imiquimod (IMQ)-treated mice and psoriasis patients. Additionally, we investigated the therapeutic effect of recombinant programmed cell death ligand-1-Fc (PD-L1-Fc) protein on IMQ-induced psoriatic inflammation. During IMQ-induced psoriatic inflammation, PD-1 is overexpressed on CD27−Vγ1− γδ T cells. Further, PD-1 expression on IL-17A+ T cells was confirmed in tissue samples from patients with psoriatic skin. In the CD27−Vγ1−γδ T cell population, Vγ4−γδ T cells with Vγ6 mRNA expression showed a high level of PD-1 expression. Further, these PD-1hiVγ4−(Vγ6+) γδ T cells were specialized for T-cell receptor (TCR)-induced IL-17A production, which was inhibited by PD-L1-Fc protein treatment. In IMQ-treated mice, PD-L1-Fc protein reduced psoriatic inflammation when given alone and enhanced the therapeutic effect of anti-p40 when given in combination. In conclusion, PD-1 is overexpressed in IL-17A-producing T cells in both IMQ-treated mice and psoriasis patients, and recombinant PD-L1-Fc protein alleviates psoriatic inflammation in IMQ-treated mice.
Constitutive expression of LAG-3 defines the exhaustion of intratumoral PD-1+ T cells in B-cell non-Hodgkin lymphoma
Authors: Yang Zhi-Zhang , Kim Hyojin , Price-Troska Tammy , Novak Anne , Ansell Stephen 
Published: January 2023

Abstract
We have previously shown that not all PD-1+ cells are exhausted and that PD-1 is differentially expressed on two distinct T-cell subpopulations, with high expression on T follicular helper cells and dim expression on exhausted T cells. We observed that while PD-1bright T cells lacked the ability to produce cytokines and granules, PD-1dim T cells from biopsy specimens of B-cell NHL were the major source of cytokine and granule production compared to PD-1− T cells, suggesting that PD-1dim are functionally active. Therefore, other markers are needed to more clearly identify exhausted intratumoral T cells. We first phenotyped intratumoral PD-1+ (brightly- and dimly-stained) T cells and found that LAG-3 was exclusively expressed on dimly-stained PD-1+ T cells, forming a defined population of intratumoral PD-1+LAG-3+ T cells. We found that LAG-3 expression was strongly upregulated on CD4+ or CD8+ T cells by IL-12, a cytokine that has been shown to induce T-cell exhaustion. Functionally, with a substantial numbers of PD-1dim T cells being cytokine- or granule-producing cells, it is PD-1dimLAG-3−, but not PD-1dimLAG-3+, T cells that were responsible for the capacity of producing cytokines and granules. Supporting this finding, we found that blockade of both PD-1 and LAG-3 signaling by using the combination of PD-1 and LAG-3 blocking antibodies increased cytokine production by T cells significantly more than PD-1 or LAG-3 antibody alone, suggesting that LAG-3 is important in the impairment of PD-1+ T cells. Taken together, these results suggest that constitutive expression of LAG-3 was critically involved in and defines the exhaustion of PD-1+ T cells in B-cell NHL.
Tumor B7-H1 regulates cancer stem cell generation and virulence
Authors: Gupta Harshita B. , Hurez Vincent , Clark Curtis A , Sareddy Gangadhara R , Vadlamudi Ratna , Li Rong , Curiel Tyler J 
Published: January 2023

Abstract
Cancer stem cells (CSC) resist chemotherapy and radiation, leading to treatment failures and reduced patient survival. Thus, CSC are clinically relevant therapy targets. B7-H1 is an immune checkpoint molecule and a target for αB7-H1 immunotherapy. We generated control (native B7-H1+) and B7-H1lo B16 melanoma lines by shRNA. B16*ctrl cultures were 4.2% CSCs (CD44+CD133+CD24+) vs. 0.19% in B16*B7-H1lo. CSC from B16*B7-H1lo cultures formed fewer and smaller (&lt;50 μm) microspheres vs. many, large (100–150 μm) microspheres from control CSC. B16*ctrl CSC were more tumorigenic in mice than B7-H1lo CSC. B7-H1 expression was 2-fold higher on CSC vs. non-CSC. αB7-H1 reduced CSC numbers in B16*B7-H1lo cultures (~4-fold) but not control cultures. PD-1 expression was 2-fold higher in B16*B7-H1lo non-CSC vs B16*ctrl non-CSC. PD-1 was 5-fold higher in B7-H1lo CSC vs. B7-H1lo non-CSC, but equal in B16*ctrl CSC vs. non-CSC, suggesting PD-1 regulation by B7-H1. αPD-1 reduced CSC only in B7-H1*ctrl cultures (18.9% to 2.4%). 0.1 ng/ml IFN-γ for 48 hours reduced total cell counts in control vs. B7-H1lo cultures (by 55% and 65%, respectively). Strikingly, IFN-γ increased B7-H1lo CSC numbers 9.5-fold vs. 5.6-fold for control CSC. In parallel work, we tested ID8agg ovarian cancer cells. CSC (CD24+CD44+) were 12.2% vs. 2.2% in ID8*ctrl vs. ID8agg*B7-H1lo culture. B7-H1 and PD-1 expression was similar to B16. RNA-seq of total ID8agg cells showed significantly lower expression of CSC associated genes c-Kit, Lin28A and CD24 in B7-H1lo cells. Classic CSC genes were too low to read. B7-H1 is a candidate CSC regulator. It affects CSC PD-1 expression and alters αB7-H1, αPD-1 and IFN-γ effects differentially in CSC vs non-CSC suggesting novel pathogenic mechanisms.
FATS deficiency in mice limits melanoma progression via converting the tumor immune microenvironment to an anti-tumor state
Authors: Zhang Rongxin , Zhang Kai , Zhang Lijuan , Wang Huafeng , Zhang Qi , Fu Shuyu , Xue Zhenyi , Li Yan , Da Yurong , Yao Zhi 
Published: January 2023

Abstract
C10orf90 is a newly identified common fragile sites gene named fragile-site associated tumor suppressor (FATS) according its tumor suppressive function in FATS overexpressive cancer cell lines. However, our FATS knockout mice data suggested that FATS may service as an important immune regulator.
In this study, we found that FATS deficiency in mice can significantly inhibit the tumor incidence and growth of melanoma implanted with B16 cells. In addition, FATS deficient mice obviously increased the numbers and frequency of tumor suppressor cells CD3+ T cell, γδT and NK cells, and decreased tumor promoter cells PMN-MDSC and Treg cell, in tumor tissues. Notably, DCs which play a key role in immune response initiation were dramatically increased in tumor tissues of the FATS deficiency mice. Furthermore, IL-2 which is crucial to the activation of T cells, significantly increased in serum of the FATS deficiency mice compared with the WT mice, meanwhile, other cytokines, such as IFN-γ and IL-12 were increased as well. Importantly, FATS deficiency increased the expression of IFN-γ in CD8+ T cells and CD4+ T cells and facilitated the activation of these T cells separated from the mice tumor tissues. Also, perforin and granzyme, the pivotal effector molecules in tumor cytotoxicity, raised in the CD8+ T cells from the melanoma tissues of FATS deficiency mice. Consistently, in the ex vitro study, DC separated from FATS deficiency mice with melanoma could significantly promote the proliferation and activation of T cells. These results demonstrate the inhibitory effect of FATS deficiency on the progression of melanoma via regulating tumor immune microenvironment and suggest that FATS could be a potential immune therapeutic target for malignant tumors.
Distinct mechanisms of B and T lymphocyte accumulation generate tumor-draining lymph node hypertrophy
Authors: Habenicht Lauren M , Iritani Brian M , Ruddell Alanna 
Published: January 2023

Abstract
Tumor-draining lymph nodes (TDLNs) enlarge and remodel in many human cancer patients and murine tumor models, featuring extensive lymphocyte accumulation, lymphatic sinus growth (lymphangiogenesis), and increased lymph flow. B cells can drive these lymphatic alterations, however the cellular and molecular mechanisms that direct lymphocyte accumulation in TDLNs are unknown. This study investigated whether lymphocyte accumulation in TDLNs is due to increased entry via high endothelial venules. The popliteal lymph node of C57Bl/6 mice bearing unilateral footpad B16-F10 melanoma tumors exhibits an 8-fold increase in B cells and 3-fold increase in T cells. Comparative measurements of labeled-lymphocyte entry into TDLNs versus the contralateral non-tumor draining lymph nodes by flow cytometry only partially explained TDLN hypertrophy. Further investigation of TDLN lymphocyte exit, proliferation, and apoptosis identified distinct mechanisms regulating B and T cell trafficking through TDLNs in response to tumors, including preferential retention of B cells. Surprisingly, B and T cell accumulation in TDLNs occurred without increased lymphocyte activation, suggesting an ineffective anti-tumor immune response. These insights could provide new therapeutic targets to manipulate lymph node lymphocyte content and immune responses to cancer and other diseases.