bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ligation of Siglec-9 inhibits FcɛRI-dependent mediator release from human mast cells
Authors: Molina Irina Miralda , Samanas Nyssa Becker , Piliponsky Adrian Martin 
Published: January 2023

Abstract
Mast cell activation is critical for the development of allergic diseases. Ligation of Sialic acid-binding immunoglobin-like lectins (Siglecs) such as Siglec-7 and 8 has been shown to inhibit mast cell activation and is considered one of the main approaches to reduce mast cell contribution to allergic disease. Recent high throughput studies provided evidence that human mast cells express Siglec-9, an inhibitory receptor that is mainly expressed by innate immune cells such as neutrophils, monocytes and eosinophils. Based on this evidence, we aimed to examine Siglec-9 expression and function in human mast cells. Flow cytometry analysis showed that Siglec-9 is expressed in the human mast cell lines LAD2, LUVA, and HMC-1, and in peripheral blood-derived cultured human mast cells (PBCMCs). The expression of Siglec-9 in PBCMCs peaks at week 5 of culture and correlates positively with the expression of the high affinity receptor for IgE (FcɛRI). Pre-treatment of human mast cells with the Siglec-9 agonists glycophorin A or high molecular weight hyaluronic acid (HMW-HA) followed by FcɛRI-dependent stimulation has an inhibitory effect on mast cell degranulation. SIGLEC9 gene disruption by CRISPR/Cas9 editing resulted in a significant reduction in Siglec-9 expression in LAD2 cells that also became impervious to inhibition by Siglec-9 agonists. Together, our data shows that human mast cells express Siglec-9 and that engaging this inhibitory receptor can reduce mast cell degranulation.
Elucidating the Immunogenic Mechanisms of Merck’s Lipid Nanoparticle (LNP) Adjuvants
Authors: Bass Antonia R , Kimball Sonya , Jenkins Anjelica , Burton Aimee , Trostle Katherine , Corcoran Halea , Allen Meredith , Gallagher Catherine , Harms Jacque , Bett Andrew , Sullivan Nicole 
Published: January 2023

Abstract
Adjuvants are crucial components of subunit vaccines, as they help induce immune responses to poorly immunogenic antigens. Merck has developed a variety of lipid nanoparticles (LNPs) that contain different cationic lipids, a critical component that is mainly responsible for the reactogenicity induced by the LNP. LNP A that incorporates cationic lipid A results in high antigen expression, robust antigen-specific humoral and cellular responses, and some dose dependent reactogenicity when combined with subunit proteins. In contrast, LNP B, which incorporates cationic lipid B which has a shorter half-life than cationic lipid A, results in high antigen expression, slightly lower immunogenicity, and improved tolerability. Previous studies have shown that mouse TLR2 may be involved in the mechanism of action of LNP A. To determine the human innate signaling pathways triggered by LNP A or LNP B, we stimulated human TLR-expressing HEK-Blue cell lines (Invivogen) with the LNPs followed by measuring SEAP activity. We did not see any activation of the human TLRs despite stimulating with high amounts of LNP. Next, we evaluated whether these LNPs generated different cytokine/chemokine profiles, indicative of differential cellular recruitment in BALB/c mice in vivo. We found that LNP A stimulation results in significant upregulation of monocyte and neutrophil-specific cytokines in the serum and quadricep muscle injection site, while LNP B leads to a lower response. Furthermore, we plan to investigate the recruitment of cells at the injection site and draining LNs by flow cytometry and imaging. Together, these experiments will help mechanistically determine how these fundamentally different LNPs can activate innate immune responses.
Supported by Merck & Co.
Engineering iPSC-derived neutrophils to fight infection
Authors: Giese Morgan , Bennin David , Johnson Chad , Klemm Lucas , Yung Ho Sun , Nett Jeniel , Slukvin Igor , Huttenlocher Anna 
Published: January 2023

Abstract
Neutrophils are the initial responders to infection and injury and are critical for host survival. Immunocompromised patients are prone to recurrent bacterial and fungal infections which may require granulocyte transfusion. However, these transfusions have minimal efficacy as donor material is limited and highly heterogeneous. Induced pluripotent stem cells (iPSCs) are a promising option for generating a defined therapeutic that can improve patient outcomes. Therefore, we aimed to engineer iPSC-derived neutrophils to improve recruitment and antimicrobial function. Prior research in the field has shown that iPSC-derived neutrophils have limited function compared to primary human neutrophils, likely due to inhibitory signaling pathways. To generate iPSC-derived neutrophils with enhanced capacity, we utilized CRISPR/Cas9 to delete the protein tyrosine phosphatase 1B (PTP1B), a negative regulator of neutrophil function. PTP1B−/− neutrophils displayed increased cellular migration and inflammatory cytokine signaling in response to bacterial stimuli versus WT cells. Additionally, upon selection of cells with mature neutrophil markers, these PTP1B−/− neutrophils show similar fungal killing ability and improved phagocytosis compared to WT. Thus, these data indicate that deletion of PTP1B can improve the antimicrobial function of iPSC neutrophils and highlights the value of our system for genetically engineering iPSC-derived neutrophils.
Supported by grants from the NIH (5R01AI134749-03 and Microbes in Health and Disease T32 NIAID: T32AI055397)
C-type lectin receptor MGL-1 in SARS-CoV-2 disease pathogenesis
Authors: Huante Matthew B , Gonzalez Orozco Maria J , Naqvi Kubra F , Martinez-Martinez Yazmin Berenice , Menachery Vineet , Endsley Janice J , Rajsbaum Ricardo , Stephens Robin , Endsley Mark 
Published: January 2023

Abstract
Pattern recognition receptors, such as the C-type lectin receptor (CLR) family, play an important role in the generation of the innate immune response against a variety of pathogens. The CLR Macrophage galactose-type lectin (MGL), also known as CLEC10, is one member of this family which we and others found can bind SARS-CoV-2. MGL is known to increase in expression during SARS-CoV-2 infection and, in concert with other CLRs, is associated with elevated proinflammatory immune responses. Dexamethasone, a clinical treatment commonly used to mitigate the hyperinflammatory response in individuals with COVID-19, is also a positive stimulus for MGL expression. We therefore sought to investigate the role of MGL in disease pathogenesis using MGL-1 receptor deficient mice. Mice were intranasally infected with mouse adapted SARS-CoV-2 strain CMA3p20 and euthanized 3- or 7-days post infection. Following acute infection at 3 days, MGL-1 deficiency did not affect the viral burden in the lungs of mice. This was supported by multiplex cytokine and chemokine data which revealed similar acute inflammatory responses in the lung of infected WT and MGL-1 knockout (KO) mice. Interestingly, MGL-1 KO mice did display a significant increase in the lung viral burden when compared to WT mice 7 days post-infection. These results suggest a role for MGL-1 in resolution of SARS-CoV-2 at later stages of infection. Further investigations to determine differences in cellular recruitment and lung pathology in the absence of MGL-1 are warranted. In addition, assessment of a role for the MGL-2 orthologue expressed by murine myeloid cells may identify roles for MGL pathways in SARS-CoV-2 disease severity and pathogenesis.
Supported by UTMB Institute for Human Infection and Immunity COVID-19 Pilot Grant
Innate training induced by subcutaneous BCG administration in pre-weaned calves
Authors: Samuel Beulah Esther Rani , Maina Teresia , McGill Jodi L L 
Published: January 2023

Abstract
Innate immunity, initially thought to lack immunological memory, has been shown to have the capacity to be trained or to mount a heightened immune response towards an unrelated second challenge. The Bacillus Calmette Guerin vaccine, administered to prevent human and bovine tuberculosis, is one treatment that can induce innate immune memory. Here we evaluated the efficacy of BCG-induced innate training on the outcome of a respiratory virus challenge in pre-weaned calves. Groups of calves were given BCG Danish strain subcutaneously (or PBS as control). PBMCs and CD14+ monocytes were re-stimulated in-vitro with E. coli LPS (1μg/mL) or Pam3CSK4 (10μg/mL) at 2- and 4-weeks post-BCG respectively, and pro-inflammatory cytokine production was measured by ELISA. Calves were challenged via aerosol inoculation with BRSV at five weeks post-BCG. PBMCs from BCG calves showed enhanced IL-1b production upon in-vitro stimulation with LPS, and CD14+ monocytes from BCG calves showed increased IL-1b and IL-6 secretion. Alveolar macrophages from BCG calves showed enhanced cytokine production when re-stimulated with LPS. No significant changes were observed in clinical scores or viral burden between groups post BRSV challenge. Subcutaneous BCG administration can train bovine innate immune cells to exhibit a “memory-like” phenotype. Current efforts are focused on the characterization of epigenetic reprogramming in trained innate immune cells.
Supported by grants from USDA, NIFA. R01 HD099104-01
The Cdkn2a gene product p19ARF (alternative reading frame) is a critical regulator of IFNβ-mediated Lyme arthritis
Authors: Li Jinze , Ma Ying , Paquette Jackie K , Richards Amanda C , Mulvey Matthew A , Zachary James F , Teuscher Cory , Weis Janis J 
Published: January 2023

Abstract
Lyme disease is caused by infection with the tick-borne spirochete Borrelia burgdorferi, with a spectrum of clinical disorders. Infected C3H mice develop severe arthritis while B6 mice display mild arthritis, allowing analysis of host genetic contribution to disease. B6 mice introgressed with the C3H allele of Bbaa1 on Chr4, B6.C3-Bbaa1, display increased severity of arthritis, and heightened expression of Type I IFN. IFNβ, was identified as the key effector for arthritis severity, acting through the muscle regulatory protein myostatin. To identify regulators of IFNβ in the Bbaa1 locus, candidate genes were subjected to siRNA silencing in macrophages. The Cdkn2a gene encoded protein p19ARF was identified as the modulator of IFNβ expression. B6 Arf−/− mice reconstituted with cells expressing the C3H allele of p19ARF developed severe arthritis, whereas mice reconstituted with cells expressing the B6 allele developed mild disease. IFNβ induction by p19ARF is inducible by other pathogens and a variety of PAMPs. P19ARF regulation of IFNβ involved the tumor suppressor p53 and transcription repressor BCL6 in myeloid cells. Indeed, targeted blocking of BCL6 enhanced IFNβ activation in the joint tissue of B6 mice and resulted in increased severity of Lyme arthritis. Similar responses in B6 Rag1−/− mice, indicate dependence on myeloid cells expression of BCL6. Thus, we have identified novel involvement of p19ARF in modulating IFNβ expression in Lyme arthritis development.
Supported by R01 AR043521
Autocrine prostaglandin feedback governs the kinetics of bacterial induced type I interferon by driving autophagic targeting of the TLR4/TRIF complex.
Authors: Perkins Darren , Schlenker Catherine 
Published: January 2023

Abstract
TLR4 is unique in its capacity to initiate two qualitatively distinct transcriptional programs from two cellular locations. Cell surface TLR4/MyD88 complexes drive pro-inflammatory cytokine transcription through NF-kB, while endosomal TLR4/TRIF complexes activate IRF3 and a type I interferon response. Feedback circuits to limit signals from both locations are necessary to balance the inflammatory response to Gram negative bacteria, yet how endosomal TLR4/TRIF signals are terminated remains an open question. It has been reported previously that engagement of TLR4 triggers activation of autophagic machinery and that macrophages defective in core autophagy genes display selective hyper activation of the TRIF versus MyD88 pathways in response to LPS. Previous studies from our lab have shown that TLR4 activation induces rapid synthesis and secretion of the immunomodulatory host lipid Prostaglandin E2 (PGE2) which selectively diminishes endosomal TLR4/TRIF signaling. In this ongoing study we find that, mechanistically, autocrine PGE2 signals downstream of TLR4 significantly enhance activation of autophagic processes to likely negatively regulate the persistence of endosomal TLR4/TRIF complexes and define the kinetics of TRIF signaling through directed autophagy.
Supported by a grant from the NIH R21AI151352
PD-L1 back signaling manipulates IL-6 signaling to coordinate innate immune responses
Authors: Shirley Jamie L , Forward Tadg , Schafer Johnathon B , Lucas Erin D , Tamburini Beth 
Published: January 2023

Abstract
Programmed death ligand-1 (PD-L1) interaction with PD-1 on T cells limits immune activation to prevent immunopathology in healthy tissues and the development of autoimmunity. While ligation of PD-1 by PD-L1 leads to T cell-intrinsic PD-1 signaling, PD-L1 back signaling into the ligand-bearing cell remains poorly defined. We recently outlined a major role for PD-L1 back signaling in regulating dermal dendritic cell (DC) migration to the lymph node following a type I interferon inducing stimulus or infection. Similar to prior studies in IFNβ treated cancer cells, we noted increased pSTAT3 post DC activation that was unchanged with addition of CCL21. Increased pSTAT3 occurred in Pdl1−/− DCs and in DCs from mice in which 3 cytoplasmic residues were mutated to alanine (Pdl1CM). Since STAT3 propagates signalling through the IL-6 receptor, we hypothesized that increased IL-6 signaling may explain elevated pSTAT3 in Pdl1−/− and Pdl1CM DCs. To test whether PDL1 back signaling contributed to STAT3-associated cytokine responses, we generated bone marrow derived DCs from Pdl1−/− and Pdl1CM mice and observed significantly higher levels of IL-6 compared to WT controls following activation. We next asked if PD-L1 back signaling is important for proper innate activation as PD-L1 is upregulated on many innate immune populations early after systemic L. monocytogenes (LM) infection. We found loss of PD-L1 back signaling following infection led to differences in circulating levels of many innate immune cells and reduced IL-6 in the serum. These findings demonstrate that PD-L1 back signaling in innate immune cells may assist in coordinating IL-6 signaling and proper activation of early innate immune responses during systemic LM infection.
Supported by grants from NIH (R01 AI155474, T32 AI074491 - “Molecular Mechanisms of Immune Tolerance”)
High-throughput Quantification of Antibody-Dependent Phagocytosis using Live-Cell Analysis
Authors: Trigg Jasmine E , Lovell Gillian , Bevan Nicola , Dale Timothy 
Published: January 2023

Abstract
The uptake and clearance of tumor cells can be promoted with monoclonal antibodies (mAbs) via antibody-dependent phagocytosis (ADCP) or through the blockade of “don’t-eat-me” signals, such as CD47. These mechanisms hold immunotherapeutic promise and are studied extensively in drug development.
High-throughput assays were conducted using pHrodo®, a pH-sensitive fluorophore, that enables live-cell imaging and kinetic quantification of phagocytosis. pHrodo® labeled target cells were incubated with mAbs and added to effector cells in 96-well plates. Images were acquired using the Incucyte® Live-Cell Analysis System and fluorescence automatically quantified with integrated software.
In line with known pro-phagocytic effects, anti-CD47 promoted phagocytosis of CCRF-CEM tumor cells by blocking CD47 “don’t-eat-me” signals. Clinical anti-CD20 mAbs Truxima® and Rituximab both promoted ADCP of Ramos target cells by primary macrophages. In addition, the ADCP response of different Rituximab isotypes were compared. Anti-CD20-IgG1 mAb and Fc mutated anti-CD20-IgG1fut (non-fucosylated) exhibited concentration-dependent responses (EC50 values of 25.1 ng/mL and 42.8 ng/mL, respectively), however anti-CD20-IgG1NQ (non-glycosylated) showed no response. ADCP was also examined in adherent target cells with varied HER2 profiles. Anti-HER2 (Trastuzumab) induced an ADCP response in HER2-positive AU565 cells but not in HER2-low MDA-MB-231 cells, consistent with established correlations between HER2 expression and ADCP response.
These data exemplify that live-cell analysis is a powerful approach that enables functional quantification of ADCP and is amenable to antibody screening for therapeutic candidates.
Myeloid cell interleukin-10 is essential for atherosclerosis protection in Apoe−/− mice.
Authors: Orecchioni Marco , Wolf Dennis , Suryawanshi Vasantika , Winkels Holger , Kobiyama Koji , Makings Jeffrey , Kiosses William B , Ley Klaus 
Published: January 2023

Abstract
Atherosclerosis, the disease underlying stroke, myocardial infarction and ischemic heart failure, is initiated by the subendothelial retention of lipoproteins and cholesterol, which then triggers a non-resolving inflammatory process driving plaque progression in the artery wall. Myeloid cells and in particular macrophages are the primary driver of this process. IL10 is a prototypic anti-inflammatory cytokine made by several immune cells such as macrophages. IL10 is also essential for the protection against atherosclerosis. However, it is not known which cells are responsible for the IL10 production mediating protection from atherosclerosis. Here, we crossed the VertX mouse model, in which an IRES-enhanced green fluorescent protein (eGFP) fusion protein was placed downstream of exon 5 of the IL-10 gene, to the atherosclerosis-prone Apoe−/− background. We found that myeloid cells express high levels of IL10 GFP in VertX-Apoe−/− mice. By scRNAseq we found that macrophages especially inflammatory macrophages in plaques are the main producers of IL10 in atherosclerosis. To address whether IL10 secreted by myeloid cells is essential for the protection, we developed a LyzMCre IL-10flox/flox mouse crossed in the Apoe−/− background and confirmed that macrophages were unable to secrete IL-10. We discovered that the LyzMCre IL-10flox/flox Apoe−/− mice developed significantly more atherosclerosis than LyzMCre IL-10+/+ Apoe−/−, both on chow and lipid-rich diet. Flow cytometry data and cytokine measurements suggest that the depletion of IL-10 in myeloid cells increases Th17 cells with elevated IL-17a, IL-17f and CCL20 in blood plasma. These data underscore the critical roles of macrophage-derived IL-10 in limiting atherosclerosis.
Supported by grants NIH HL 115232, 145241, and HL088093.
Hypoxia-inducible factor regulation of NK cell dysfunction in lupus
Authors: Feldman Harrison Alexander 
Published: January 2023

Abstract
Natural Killer (NK) cells are innate antiviral and antitumor effector cells that have emerged as potent regulators of adaptive immune responses. However, NK cells are known to be dysfunctional in many diseases including systemic lupus erythematosus (lupus), where inability to restrain autoreactive T and B cells leads to potentially irreversible organ damage. To determine mechanisms driving NK-cell dysfunction in autoimmunity, we performed transcriptomic analysis of these cells in pediatric patients with lupus. Analysis of the differentially expressed genes revealed a putative gene regulatory node controlled by the heterodimeric pairing of the transcription factors EPAS1 and ARNT that is active in NK cells from patients with lupus, but not controls. Cytokines linked to lupus promote up-regulation of the protein encoded by EPAS1 in NK cells. Small molecule inhibitors of EPAS1 (PT2385) enhanced the capacity of NK cells to kill K562 cells. These results suggest that a hypoxia-related transcriptional program contributes to functional restraint of NK cells in lupus.
Supported by T32GM063483, AR073228, AR47363 (Flow Cytometry Core), DK78392 (Flow Cytometry Core), DK90971 (Flow Cytometry Core)
A critical role of energy sensor AMPKα1 in rapamycin dependent memory CD8+ T cell survival
Authors: Ara Anjuman , Xiang Jim 
Published: January 2023

Abstract
Energy sensors mTORC1 and AMPKα1 regulate T-cell metabolism and differentiation, while rapamycin (Rapa), an inhibitor of mTORC1, supports T-cell memory. However, the underlying pathway, the exact mechanisms, and the role of AMPKα1 in Rapa-induced T-cell memory is not well understood. Using the Listeria monocytogenes (rLmOVA) infection model and genetic and pharmaceutical tools, we demonstrate that Rapa promotes T-cell memory in mice post-rLmOVA infection. When T-cells stimulated in vitro in the presence of IL-2 without rapamycin (IL-2/T) or with rapamycin (IL-2+Rapa /T) and transferred into mice, it differentiated into short-term effector T (TE) [IL-7R− CD62L− KLRG1+] and long-lived memory T (TM) [IL-7R+ CD62L+ KLRG1−] cells, respectively. We determined that rapamycin-treated T cells activated transcriptional factors, FOXO1, TCF1 and Eomes and metabolic pAMPKα1(T172), pULK1(S555), and ATG7 molecules and promoted mitochondrial biogenesis and oxidative phosphorylation (OXPHOS). Using Seahorse-real time metabolic analyzer, we found that rapamycin-treated AMPKα-deficient TM cells up-regulated transcription factor HIF-1α and induced a metabolic switch from OXPHOS to glycolysis. Interestingly, despite the rapamycin treatment, AMPKα-deficient TM cells lost their cell survival capacity. Altogether, our data provide a mechanistic explanation for enhanced T-cell survival after rapamycin treatment and suggest that rapamycin promotes T-cell memory via transcriptional FOXO1-TCF1-Eomes programs and AMPKα1-ULK1-ATG7 metabolic axis. And AMPKα1 plays a critical role in rapamycin-induced increased survival and metabolic switching of CTLs during the transition of effector to memory T cells.
Supported by Canadian Institutes of Health Research grant (409228). Saskatchewan Health Research Foundation (SHRF) postdoctoral fellowship.
Increased proliferation of epidermal gamma delta T cells and expression of the transmembrane protein, BST2, in Alopecia areata
Authors: Hagan Yosiris , Gonzalez Alex , Kasler Antolette , Macedo Matilde , Rincon Anthony , Jameson Julie 
Published: January 2023

Abstract
Alopecia areata is an autoimmune disease characterized by patchy non-scarring hair loss in affected individuals. Typically, research has focused on hair follicle infiltration and disruption via CD8+ and CD4+ T cells. Our research attempts to elucidate the role of resident epidermal γδ T cells in disease. Analysis of publicly available bioinformatics data shows a statistically significant downregulation in a number of immune-related genes after patients were treated with a JAK inhibitor, Tofacitinib. We have focused on one of these proteins, bone marrow stromal antigen 2 (BST2), in vitro and in vivo using the C3H mouse model of alopecia areata. We have identified populations of epidermal γδ T cells expressing BST2 in the follicle using immunofluorescence microscopy. In addition, BST2 expression is upregulated in unaffected skin from mice with active alopecia areata suggesting BST2 expression may precede loss of hair follicle immune privilege. Furthermore, epidermal γδ T cell number is increased in skin affected by alopecia areata. Together our findings suggest that BST2 is expressed by epidermal T cells prior to hair loss and is followed by an increase in epidermal γδ T cell number. Our lab is focused on how these changes in epidermal γδ T cell number and BST2 expression impacts disease.
Supported by grants from NIH Supported by grants from NSF