bluuebunny/crossref_metadata_2025_split huggingface dataset:
G protein Coupled Receptor Kinase 2 Levels Regulate Differential Internalization of C-C Chemokine Receptor 7 (CCR7) in Primary Mouse T Cells
Authors: Vines Charlotte M. , Winebrenner Caitlin T. , Bill Charles A. , Sanchez Anahi , Wagener Brant , Bill Colin A. 
Published: January 2023

Abstract
G protein-coupled receptor (GPCR) signaling can be regulated by phosphorylation via GPCR kinases (GRKs) and recruitment of b-arrestins. The C-C chemokine receptor 7 (CCR7) GPCR, has two ligands, CCL19 and CCL21 that promote chemotactic migration of CCR7 expressing immune cells, including naïve T cells. Distinct signaling pathways are activated in T cells depending upon which ligand is bound to the CCR7. It remains unclear to what extent GRKs contribute to differential internalization of ligand-bound CCR7 and subsequent signaling that controls T cell migration.
We used naïve primary T cells from wild type C57 BL/6 mice (WT) or syngeneic mice heterozygous for the GRK2 gene that express 50% GRK2 protein levels compared to WT mice. T cells were incubated with 40 nM of CCL19 or CCL21 for up to 8 minutes. Incubation with CCL19, but not CCL21, led to CCR7 internalization; however, both ligands facilitated rapid, sustained CCR7 internalization in GRK2+/− T cells. The reduced GRK2 levels in the heterozygote T cells had no significant effect on migration to CCL19 or CCL21 compared to WT T cells. Titration transfection experiments in HEK293T cells or CRISPR-Cas9 knockdown of GRK2 in CEM T cells suggested that GRK2 is required for ligand-mediated CCR7 internalization, while higher GRK2 levels inhibit the internalization process. CCR7 internalization was blocked following transfection of a kinase dead GRK2 mutant indicating that GRK2 phosphorylation is required for CCR7 internalization. Immunomicroscopy suggested arrestin-3 formed clusters, independent of the ligand used. Overall, these data suggest that cellular concentration of GRK2 plays an important role in the internalization of ligand/CCR7.
The role and regulation of cell-intrinsic glycogen metabolism in dendritic cell immune responses.
Authors: Thwe Phyu M. , Pelgrom Leonard , Cooper Rachel , Beauchamp Saritha , Reisz Julie A. , D’Alessandro Angelo , Everts Bart , Amiel Eyal 
Published: January 2023

Abstract
Stimulation of dendritic cells (DCs) through toll-like receptors (TLRs) is accompanied by an increased metabolic demand, which provides metabolites and energy required for DC activation. This metabolic requirement is fulfilled by TLR-driven rapid glycolytic burst, and inhibition of glycolysis impairs the survival and effector function of DCs. Although previous studies have shown an important role for extracellular glucose uptake via glucose transporter 1 (Glut1) in supporting TLR-driven glycolysis, the contributions of intracellular glucose stores to these processes have not been well-defined. While non-immune cells store glucose as glycogen, the role of glycogen in DCs has not been identified. Here, we demonstrate that cell-intrinsic glycogen metabolism in DCs supports the early glycolytic reprogramming essential for TLR activation, particularly before Glut1 upregulation. DCs express key enzymes for glycogen metabolism, possess intracellular glycogen stores, and inhibition of glycogen breakdown severely impairs the effector function of DCs, including the ability to stimulate T cells. Our preliminary data show a time-dependent increase in intracellular glycogen levels upon TLR stimulation, implying that intricate regulatory mechanisms of glycogen metabolism are at play for proper activation responses. Interestingly, our metabolomics data indicate that the glycogen metabolism in DCs generates both glycolytic and TCA cycle intermediates and that glycogen-derived carbons preferentially engage in metabolic pathways distinct from free glucose catabolism. Our work reveals a novel metabolic regulatory pathway by which DCs differentially utilize glycogen and glucose metabolism to support their activation.
Effects of serum amyloid protein A on influenza A virus replication and viral interactions with neutrophils
Authors: White Mitchell R. , Hsieh I-Ni , De Luna Xavier , Hartshorn Kevan L. 
Published: January 2023

Abstract
Innate immunity is vital for the early control of influenza A virus (IAV) infection. Serum amyloid A (SAA1) is an acute phase reactant produced in the liver and lung that rises dramatically during IAV infection. SAA has been reported to directly activate neutrophils and to recruit them to the lung during infectious and inflammatory processes. In this study we characterized the interactions of several forms of SAA with IAV and with neutrophils. The complex of SAA with HDL had some neutralizing activity against IAV. Similarly purified SAA containing amino acids 19–94 prepared from human serum had some viral neutralizing activity. In contrast recombinant SAA containing amino acids 19–123 (rSAA) did not. SAA preparations, including rSAA, increased various responses of neutrophils to the virus. Neither serum nor rSAA directly stimulated neutrophil respiratory burst responses on their own; however, both preparations increased these responses to IAV. rSAA also increased neutrophil uptake of IAV. rSAA alone did trigger neutrophil IL-8 production but modestly increased the strong neutrophil IL-8 response triggered by IAV. rSAA alone did trigger a neutrophil intracellular calcium response and also increased the peak calcium response to IAV. Combining rSAA with surfactant protein D (a known lung inhibitor of IAV) resulted in potentiation of viral neutralizing and aggregating activity of IAV. We provide evidence that the effects of SAA on neutrophil responses to IAV were not mediated by formyl peptide receptor 2 but were in part mediated by TLR2. These results suggest that SAA produced in the lung may significantly modulate the early neutrophilic host response to IAV.
T-bet-expression in dendritic cells is essential for T. gondii clearance
Authors: Lopez-Yglesias Americo H. , Burger Elise , Araujo Alessandra , Martin Andrew T. , Yarovinsky Felix 
Published: January 2023

Abstract
The host’s type I immune response is essential for resistance to diverse groups of intracellular pathogens, including Mycobacterium, Leishmania, Salmonella, and Toxoplasma gondii. Type I immunity is traditionally characterized by the transcription factor T-bet, encoded by Tbx21, which has been considered the master regulator of determining the lineage commitment of CD4+ TH1 cells and their production of the effector molecule IFN-γ. However, recent studies by our and other groups have shown that T-bet expression in CD4+ T cells is dispensable for IFN-γ production, and that T-bet-independent T cell-derived IFN-γ is sufficient to cause immunopathology during infection. Despite comparable levels of IFN-γ generated by Tbx21−/− mice, they remain highly susceptible to infection. Furthermore, Tbx21−/− mice succumb significantly faster to T. gondii infection than animals lacking B and T cells, yet retain an intact myeloid innate compartment, suggesting that T-bet deficiency in innate immune cells results in loss of host resistance to intracellular parasite, and rapid host mortality during infection. Thus, we investigated T-bet expression levels in myeloid innate immune cells and observed that T-bet expression in monocyte-derived CD11c+ DCs occurs in an IFN-γ-dependent manner. We also established that T-bet expression in DCs, but not macrophages, is required for resistance to intracellular T. gondii infection. Overall, we revealed an essential role for T-bet expression in DCs for controlling host immunity to intracellular parasite infection.
Prolonged cell survival may contribute to the cytokine storm observed during the coinfection of influenza A virus (IAV) and Streptococcus pneumoniae.
Authors: Rodriguez Angeline E. , Lupfer Christopher R. 
Published: January 2023

Abstract
Coinfection with influenza A virus (IAV) and Streptococcus pneumoniae is known to cause severe pneumonia. Numerous reports suggests an inappropriate immune response plays an important role in this disease. Many reports have examined signaling pathways responsible for the excessive immune response to coinfection, but the role for cell death in mediating inflammation during coinfection is not well understood. The inflammasome is a caspase-1 containing protein complex that activates interleukin-1β (IL-1β) and induces a pro-inflammatory form of cell death termed pyroptosis. We have discovered enhanced IL-1β levels during coinfection of macrophages with IAV and S. pneumoniae. Furthermore, inflammatory macrophages/monocytes, but not alveolar macrophages, are the major cell type producing IL-1β in the lung. However, this is not due to increased inflammasome activation. Intriguingly, coinfected macrophages are less prone to cell death than those singly infected with S. pneumoniae. Thus, we have elucidated a mechanism whereby prolonged cell survival may contribute to the cytokine storm observed during coinfection.
A systematic approach integrating genetics, computational biology, chemical genomics and gene editing to identify novel targets in the Type I interferon signaling pathway for autoimmune and infectious diseases
Authors: Zeng Xin , MacNamara Aidan , Xie Qing , Lu Quinn , Hou Wangfang , Rao Yanhua , Maratuo Klio , Rea Ceara , Madsen Heather , Larminie Chris 
Published: January 2023

Abstract
The type I interferon (IFN a/b) signaling pathway plays a critical role in microbial host defense, including mediating the interaction between innate and adaptive immunity. A growing body of evidence suggests that IFN is aberrantly regulated in human autoimmune diseases and chronic infections, such as Systematic Lupus Erythematosus, HIV infection and the rare Mendelian diseases, Aicardi-Goutier Syndrome and the Singleton Merton Syndrome (also called “interferonopathies”). This has raised significant interest in exploring the cellular signaling associated with type I IFNs for potential targets for autoimmunity and infectious disease. In this study, we systematically interrogated the type I IFN signaling pathway to identify and validate potential therapeutic targets based on the following workflow: 1. Perform computational biology analysis using genetics, interaction networks and gene expression in human diseases to generate a list of targets that have an association with increased type I IFN. 2. Establish the THP-1 cell-based type I IFNbeta release assay to screen a focused set of target annotated tool compounds. 3. Integrate the inferred targets from the tool compound hits into the in-silico network analysis. 4. Generate a short list of targets (10–20) with high confidence to perform CRISPR analysis in THP-1 cells. 5. Generate a long list of targets (~200) to perform a pooled CRISPR screen using single cell technology and reporter-based flow sorting and enrichment. Here we present validation data on a few representative targets and a summary of the outcomes and learning from this study.
Commensal Bacteria Suppress Vitamin A Metabolism in the Intestinal Epithelium to Modulate IL-22 Activity in the Gut.
Authors: Grizotte-Lake Mayara , Vaishnava Shipra 
Published: January 2023

Abstract
Antimicrobials secreted by the intestinal epithelium are critical for regulating host-microbe interactions in the gut. However, certain pathogens can exploit excessive antimicrobial response during infection to outcompete commensal bacteria. We have uncovered a novel intestinal epithelial cell intrinsic mechanism by which commensal bacteria modulate Interleukin (IL)-22 induced antimicrobials to provide colonization resistance against pathogens. Vitamin A metabolite, retinoic acid (RA), is critical for coordinating multiple immune responses in the intestine, including IL-22 production, yet, we barely understand how conversion of vitamin A into RA is regulated in the intestinal tissue. Our work shows that commensal bacteria limited RA by suppressing expression of retinol dehydrogenase 7 (rdh7) in the intestinal epithelium. Deletion of rdh7 resulted in reduced RA signaling in immune cells, lower levels of IL-22, and an enhanced resistance to colonization by pathogen that exploits IL-22 induced antimicrobials to thrive in the gut. Supplementing mice deleted for rdh7 with RA reversed the colonization resistance, revealing the significance of downregulation of rdh7 by commensal bacteria. Our findings thus provide a novel insight into molecular mechanism by which commensal bacteria promote resistance to colonization by pathogenic bacteria. More importantly, we are demonstrating for the first time that bacterial regulation of RA provides a crucial feedback loop to curb excessive IL-22 activity in the gut.
Vasodilator Stimulated Phosphoprotein (VASP)-Mediated Actin Polymerization Drives Natural Killer Cell Granule Convergence
Authors: Wilton Katelynn Marie , Billadeau Daniel D. 
Published: January 2023

Abstract
Natural killer (NK) cells eliminate transformed and malignant cells through a highly orchestrated series of actin and microtubule cytoskeletal rearrangements, culminating in the secretion of preformed lytic granules. Despite the importance of actin and microtubules, the roles of many molecular cytoskeletal regulators in this process have not yet been ascertained. In this study, we investigated the role of Vasodilator Stimulated Phosphoprotein (VASP), an actin regulatory protein, in NK cell-mediated cytotoxicity. Interestingly, we found that depletion of VASP inhibits NK cell cytotoxicity and that VASP co-localizes with F-actin at the NK cell – target cell cytotoxic synapse. Surprisingly, despite their co-localization, VASP depletion did not affect either F-actin accumulation at the cytotoxic synapse or conjugate formation. Instead, we found that a minority of VASP localized and biochemically co-purified with cytolytic granules. Although VASP knockdown did not affect microtubule organizing center (MTOC) polarization, it did dramatically impact lytic granule convergence to the MTOC. Significantly, VASP depletion decreased F-actin accumulation on cytolytic granules and depolymerization of F-actin with Latrunculin A impaired lytic granule convergence. Taken together, these results demonstrate a novel requirement for VASP-mediated actin polymerization in NK cell granule convergence and cytotoxicity and highlight a role for F-actin in lytic granule convergence.
Foreign antigen-specific Foxp3+ regulatory T cells expand with immunization and limit Th1 polarization by Foxp3− conventional cells specific for the same antigen
Authors: Krueger Peter D. , Jenkins Marc K. 
Published: January 2023

Abstract
The CD4+ T cell repertoire contains thousands of populations, each with antigen receptors specific for a different major histocompatibility complex class II (MHCII)-bound foreign peptide. Before exposure to the foreign peptide, an average population in a mouse consists of about 100 naïve Foxp3− conventional cells and 10 Foxp3+ regulatory T (Treg) cells. After infection containing the peptide, the population increases several hundred-fold and contains Foxp3− and Foxp3+ effector cells. Because conventional T cells have been reported to generate Treg cells and vice versa, it is not clear which precursors generate which effector cells. Using progeny-tracing systems, we found that Foxp3− conventional cells and Foxp3+ Treg cells specific for the same peptide:MHCII ligand proliferated at the same rate after immunization. Foxp3− naïve cells generated only Foxp3− effector cells, and Foxp3− naïve cells generated primarily Foxp3+ effectors cells. The Treg cells suppressed the Th1 differentiation of the Foxp3− cells but did not cause them to become Treg cells. These results demonstrate that Foxp3− and Foxp3+ naïve T cells are remarkably lineage-committed during the primary response to infection, and although their progeny interact, this interaction does not result in lineage instability.
Negative costimulation constrains T cell differentiation
Authors: Wei Spencer C. , Sharma Roshan , Anang Nana-Ama , Levine Jacob , Zhao Yang , Wang Jing , Pe’er Dana , Allison James P. 
Published: January 2023

Abstract
T cell costimulation is a principal mechanism by which T cell activation is regulated, but it remains unclear whether costimulatory pathways also control T cell differentiation. This unresolved question is germane to our understanding of how T cell function is controlled and has therapeutic implications in the context of cancer immunotherapy. Using a mass cytometry based systems approach and murine models we investigated whether negative costimulation regulates T cell differentiation as well as activation. Unsupervised population identification reveals 30 T cell subsets in Ctla-4−/− and littermate control mice including 6 CD8, 3 Treg, 14 CD4 effector T cell populations. Expectedly, loss of CTLA-4 led to dramatic changes in the relative frequency of CD8 and CD4 T cell subsets. Strikingly, however, loss of CTLA-4 led to the generation of multiple non-canonical T cell populations outside of the normal boundaries of T cell phenotypes, which interestingly, were all in the CD4 T compartment. To determine if the role of negative costimulation in differentiation may be a generalizable phenomenon, we performed similar analyses of Pdcd-1−/− mice. This profiling revealed that loss of PD-1 leads to a subtle expansion of CD8 T cell phenotypes in addition to changes in the relative frequencies of CD4 and CD8 T cell populations. These observations indicate that CTLA-4 restricts CD4 T cell phenotypes while PD-1 restricts CD8 T cell phenotypes. Consistent with this model, loss of CTLA-4 differentially affects the expression of individual markers in CD4 and CD8 T cells. Together these findings indicate that in addition to attenuating T cell activation, negative costimulation acts to constrain T cell differentiation.
Differential surface phenotype and context-dependent reactivity of functionally diverse NKT cells
Authors: Cameron Garth , Godfrey Dale I. 
Published: January 2023

Abstract
Natural Killer T (NKT) cells are a functionally diverse population that recognize lipid-based antigens in association with the antigen-presenting molecule CD1d. Here we define a technique to separate the functionally distinct NKT1, NKT2 and NKT17 cell subsets by their surface expression of CD279 (ICOS) and the activation-associated glycoform of CD43, enabling the investigation of subset-specific effector-functions. We report that all three subsets express the transcription factor GATA-3 and the potential to produce IL-4 and IL-10 following activation. This questions the notion that NKT2 cells are the predominant source of IL-4 within the NKT cell pool, and suggests that IL-10-production may be more indicative of NKT cell plasticity than the existence of a distinct regulatory lineage or subset. We also show that many NKT17 cells are CD4+ and are biased towards Vβ8.3 TCR gene usage. Lastly, we demonstrate that the toll-like receptor (TLR) ligand lipopolysaccharide (LPS) can induce a NKT17 cell-biased response, even in the absence of exogenous antigen, and that combining LPS with α-GalCer resulted in enhanced IL-17A-production, and reduced levels of the immunosuppressive cytokine IL-10. This study provides a novel means to examine the context-dependent reactivity of the functionally heterogeneous NKT cell population and provides important new insight into the functional biology of these subsets.
CD8+ T-cell effector molecules are differentially expressed in blood and gastrointestinal mucosa in chronic HIV-1 infection
Authors: Shacklett Barbara L. , Kiniry Brenna E. , Somsouk Ma , Hunt Peter W. , Deeks Steven G. 
Published: January 2023

Abstract
CD8+ T-cells in human gastrointestinal mucosa exhibit reduced perforin expression and weak cytotoxicity compared to their counterparts in blood. We previously reported that these cells are predominantly T-bet-low and Eomesodermin-negative. Nevertheless, in response to antigenic stimulation these cells degranulate and express cytokines and chemokines. The goal of this study was to elucidate expression patterns of granzymes (Gzm) A, B and K in conjunction with perforin in rectosigmoid CD8+ T-cells during chronic HIV-1 infection and determine whether these patterns vary with disease status. We used flow cytometry to compare intracellular Gzm A, B, K and perforin in resting CD8+ T-cells from blood and rectosigmoid biopsies of HIV-1+ controllers; viremic individuals; virologically suppressed individuals on antiretroviral therapy; and seronegatives. Expression of perforin and GzmB, but not GzmA or GzmK, was reduced in mucosa compared to blood. A large fraction of rectosigmoid CD8+ T-cells did not express granzymes or were single-positive for GzmA. Rectosigmoid CD8+ T-cells appeared skewed towards cytokine production rather than cytotoxicity, with cells expressing multiple cytokines/chemokines lacking perforin and granzyme expression. Percentages of mucosal CD8+ T-cells expressing cytotoxic effectors were higher in HIV-1+ groups, compared to seronegatives. These data support the interpretation that perforin and granzymes are differentially regulated, and display distinct expression patterns in blood and rectosigmoid T-cells. These findings also reinforce our previous work, suggesting that rectosigmoid CD8+ T-cells are programmed for cytokine polyfunctionality rather than cytotoxicity.
TRIM34 attenuates colon inflammation and tumorigenesis by promoting Muc2 exocytosis from goblet cells
Authors: Sun Bing 
Published: January 2023

Abstract
Loss of colonic inner mucus layer leads to spontaneously severe colitis and colorectal cancer. However, it is rarely reported whether there are some key host factors to control the generation of inner mucus layer. Here, we identify a novel function of TRIM34 in goblet cells (GCs) in controlling the inner mucus layer generation. Upon DSS treatment, TRIM34 deficiency led to a reduction in Muc2 secretion by GCs and subsequent defects in the inner mucus layer and massive commensal bacterial penetration. This rendered TRIM34-deficient mice more susceptible to DSS-induced colitis and colitis-associated colorectal cancer. Mechanistic experiments demonstrated that TRIM34 controlled TLR signalling-induced, Nox/Duox-dependent ROS synthesis, thereby promoting the compound exocytosis of Muc2 by colonic GCs that were exposed to bacterial TLR ligands. Clinical analysis revealed that TRIM34 levels in patient samples were highly correlated with the outcome of ulcerative colitis (UC) and the prognosis of rectal adenocarcinoma. This study indicates that TRIM34 expression in GCs plays an essential role in generating inner mucus layer and preventing excessive colon inflammation and tumorigenesis.