bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alternative splicing of Bax controls cell death and innate immune responses in macrophages
Authors: Wagner Allison , Weindel Chi , Watson Robert , Patrick Kristin 
Published: February 2023

Abstract
To defend against invading pathogens, the macrophage innate immune response requires rapid, robust gene expression reprogramming. Innate immune genes that are induced as part of this response are regulated at many steps. While transcriptional activation of innate immune genes has been extensively studied, the mechanisms driving their post-transcriptional regulation (i.e. alternative splicing) needs further investigation. To begin learning how pre-mRNA splicing changes impact the innate immune response in macrophages, we examined how loss of a splicing regulatory protein called SRSF6 (serine arginine rich splicing factor 6) impacts gene expression. We discovered a novel phenotype whereby loss of SRSF6 increased basal expression of many interferon stimulated genes (ISGs). Consistent with mitochondrial dysfunction, a phenotype that we and others have previously associated high basal ISG expression, we measured an increase in apoptosis and macrophage depolarization in SRSF6 knockdown macrophages compared to scramble controls. Subsequent bioinformatic analysis of RNA-seq data from SRSF6 knockdown macrophages identified an alternative splicing event in the apoptosis activation factor BAX. While Bax isoforms have previously been linked to cell death regulation, no one has reported a role for Bax isoforms in modulating mtDNA-driven innate immune responses. Our data is consistent with a model whereby upregulation of a Bax isoform promotes pore formation in mitochondria which 1) causes leakage of mtDNA into the cytosol where it is detected by cGAS, eliciting type I interferon production and 2) primes macrophages to undergo programmed cell death.
MER Tyrosine Kinase Mediates Efferocytosis Through a Novel β2 Integrin-Activating Signalling Pathway.
Authors: Heit Bryan , Tasnim Tarannum , Le Lam Austin 
Published: February 2023

Abstract
Efferocytosis – the phagocytic removal of apoptotic cells – is a central component of tissue homeostasis and contributes to inflammatory and autoimmune conditions including atherosclerosis and multiple sclerosis. MER tyrosine kinase (MERTK) is the predominant or sole efferocytic receptor in multiple tissues, and is the predominant efferocytic receptor on macrophages. MERTK is an opsonin-dependent receptor tyrosine kinase that triggers a poorly understood signaling pathway to mediate apoptotic cell engulfment. Using immunoprecipitation, mass spectrometry, and super-resolution microscopy, we have identified a pre-formed receptor complex on the macrophage plasma membrane comprised of ~100 nm clusters of MERTK, β2 integrins, and multiple signaling molecules including Src-family kinases, PI3-kinases, and the integrin regulatory proteins ILK and FAK. MERTK is unable to mediate efferocytosis in the absence of β2 integrins or their opsonins, while β2 integrins require activation via MERTK signaling to induce the engulfment of apoptotic cells. Using FRET microscopy, we determined that MERTK directly induces the conformational change of β2 integrins from the low to high-affinity form via a PI3-kinase, FAK and ILK-dependent signaling pathway. Activation of this pathway then drives the coalescence of the MERTK/β2 integrin clusters into a larger synapse through which efferocytosis occurs. The identification of the MERTK signaling pathway and the role of β2 integrins in this pathway provides new insights into the function of this critical homeostatic receptor and provides new insights into how MERTK mutations and signaling defects may contribute to inflammatory and autoimmune diseases.
Specialized pro-resolving lipid mediators stimulate mitochondrial metabolism in macrophages
Authors: Calderin Ernesto Pena , McNally Lindsey , Hill Bradford G. , Hellmann Jason 
Published: February 2023

Abstract
Chronic inflammation and sustained recruitment of classically activated macrophages (CAMs) with unchecked pro-inflammatory cytokine production is associated with disease development (e.g., atherosclerosis). Pro-inflammatory actions of CAMs are fueled by enhanced glycolysis and disruptions in the TCA cycle. Conversely, alternatively activated macrophages (AAMs) display an intact TCA cycle and mitochondrial oxidative phosphorylation (OXPHOS). Specialized pro-resolving lipid mediators (SPMs) stimulate resolution, in part, through actions on specific G-protein coupled receptors (GPCRs) in macrophages (e.g., RvD1□Fpr2). Whether SPMs exert pro-resolving effects by altering macrophage metabolism is unknown. We found that 1 hr treatment of SPMs (e.g., RvD1, RvD2, RvE1, and MaR1) enhanced basal (vehicle vs 1nM SPM; 6.11±0.1 vs RvD1 7.76±0.6 vs RvD2 9.48±0.9 vs RvE1 7.20±0.5 vs MaR1 8.73±0.9 pmol/min/μg protein; P&lt;0.05; n=3), maximal (vehicle vs 0.1nM SPM; 9.06±1.1 vs RvD1 14.37±1.1 vs RvD2 16.54±2.7 vs RvE1 12.32±1.5 vs MaR1 13.28±1.3 pmol/min/μg protein; P&lt;0.05; n=3), and ATP-linked respiration (vehicle vs 1nM SPM; 2.83±0.2 vs RvD1 4.50±0.3 vs RvD2 4.39±0.2 vs RvE1 4.71±0.3 vs MaR1 4.20±0.2 pmol/min/μg protein; P&lt;0.05; n=3). Stimulation of mitochondrial metabolism with RvD1 was receptor mediated as Fpr2-deficient macrophages display no significant changes in metabolism following treatment (vehicle vs 1nM RvD1; basal 6.74±0.6 vs 6.52±0.2; maximal 11.54±1.2 vs 13.26±1.0; ATP-linked 5.84±0.8 vs 5.33±0.2 pmol/min/μg protein). Collectively, these data suggest that SPMs, despite having unique GPCRs, stimulate OXPHOS in macrophages, which could underlie their common pro-resolving properties.
Mast cell-specific inactivation of Fosb exacerbates release of pro-inflammatory mediators in models of systemic anaphylaxis and lipopolysaccharide-induced sepsis
Authors: Duque-Wilckens Natalia , Maradiaga Nidia C , Szu-ying Yeh , Nestler Eric J. , Robison Alfred J. , Moeser Adam J. 
Published: February 2023

Abstract
Mast cells (MC) are innate immune cells that can orchestrate diverse physiologic and pathologic responses. This is due to the MC capacity to respond to a variety of stimuli and selectively release a diverse repertoire of mediators including prestored granule mediators, cytokines, growth factors and neurotransmitters. This vast heterogeneity in MC functions implies that their transcriptional profile needs to be extensively regulated, but the underlying mechanisms are not understood. Our previous data showed that MC expression of FosB and ΔFosB, two transcription factors formed by alternative splicing of the FosB gene, is markedly upregulated in response to a variety of stimuli, including IgE-antigen activation and stress, but the role of MC-specific FosB and ΔFosB in MC function remains unexplored. Here we crossed Mcpt5-Cre mice with a Cre-dependent floxed FosB mouse strain (Mcpt5-Cre;FosB (fl/fl)) to assess the role of MC-specific FosB expression. Bone marrow derived mast cells from Mcpt5-Cre;FosB (fl/fl) mice exhibited increased IgE-antigen mediated histamine release. Using a passive systemic anaphylaxis model, we next found that Mcpt5-Cre;FosB (fl/fl) mice exhibited greater serum histamine levels, and more severe hypothermia and clinical scores, compared with Mcpt5-Cre-negative controls. Mcpt5-Cre;FosB (fl/fl) mice also showed exacerbated hypothermia and serum IL-6 levels in response to lipopolysaccharide (LPS) (1 mg/Kg) injection. Together, these data demonstrate that MC Fosb expression exerts a negative regulation on MC mediator release and associated inflammatory responses. Ongoing studies using CUT&RUN-sequencing aim to identify the down_stream targets of FosB and ΔFosB in MCs.
STAT3 and IL-10 inhibit interferon signaling in type 1 conventional dendritic cells
Authors: Chrisikos Taylor T , Zhou Yifan , Denne Nina , Brooks Athena , Watowich Stephanie S 
Published: February 2023

Abstract
Conventional dendritic cells (cDCs) are the primary antigen presenting cells of the immune system, and thus control adaptive immunity. Type 1 cDCs (cDC1s) are crucial for mounting protective T cell responses against tumors and viruses. Recently, we reported that interleukin (IL)-10 inhibits polyinosinic-polycytidylic acid (poly I:C; a toll-like receptor 3 agonist) induced maturation of cDC1s in a signal transducer and activator of transcription (STAT) 3-dependent manner. In addition, using a tumor vaccine approach, we demonstrated that STAT3-deficient (Stat3Δ/Δ) cDC1s are more capable of inducing anti-tumor immunity than STAT3-sufficient (Stat3fl/fl) cDC1s. To further investigate how STAT3 and IL-10 inhibit cDC1 function, we performed RNA-sequencing using Stat3Δ/Δ and Stat3fl/fl cDC1s treated with IL-10, poly I:C, or both IL-10 and poly I:C. Gene Set Enrichment Analysis revealed that although many inflammatory signaling pathways are enriched in cDC1s upon treatment with poly I:C, only the interferon-alpha (IFN-α) response pathway is inhibited by IL-10 and STAT3. Furthermore, Ingenuity Pathway Analysis identified IFNs, and components of IFN receptor signaling, such as STAT1, as the primary targets of IL-10/STAT3-mediated inhibition. Therefore, pathway analyses suggest IL-10 and STAT3 inhibit poly I:C-induced IFN signaling in cDC1s. Indeed, IL-10 was found to inhibit poly I:C-induced accumulation of phosphorylated STAT1 by immunoblot. Moreover, investigations into specific IFNs determined that IFN-β and IFN-γ, but not IFN-λ, induce IFN-stimulated genes in cDC1s. Currently, studies are underway to further dissect the role of IFNs in cDC1 function and IL-10/STAT3-mediated inhibition of poly I:C-induced maturation.
Hypoxia-inducible factor 1α is a negative regulator of IRF5 in hypoxic macrophages
Authors: Son Myoungsun , Peng Travis , Du Shin-Yi , Diamond Betty 
Published: February 2023

Abstract
Patients with severe Covid-19 exhibit a low level of oxygen in affected tissue and blood and a low interferon level in blood. It is necessary to understand cell function during hypoxia to understand the pathophysiology of Covid-19 infection. We investigated aspects of human monocyte activation under hypoxic conditions. HMGB1 is an alarmin released by stressed cells. Under normoxic conditions, HMGB1 activates IRF5 and NF-κB in monocytes, leading to type 1 interferon expression and the inflammatory cytokines TNFa and IL-1b, respectively. When hypoxic monocytes are activated by HMGB1, they produce pro-inflammatory cytokines but fail to produce type 1 interferon as HIF-1α functions as a direct transcriptional repressor of IRF5. Moreover, exposure to Dimethyloxaloylglycine (DMOG), which stabilizes HIF-1α, exhibits the same transcriptional program as hypoxia. These findings have implications for the pathogenesis of Covid-19 showing that a hypoxic microenvironment that favors macrophage production of inflammatory cytokines but not interferon.
Mechanism of differentiation and unique repertoire of IgM secreting B-1 plasma cells
Authors: Smith Fauna Leah , Savage Hannah P , Baumgarth Nicole 
Published: February 2023

Abstract
Natural antibodies are broadly reactive, mostly IgM antibodies that provide a first line of defense against pathogens. Distinct populations of fetal/neonatal-derived IgM-secreting B-1 cells, including non-terminally differentiated B-1 and B-1 plasma cells (PC) are present in bone marrow (BM) and spleen. B-1PC are transcriptionally and phenotypically similar to foreign antigen-induced, long-lived conventional PC, prompting us to hypothesize that both receive similar differentiation signals. To determine the extent to which natural IgM-producing B-1PC depend on T cell help, we studied TCRβδ−/− mice lacking either all T cells, or depleted of CD4 T cells by anti-CD4 mAb treatment. Both showed significantly reduced numbers of BM but not spleen B-1PC and slightly increased populations of B-1 cells. Consistent with those data, reconstitution of RAG-deficient mice with B-1 and CD4+ T cells, but not with B-1 cells alone, restored the BM B-1PC compartment and significantly enhanced serum IgM levels. B-1PC did not bind to PtC-containing liposomes and depletion of B-1 PC had minimal effect on the number of anti-PtC IgM ASC. In contrast, most anti-influenza natural IgM ASC were B-1PC, consistent with reduced serum levels influenza-binding natural IgM in TCRβδ−/− mice. Together, the study reveals that in contrast to previous assumptions, some natural IgM production is dependent on CD4 T cell help, suggesting that multiple mechanisms regulate induction of natural IgM secretion.
Sex dictates organ-specific differences in the production and activation of plasmablasts and plasma cells
Authors: Pioli Peter , Crone Michael , Kornas Matthew , Renner Patrick , Pioli KimAnh 
Published: February 2023

Abstract
In humans and mice, sex dichotomies exist in regards to physiological immune responses as well as pathological autoimmune responses. However, very little information exists in regards to how sex patterns the production and function of antibody-secreting plasmablast (PB) and plasma cell (PC) populations. Using the Prdm1-enhanced yellow fluorescent (eYFP) reporter mouse strain, we compared the percentages and numbers of PBs and PCs in the bone marrow (BM), spleen (SPL) and thymus (THY) of young (3 months old) female and male mice. While PB/PC generation was equivalent in the BM and SPL of both sexes, the female THY had significantly increased percentages and numbers of both PBs and PCs when compared to males. This correlated with the overall increase in thymopoiesis present in females. Characterization of THY PBs/PCs demonstrated increased expression of canonical B cell activation markers such as CD69 and MHC II when compared to their BM and SPL counterparts. In some aspects, these differences were sex-based in origin. αCD45 intravenous antibody labeling suggested that THY PBs/PCs were locally generated and not a consequence of immigration from the periphery. As such, female THY demonstrated increased numbers of a germinal center B (GCB) cell-like population expressing both GL7 and CD95(Fas). THY B cells have been previously shown to participate in the regulation of T cell selection and we hypothesized that THY PBs/PCs would be generated in a T cell dependent manner. Indeed, administration of αCD40L blocking antibodies ablated THY GCB, PB and to some extent PC production. In summary, female mice are skewed towards increased THY PB/PC generation. The production of these cells happens locally and is dependent on CD40L-based T cell interactions.
Meta-analysis of the CD56-negative NK cell subset indicates altered functional responses and unique KIR regulation
Authors: Cocker Alexander , Parham Peter 
Published: February 2023

Abstract
Chronic infections such as HIV-1, HCV, and some herpesviruses, expand a subset of NK cells that lack CD56 expression (CD56neg) and are less responsive to HLA-negative targets than CD56+CD16+ (CD56dim) cells. The 39 research papers that have reported on CD56neg NK cells have considered that this subset comprises anergic, dysfunctional or adaptive NK cells. Our objective was to assess these possibilities through meta-analysis of these papers, with inclusion of new data extracted from openly deposited CyTOF datasets (n=7).
Data on CD56neg NK cells was extracted from papers. CyTOF datasets were downloaded and markers expressed by CD56neg cells identified. Meta-analysis was undertaken following the Cochrane guidelines using Random Effect Model assuming study variability.
We found through analysis of “healthy” control groups (n=30) that the CD56neg subset represents 5.42% of peripheral blood NK cells, and confirmed that HIV-1 infection expands this population by 9.00%. Four studies presenting comparable functional data report decreased IFNγ and CD107a CD56neg cell responses to HLA-lacking targets compared to CD56dim cells (−5.99% and −4.24% respectively). Comparison to CD56dim marker expression showed no difference in Ki67 implying CD56neg cells are not anergic. The CD56neg subset also has a decreased proportion of CD57+NKG2C+ cells so are not adaptive NK cells. Lower KIR expression and range of KIR combinations were seen in CD56neg cells indicating their reduced responsiveness is linked to altered KIR:HLA signalling capacity. The expansion of this subset may be due to disrupted HLA expression induced by chronic viral infections, or may be an adaptation to long-term infection to mediate ongoing activation of the NK compartment.
Reciprocal regulation of mTORC1 and ribosomal biosynthesis determines cell cycle progression in activated T cells
Authors: Rosenlehner Teresa , Pennavaria Stefanie , Straub Tobias , Kranich Jan , Obst Reinhard 
Published: February 2023

Abstract
Anabolic metabolism of antigen-stimulated T cells is coordinated by mTOR which is activated by TCR and IL-2 signals. We show that mTORC1 regulates both the exit of quiescence and later divisions as Raptor-deficient T cells proceed 3x slower through each cell division in vivo. We noticed that following stimulation the amount of RNA per cell increases dramatically (10–40x) and is reduced to ~50% in Raptor-, but not Rictor-deficient T cells. RNAseq data indicate that this applies to all RNA polymerase I- and II-dependent biotypes. Using Coulter volume measurements, RNA-, FISH- and imaging-flow cytometry we determined that following stimulation the cell volume increases five-fold. It consists of the increase of nuclear volume and nucleolar rRNA synthesis on day 1 and a further increase of cytoplasmic volume on day 2. Raptor-deficiency delays and diminishes the growth of these cellular compartments, decreases ribosomal biosynthesis and thus the cell volume. Inhibitors of rRNA transcription block cell cycle progression at two checkpoints, of which only the G1/S transition is regulated by Raptor. We found that mTORC1 activity depends on ongoing rRNA synthesis in early activation and regulates the further synthesis rate. Altogether our data show that mTOR-adjusted cell physiology and the biosynthesis of ribosomal ‘hardware’ are reciprocally regulated during clonal T cell expansion.
Circulating KLRG1+ memory CD8 T cells support immune responses in non-lymphoid tissues
Authors: Huggins Matthew A , Peng Changwei , Wanhainen Kelsey M , Gress Abigail R , Jameson Stephen C , Hamilton Sara E 
Published: February 2023

Abstract
The memory T cell population must be durable and capable of responding with rapid effector functions during re-encounter with pathogens. This is accomplished by forming a memory CD8 T cell pool containing multiple distinct subsets of cells that combine varying functional and homing characteristics. At steady-state, memory T cells expressing KLRG1 are restricted to circulation and patrol the host from within the vasculature. These cells convey robust protection during acute rechallenge with pathogens delivered intravenously. Given their localization pattern, we questioned whether KLRG1+ memory T cells participate in immune responses within non-lymphoid tissues. Here, we find that during local and systemically administered infections, KLRG1+ memory cells alter their trafficking pattern and enter infected non-lymphoid tissues. Following intranasal influenza infection, antigen-specific KLRG1+ memory cells proliferate and extravasate into the lung parenchyma. Additionally, during acute LCMV infection, similar findings were observed in all non-lymphoid tissues assayed, including small intestine, kidney, and salivary gland, indicating this is not a unique phenomenon, but rather a consistent trait of these cells. Unexpectedly, progeny from KLRG1+ cells remained within non-lymphoid tissues after acute infection is cleared, downregulating markers of circulation (CX3CR1, KLRG1) and acquiring markers of tissue residency (CD69, CD103). This work challenges the concept that KLRG1+ memory cells are terminally differentiated and demonstrates unappreciated plasticity within this subset to support immunity within non-lymphoid tissues.
Signature of Enhanced Effector Function Defines Early Life Regulatory T Cells
Authors: Connors Thomas , Bradley Marissa , Verma Shivali , Waldman Erik , Jang Minyoung , Grunstein Eli , Maurrasse Sarah E , Pethe Kalpana , Dogra Pranay , Farber Donna L 
Published: February 2023

Abstract
The induction of self-tolerance and regulation of inflammatory immune responses is critical during early life when a myriad of primary antigenic challenges are encountered. Regulatory T Cells (Tregs) promote self-tolerance and function to modulate immune responses utilizing a variety of immunosuppressive mechanisms. Tregs are known to be disproportionately more abundant during early life, suggesting an essential role during immune development. We performed bulk RNA sequencing and multi-parameter flow cytometry on Tregs isolated from adult and pediatric (1 – 3 years of age) tissue (tonsils) and blood to discern the defining characteristics of early life Tregs. Early life Tregs were found to have an increased expression of genes associated with cell cycle (Ki67), effector and suppressive function (ICOS/CTLA4/IL10/GZMB/GZMA) and chemokine signaling (CCR5/CCR9) compared to adults. Importantly, distinctions in canonical effector function genes were apparent across all early life Tregs and not restricted to tissue site. Interestingly, CCR9, essential in gut-specific homing, was upregulated in both tissue and blood early life Tregs, suggesting a biasing towards intestinal immune homeostasis during childhood. We further explored the upregulation of genes related to cell cycle and found that early life Tregs exhibit higher proliferative capacity compared to both adult Tregs and early life CD4 conventional T cells, revealing a potential effector mechanism unique to early life. Early life Tregs are defined by a signature of augmented effector functionality enhancing their ability to respond to the abundant immunological challenges of infancy and childhood.
Unique abilities of human memory T cell subsets are associated with both specialized gene expression modules and epigenetic priming.
Authors: Rose James R , Rahmberg Andrew , Scharer Christopher , Boss Jeremy M 
Published: February 2023

Abstract
Memory T cells are a diverse set of cells that respond to immunologic challenge more quickly and with greater efficacy than their naïve counterparts. Their potential in the resting and stimulated state has not been explored for each of the subsets. To define their capabilities, RNA-seq and ATAC-seq data assessing the transcriptome and chromatin accessibility potential of both resting and activated human memory T cells was analyzed. Clustering gene expression modules identified a core set of genes highly expressed in both resting central memory and effector memory cells (but not in naïve or terminally differentiated effector cells). Memory subsets also expressed unique gene modules enabling specialized functions such as cell cytotoxicity, migration, or self-renewal. Subset differences in gene expression were reflected in differentiated chromatin accessibility and enhanced variation around binding motifs for key T cell differentiation transcription factors such as Tbet, EOMES, and LEF1. Upon activation memory T cells showed substantial rapid upregulation of genes including cytokines which were absent in activated naïve T cells. These differences in gene expression from naïve T cells were often preceded by areas of open chromatin in a resting memory state, suggesting mechanisms of epigenetic priming for rapid recall or augmentation of effector T cell function in memory. In summary, memory T cell subsets are distinct in both resting gene expression functionalities and differentiated epigenetic landscape—both of which enable unique, rapid, and effective response to antigen.