bluuebunny/crossref_metadata_2025 huggingface dataset:
Histone methyltransferase, Setdb2, regulates wound healing in a diet-induced obesity model of diabetes (IRM9P.601)
Authors: Joshi Amrita , Allen Ronald , Kroetz Danielle , Schaller Matthew , Dalton Jennifer , Kunkel Steven , Gallagher Katherine
Published: January 2023

Impaired wound healing in Type 2 diabetes (T2D) is associated with a chronic inflammatory response mediated by accumulation of macrophages producing pro-inflammatory cytokines. While recent work has shown that epigenetic regulation plays a key role in influencing macrophage function, the specific alterations that occur in T2D macrophages and their effects on gene transcription have not been elucidated. Work in our laboratory has shown that Histone H3 Lys9 methylation that results in transcriptional silencing is mediated by Setdb2 (SET domain, bifurcated2) and plays a critical role in lung inflammation. Thus, we examined the role of Setdb2 in wound healing and macrophage activation in a murine model of T2D. Superficial wounding resulted in increased transcript levels of Setdb2 in wound macrophages of control mice compared with diabetic mice. Specific deletion of Setdb2 in macrophages (Setdb2lyz2) using cre recombinase resulted in delayed wound healing. Furthermore, bone marrow derived macrophages from Setdb2lyz2 cre+ mice show reduced expression of key regulatory mediators such as IFN-β. In summary, our data indicate that Setdb2 may play a protective role in wound healing by regulating inflammation.
Weight loss induced by blocking CB1 cannabinoid receptors is associated with an altered microRNA profile and suppression of inflammation in adipose tissue (IRM9P.458)
Authors: mehrpouya pegah , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Obesity is associated with chronic inflammation. Cells of the immune system express cannabinoid CB1 and CB2 receptors, the activation of which triggers anti-inflammatory effects. We investigated the effect of treatment of mice with SR141716A, a cannabinoid receptor1 (CB1) antagonist, on Diet-Induced Obesity (DIO), specifically whether such a treatment can induce anti-inflammatory state in adipose tissue. DIO model was generated by feeding C57BL/6J mice with high-fat diet (HFD) whereas their lean, age-matched controls were fed low-fat diet (LFD). HFD-fed mice were treated with either SR141716A (10mg/kg/day) or vehicle by daily oral gavage for 4 weeks starting at week 12. To assess the effect of SR141716A beyond its effect on weight loss and diet intake, pair-feeding was conducted in diet-intake matched controls and diet intake was adjusted in body-weight-matched controls. Our data demonstrated that SR141716A suppressed diet intake transiently however weight loss and reduction in fat mass was persistent. High throughput miR analysis showed that SR141716A skewed adipose tissue macrophage balance to more anti-inflammatory macrophages. Induction of myeloid-derived-suppressive cells and TH2 cells followed by suppression of pro-inflammatory macrophages were observed in SR141716A-treated HFD-fed group. Together, this study demonstrates that CB1 receptor blockade affects miR expression that induces anti-inflammatory state in adipose tissue independent of calorie intake inhibition.
An insight on antibody-mediated immune-metabolism in inflammation and homeostasis: the role of Fc gamma receptors in regulation of indolemaine 2,3 dioxygenase activity in dendritic cells (IRM9P.460)
Authors: Morsi Hadeil , Salazar Fabian , Ghaemmaghami Amir
Published: January 2023

Indoleamine2, 3-dioxygenase (IDO) is a key immune-modulatory enzyme in Tryptophan catabolism that is highly expressed by Dendritic cells (DCs). Notably, DCs are the main mediators of Fc-dependent anti-inflammatory effects of Intravenous Immunoglobulins (IVIgs) as well as Immune Complexes (ICs)-induced inflammation. Hence, we aimed to study the role of FcγRs in regulation of IDO activity in human DCs from healthy individuals, which remains elusive compared to the well-established role of TLRs. Plasma-pooled human IgG showed no effect on IDO activity in un-stimulated monocyte-derived DCs (Mo-DCs). However, on TLR4 stimulation, IgG down-regulates LPS-induced IDO activity. This counter-regulation was enhanced further, by blocking FcγRIIA, the highest expressed FcγR on Mo-DCs. Interestingly, this effect was elicited by both soluble and plate-bound IgG, resembling IVIgs and ICs, respectively, but with higher potency with soluble IgG. Strikingly, mouse IgG demonstrated a similar effect to human IgG. Herein, we propose a threshold model for IDO regulation through a crosstalk between TLR and FcγRs in homeostatic and inflammatory conditions. IgG was only able to down-regulates TLR-4-induced IDO activity in human DCs, with no effect in the steady state. Furthermore, data suggest that recognition of conserved proteins or glycan moieties of IgG by gamma associated receptors modulate IDO activity. Such insight has potential therapeutic implications for antibody-based immunotherapy.
Tripeptidyl peptidase II links intracellular amino acids homeostasis with immune response through glycolytic pathway (IRM9P.463)
Authors: Lu Wei , Zhang Yu , Hambleton Sophie , Lenardo Michael
Published: January 2023

Proteasomes and lysosomes constitute the major cellular proteolytic systems that not only remove unfolded, damaged, or unnecessary proteins, but also provide an essential supply of free amino acids for energy and new protein synthesis. Tripeptidyl peptidase II (TPP2) is a required component of the proteasome-ubiquitin protein degradation pathway. The patients lack of TPP2 manifest a combined immunodeficiency with recurrent infections, severe autoimmunity, and neurodevelopmental delay. We observed a marked increase in lysosome number and lysosomal proteolytic activity in patient cells lacking TPP2 activity. Upregulated lysosomes compensated for deficient amino acid homeostasis when TPP2 was absent. We confirmed these results in Tpp2 knockout mice and in normal human cells treated with butabindide, a selective chemical inhibitor of TPP2. However, we also found that the overabundant lysosomes exerted a pathogenic effect in which key glycolytic enzymes including hexokinase II (HKII), were consumed. The resulting decreased glycolytic potential in TPP2 defective cells selectively attenuated the production of several immunomodulatory cytokines such as IFN-γ and IL-1β, whose production require aerobic glycolysis. Taken together, our data reveal the vital role of TPP2 in amino acid supply and unveil the intimate homeostatic balance between lysosome number, amino acid level, and glycolysis in mammalian cells.
Expression pattern of hepcidin in E.coli challenged obese mice (IRM9P.600)
Authors: Lai DengMing , Chen QingHua , Zeng CongLi , Chen QiXing , Fang XiangMing , Shu Qiang
Published: January 2023

Background:Hepcidin is a master regulator of iron metabolism and plays important roles in the pathophysiology of sepsis. Obesity leads to host immune changes and is associated with the pathogenesis of sepsis. Increased hepcidin expression has been observed in adipose tissue. However, the expression pattern of hepcidin in obese mice after sepsis challenge remains unknown. Methods:Mice were fed with high-fat-diet (HFD) containing 60% energy from fat for 12 weeks. The control mice were supplied with a diet with 10% energy from fat. Sepsis was induced by intraperitoneal injection of E. coli. Hepcidin mRNA expression was studied in peripheral blood mononuclear cell (PBMC), liver, spleen, adipose tissue and peritoneal macrophage. Results:HFD-fed mice showed higher weight and increased total cholesterol and triglyceride levels in serum than controls. Obese mice, as compared with controls, exhibited increased mortality following E.coli administration. Bacterial load and IL-6 levels in blood and peritoneal lavage fluid in the HFD group were higher than controls. The hepcidin mRNA levels were increased both in HFD and LFD groups. Especially, in septic obese mice, the hepcidin mRNA levels in PBMC, spleen and peritoneal macrophage, but no in liver and adipose tissue, were higher than those in controls. Conclusions:The different expression pattern of hepcidin after E.coli challenge suggests that tissue specific hepcidin may take a diverse role in the pathogenesis of sepsis in obese mice.
Significance of IL-15 in IL-4 induced B cell activation, immunoglobulin isotype class switching and IgE production (IRM10P.613)
Authors: Shukla Anshi , Rajavelu Priya , Upparahalli Venkateshaiah Sathisha , Jalahalli Mariswamy Siddesha
Published: January 2023

IgE-mediated immune responses contribute to the pathogenesis of allergic diseases. Despite their significant role in immune biology, the factors that produce and regulate IgE responses are yet to be established. Notably, IL-4 is a well-established cytokine involved in immunoglobulin (Ig) isotype class switching and IgE production. However we recently observed that IL-15 over expression in mice (pharmacologically or by transgene insertion) induces IgE levels. Therefore we hypothesized that IL-15 might have some role in B cells proliferation, activation, Ig isotype class switching and IgE production. Accordingly we examined the level of IgE, B cells and Ig isotype class switching in response to IL-15 using ELISA, real time qPCR and flowcytometric analysis. Our initial in vitro data show proliferation, activation and induced Ig class switching genes in purified B cells following rIL-15 treatments. Further, we show reduced IgE levels in IL-15 gene-deficient mice compared to Balbc mice following Aspergillus fumigatus exposure. Furthermore we examined the role of IL-15 in IL-4 mediated IgE production. We observed that IL-4 gene-deficient mice have increased IgE levels and B cell activation following intravenous administration of IL-15 compared to saline. However, significant IgE levels were observed following IL-15 treatment in Balbc mice compared to IL-4 gene-deficient mice.Together we conclude that IL-15 is required for the IL-4 induced B cell activation and allergen induced IgE.
The development og CD138high Breg in AAD is dependent on Semaphorin 4C (IRM10P.614)
Authors: Xue Di , Divangahi Maziar , Mazer Bruce , Fritz Jorg , Mindt Barbara , Beland Marianne , Fiter Ryan
Published: January 2023

We have previously found that mice with Semaphorin 4C (Sema4C) deficient B cells exhibited exaggerated airway damage and pulmonary inflammation during allergic airways disease (AAD). We aimed to determine the mechanisms by which Sema4C on B-cells regulates AAD. Following allergen exposure, Sema4C-/- AAD mice had significantly fewer CD138high IL-10+ B cells (CD138high Breg) and significantly increased CD138high IL4+ B cells in spleen. In vitro, Sema4C-/- B cells consistently failed to differentiate into CD138high Breg under Th2 culture conditions. Adoptive transfer of Sema4C-/- plasma cells induced exacerbated Th2 responses, with airway eosinophilia and increased IL-4+ inflammatory cell infiltrates in the lungs. In contrast, transfer of CD138high Breg to WT mice with AAD dramatically alleviated airway inflammation. Co-culturing Sema4C-/- B cells with Sema4C transfected cells (Hek293-Sema4C) corrected impaired CD138high Breg development. Co-culture of B cells with Hek293-Sema4C decreased IgE production for both WT and Sema4C-/- B cells. Sema4C is essential for the development of CD138high Breg and may play a crucial role in the auto-regulation of B cells in allergic inflammation.
Control of mammalian hematopoiesis and humoral immune response by microRNA-142 (IRM10P.617)
Authors: Boldin Mark , Kramer Nicholas , Wang Wei-Le , Reyes Estefany , Kumar Bijender , Chen Ching-Cheng , Chandran Ramakrishna , Cantin Edourad , Taganov Konstantin , Chau Nelson
Published: January 2023

MicroRNAs (miRNAs) are a class of powerful post-transcriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is specifically and abundantly expressed in immune cells, we created a mouse line with targeted deletion of this gene. Our analysis of miR-142-/- mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, while the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142-/- animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142-/- B cells express elevated levels of BAFF receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142-/- mice rescued the B cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphocyte ontogenesis and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.
FCRL5 regulates innate-like B cell natural antibody production and T cell-independent responses (IRM10P.607)
Authors: Becker Eugene , Zhu Zilu , Won Woong-Jai , Dalla-Favera Riccardo , Davis Randall
Published: January 2023

At homeostasis and during primary responses to pathogens, humoral host defense is chiefly coordinated by body cavity-derived B-1 and splenic marginal zone (MZ) B cells. These early responders have been termed “innate-like” due to their broadly-reactive natural antibody repertoires and rapid responses to T cell-independent (TI) antigens. However, what regulates these functions in these specialized cells remains unclear. Fc receptor-like (FCRL) molecules have homology to the classical FCRs for IgG and IgE, exhibit complex tyrosine-based regulation, and are preferentially expressed by B cells. Notably, these receptor-genes have been increasingly associated with immune disorders and human (h) FCRL3 has emerged as a risk factor in autoimmunity. To better understand hFCRL3’s role in B cells, we investigated its closest relative, mouse FCRL5 (mFCRL5), which shares homologous extracellular domains, binary tyrosine-based signaling, and innate-like B cell expression. Here we characterize the first in vivo model of FCRL function by examining Fcrl5 deficient mice. Our findings demonstrate that at homeostasis Fcrl5 deficient mice have normal total B cell numbers and subset frequencies, but enhanced natural antibody production. By contrast, Fcrl5 deficiency was found to impair TI humoral responses, but T cell-dependent responses remained intact. These data indicate a critical role for mFCRL5 in regulating innate-like B cell function at the interface of host defense and tolerance.
The negative regulatory role of ORMDL3 in antigen-induced cytokine production, cyclooxygenase-2 expression, and chemotactic response in mast cells (IRM11P.637)
Authors: Draber Petr , bugajev Viktor , Halova Ivana , Stegurova Lucie , Draberova Helena , Bambouskova Monika , Paulenda Tomas , Draberova Lubica
Published: January 2023

Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where human ORMDL3 gene is localized, to the risk of asthma and other inflammatory diseases. Although mast cells are involved in development of these diseases, the contribution of ORMDL3 to mast cell physiology is unknown. In this study we examined the role of ORMDL3 in mouse bone marrow-derived mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation, calcium response, and sarco/endoplasmic reticulum Ca2+ ATPase 2b expression, but significantly enhanced phosphorylation of the IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were followed by an increased expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2, an enzyme involved in the synthesis of prostanoids. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas adhesion and spreading on fibronectin were decreased. On the other hand, increased expression of ORMDL3 had no significant effect on mast cell signaling events, except for reduced antigen-mediated chemotaxis. Our data provide the evidence that ORMDL3 in mast cells acts as a non-inducible suppressor of NF-kB-directed signaling pathways and chemotaxis.
Lipoteichoic acid of Lactobacillus plantarum inhibits Staphylococcus aureus-induced IL-8 expression in human intestinal epithelial cells (IRM11P.641)
Authors: kang Seok Seong , Noh Su Young , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Lactobacilli as probiotics are well known to alleviate intestinal inflammation. However, the molecular basis for the mechanism has not been well understood. Here, we investigated the inhibitory effect of Lactobacillus plantarum lipoteichoic acid (Lp.LTA) on the induction of interleukin-8 (IL-8), which plays an important role in inflammatory responses by recruiting various immune cells, in human intestinal epithelial cells, Caco-2. Staphylococcus aureus but not the lipoprotein-deficient S. aureus mutant, induced IL-8 expression, implying that the lipoproteins are crucial in the induction of IL-8 by S. aureus. Pam2CSK4, a synthetic lipopeptide that is known as a mimic of Gram-positive bacterial lipoproteins, also induced IL-8 expression in Caco-2 cells. While heat-inactivated L. plantarum or peptidoglycan of L. plantarum did not inhibit Pam2CSK4-induced IL-8 expression, Lp.LTA significantly inhibited its expression. Deacylated or dealanylated Lp.LTA failed to inhibit Pam2CSK4-induced IL-8 expression, suggesting that both lipid and D-alanine moieties are responsible for the inhibitory potency of Lp.LTA. Furthermore, Lp.LTA inhibited the activation of Toll-like receptor 2 sensing bacterial lipoproteins and the intracellular signal transduction involving p38 kinase, JNK and NF-κB in Pam2CSK4-stimulated Caco-2 cells. Taken together, these results suggest that Lp.LTA alleviates S. aureus-induced intestinal inflammatory responses by inhibiting IL-8 expression.
TNFα-mediated I kappa kinase phosphorylation of ITCH impairs ubiquitin ligase activity (IRM11P.625)
Authors: Perez Jessica , Abbott Derek
Published: January 2023

The I Kappa Kinase (IKK) signalosome serves as a central hub for the relay of inflammatory signaling. Once activated, the kinase components of this hub, IKKα and IKKβ, can phosphorylate substrates outside of the known NF-κB inflammatory cascade. Additionally, two other closely related kinases, IKKε and TBK1, share kinase homology with IKKα and IKKβ, but phosphorylate substrates related to IFN regulatory factor (IRF) activation. Despite their well-known roles in NF-κB and IRF activation, we and others have found substrates outside of these signaling cascades. Given this, we have devised a proteomic and bioinformatic approach to identify novel IKK substrates. In the present work, we show IKK-mediated phosphorylation of ITCH, an E3 ubiquitin ligase implicated in the maintenance of mucosal immunologic homeostasis. We identify the site of phosphorylation, and demonstrate the inactivating effect of IKK phosphorylation on ubiquitin ligase activity. Given that mice null for ITCH develop pulmonary interstitial fibrosis and gastritis and given that TNF strongly activates the IKKs and this phosphorylation event, we bred ITCH-/- mice with TNFR1-/- mice to determine if loss of TNF activation of IKKα/β could reverse this phenotype. Both mortality and lung inflammation were improved in TNFR1-/-ITCH-/- when compared with ITCH-/- mice. These results show that bioinformatic and proteomic studies can be coupled with mucosal immunology for novel insights.
Measurement of IL-6 levels in live cells using RNA detection probe with Imaging Flow Cytometer
Authors: Vaidyanathan Shobana , Friend Sherree , Weldon Don , Morrissey Philip
Published: January 2023

Acute inflammatory responses to infection and tissue damage are orchestrated by release of cytokines, primarily from macrophages and mast cells, but also by endothelium and tumor cells. A key initiator of the inflammatory response is tumor necrosis factor alpha (TNF-alpha), which binds to its receptor on target cells and activates a pro-inflammatory transcriptional program via the transcription factor NFkB. A critical downstream target of NFkB is the gene encoding interleukin-6, which stimulates an array of anti-infection processes, including synthesis of acute-phase proteins by the liver, proliferation of B cells, neutrophil production in bone marrow, and differentiation of Th17 helper T cells. Although beneficial in the context of attacking infection, dysregulation of IL-6 plays a deleterious role in autoimmune disease and cancer. In this study, we introduce a new method for measuring NFkB translocation by looking at the increase in IL-6 mRNA levels in the THP-1 suspension cell line using SmartFlare™ RNA detection probes. The ability to detect RNA in live cells and to acquire multi-spectral images of large numbers of cells allows for the accurate assessment of IL-6 mRNA levels in THP-1 cells.
In vivo consequences of inducible cell death via direct activation of RIPK3
Authors: Olsen Tayla M , Orozco Susana , Oberst Andrew
Published: January 2023

Necroptosis is a form of programmed cell death and is distinct from apoptosis in its mechanism and morphology; apoptosis is thought to be a non-inflammatory form of cell death whereas necroptosis is thought to be inflammatory. Necroptosis occurs in response to viral infection and is important for control of DNA and RNA viruses in vivo. While much of the necroptotic pathway has been characterized in vitro, the effects of necroptosis are poorly understood in vivo.

We recently described a system by which necroptosis can be triggered using a version of the pro-necroptotic kinase RIPK3 fused to a ligand binding domain. Here, we characterize a novel transgenic model in which this inducible form of RIPK3 is expressed in vivo, allowing induction of necroptosis in primary tissues. We find that widespread activation of necroptosis in vivo using this model triggers sepsis-like systemic inflammation.
Epigenetic regulation of histone H3 lysine 27 (H3K27) controls the division-specific timing of gene expression during B cell differentiation
Authors: Scharer Christopher , Barwick Benjamin , Bally Alexander , Boss Jeremy M
Published: January 2023

The differentiation of B cells to plasma cells is required for the generation of a protective antibody response. Following activation, B cells undergo multiple rounds of cell division that result in progressive differentiation and the acquisition of plasma cell phenotypic traits. However, how the epigenetic program is remodeled to facilitate differentiation, what the cis-regulatory elements that participate in this process are, and the molecular timing of these events during cellular division are poorly understood. To dissect B cell differentiation in vivo, five distinct divisions, representing unique stages of B cell differentiation were isolated by FACS. The changes in chromatin landscape were identified by the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and the effects on gene expression measured by RNA-seq. In parallel, histone modification profiles in naïve B cells and plasmablasts were defined. Interestingly, the loci with losses and gains in the histone modifications H3K27ac and H3K27me3 were highly correlated with enhancer activity and gene expression changes. To investigate the role of H3K27me3, B cells were differentiated in the presence of GSK343, a small molecule inhibitor of H3K27me3. Plasma cells differentiated at earlier divisions in the absence of H3K27me3 and a three-fold increase total plasma cells was observed. Together these data define the division coupled epigenetic events that regulate B cell differentiation, provide the first map of plasma cell cis-regulatory elements, and identify epigenetic processes that regulate the timing of differentiation during division.
Transcriptomic analyses reveal senescent and SnoRNA changes in B cells with age irrespective of vaccine-stimulation in mice.
Authors: Baudier Robin L , Czarny-Ratajczak Malwina , Eastwood James R , Zwezdaryk Kevin J , Norton Elizabeth B
Published: January 2023

Aging is associated with a decline in immune function that is not fully understood. Transcriptomic analysis is a powerful tool to evaluate age-related changes, but has not yet been reported for B-cells. To address this, we performed RNA-seq analyses on purified B-cells from naïve or vaccinated BALB/c mice of varying ages with the goal of expression profiling by age as opposed to any ongoing immunostimulation. Mouse age groups were Y (young, 15–20 wks), MA (middle-aged, 68–81 wks), and O (older, 103–111 wks) ± intramuscular Flulaval injection (n=5). Sequencing was performed using Ion Torrent/Ion Proton System. PCR and phenotype analyses were performed to validate selected results. In naïve groups, 230 genes were significantly differentially expressed by MA and O compared to Y. Nine of these were also significant comparing MA and O to Y in vaccinated groups. These included protein-coding genes: senescence-associated Cdkn2a; NFκB-mediator Edaradd; Klk4, a kallikrein implicated in cancer; and 4921504E06Rik, a gene region linked to inflammatory disease. Non-coding genes included: pseudogenes Gm2619 and RP24-374C7.3; antisense Gm16028; and box C/D small nuclear (sno) RNAs, Snord123 and Snord1a. The latter genes were particularly interesting as box C/D snoRNAs regulate rRNAs through methylation, and changes in their expression levels occur with neurodegenerative disorders, inflammatory diseases, and cancer. Altered DNA methylation or epigenetic drift is implicated in both healthy aging and age-related dysfunction but exact mechanisms are unclear. Our results suggest that Snord123 and Snord1a play a previously unappreciated role as regulators of altered methylation patterns contributing to age-related immune decline in B-cells.
Two different nonclassical MHC class I-restricted invariant T cell lineages with non-overlapping antiviral and anti-mycobacterial immune functions in the amphibian Xenopus
Authors: Edholm Eva-Stina , Park Jules , Robert Jacques
Published: January 2023

Unconventional T cells, including disparate populations of MHC class Ib-reactive innate T (iT) cells are emerging as key factors in the immune system partly due to their public antigen specificities and rapid effector responses. The biological relevance and evolutionary conservation of iT cells have, over the past few years, been strengthened by studies in the amphibian Xenopus, which have revealed an overrepresentation of several invariant TCRs in tadpoles and identified a prominent subset of iT cells (invariant Vα6 [iVα6]) restricted by the nonclassical MHC class Ib molecule XNC10. Recently, we showed that similar to CD1d restricted iNKT cells in humans and mice Xenopus iVα6 T cells are critical for early antiviral immunity. Here, using RNAi loss-of-function by transgenesis targeting another Xenopus nonclassical gene, XNC4, we have identified a different XNC4-dependent iT cell population expressing one of the 6 previously identified overrepresented TCRα rearrangements (TRAV45 joined to TRAJ1.14). We show that this invariant Vα45 [iVα45] T cell population is critical for antibacterial immunity against Mycobacterium marinum. These data suggest that functionally distinct populations of MHC class Ib-reactive iT cell populations play a prominent role in amphibian immune defense and as such may represent a more primordial immune cell type that previously thought.
Time-assessed modulation of SOCS1 expression through TLR4 and IFNγ signaling in RAW 264.7 macrophages.
Authors: Veal Antia E , Ganesh Bhagyalaxmi Sukka , Larkin Joseph
Published: January 2023

Innate immune cells, such as macrophages (MΦ), play a critical role in the initiation and duration of inflammatory responses directed against foreign pathogens. The elaboration of immune effector by MΦ are coordinated by interactions of microbial stimuli, such as LPS, and critical immuno-modulatory cytokines with receptors present on the MΦ. The interactions mediate a cascade of signal transduction events that ultimately culminate with the activation and nuclear translocation of transcription factors which facilitate appropriate immune responses. Notably, TLR4 stimulation by LPS mediates the activation of NF-κβ , while the immuno-modulatory cytokine IFNγ mediates nuclear translocation of phosphorylated STAT1 dimers. In addition to mediating pro-inflammatory signals, NF-κβ and IFNγ also modulate a critical limiter of pro-inflammatory signals, suppressor of cytokine signaling-1 (SOCS1). Notably the regulation of SOCS1 molecules in response to multiple external stimuli, such as LPS and IFNγ are not well understood. In this study we investigate the mechanisms through time kinetics of SOCS1 upregulation through TLR4 and IFNγ signaling in RAW 264.7 MΦ as it relates to its transcription regulators, NF-κβ and pSTAT1. We found LPS treatment mediated the phosphorylation and subsequent degradation of Iκβ, which is indicative of NF-κβ nuclear translocation, at 0.5–3 hrs, with reaccumulation at 6–12 hrs. LPS-mediated pSTAT1 was detected at 3–6 hrs. In contrast, basal levels of SOCS1 were reduced subsequently to at 0.5–3 hrs, with reaccumulation at 12–48 hrs. While IFNγ stimulated pSTAT1 at 0.5–12 hrs and basal protein levels of SOCS1 were initially reduced further showed enhanced at 6–24 hours.
Studying human hepatic immunology through the isolation and culture of liver slices and purified cellular subpopulations
Authors: Roberto Jessica Brownell , Knupp Allison , Kenerson Heidi , Yuen Sebastian , Brempelis Katherine , Crispe Ian Nicholas
Published: January 2023

Although advances in culture techniques have allowed us to better understand hepatocyte inflammatory signaling under both normal and diseased conditions, still little is known about how non-parenchymal cells (NPCs) such as liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and hepatic macrophages (i.e. Kupffer cells) impact these events in the intact human liver. In order to investigate this network of cellular cross-talk, we have developed two novel ex vivo culture systems. In Method A, 250 μm slices of human liver tissue were cultured in a transwell system. These slices induced inflammatory and tissue repair signals following both physical injury and challenge with pro-inflammatory ligands (polyI:C, LPS, and LTA). Responses following physical injury were consistent with an acute phase response, while pro-inflammatory ligands induced responses consistent with TLR activation and production of type I interferon. In Method B, we perfused wedges of liver tissue and isolated purified hepatocyte and NPC populations. Hepatocytes isolated by this procedure were successfully maintained in culture for up to 14 days on collagen-coated plates. Isolated NPCs were further purified by FACS analysis into individual populations of LSECs, HSCs, and Kupffer cells. Purified Kupffer cells recovered from sorting were also successfully maintained in culture. Together, these systems will ultimately allow us to examine how the presence of intact liver structure and/or various NPC populations impacts liver cellular signaling in response to different immune stimuli.
TCF-1 regulates effector T cell responses upon FTY720 treatment in multiple sclerosis
Authors: Gandhi Roopali , Mazzola Maria , Raheja Radhika , Gopal Murugaiyan , Rajabi Hasan , Kumar Deepak , Pertel Thomas , Regev Keren , Griffin Russell , Aly Lilian , Kivisakk Pia , Nejad Parham , Patel Bonny , Gwanyalla NGuen , Hey Hillary , Glanz Bonnie , Chitnis Tanuja , Weiner Howard L , Gandhi Roopali
Published: January 2023

Background

Cytokine producing effector T helper cells are the prime mediators of inflammation during multiple sclerosis (MS) and other autoimmune diseases. The basic immune mechanisms associated with increased pro-inflammatory effector T cell function in MS are not understood. TCF-1 (T cell factor 1, also known, as TCF-7) is a transcription factor that plays an important role in T cell biology in mice.

Methods

T cells from MS patients and healthy controls were isolated to measure gene expression profiles using nanostring and qPCR. Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying shRNA was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess TCF-1 binding to promoter regions. Luciferase assays were performed to test the regulation of IFN-γ and granzyme B by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3β.

Results

We found that TCF-1 expression is decreased in MS T cells, and FTY720 increased the expression of TCF-1 in treated T cells. We found that FTY720 treated T cell have decreased effector phenotype as measured by decreased T cell proliferation, decreased cytokine and granzyme B production. The decreased effector phenotype of the T cells is dependent upon regulatory transcription factor, TCF-1. TCF-1 blocked T cell effector function by directly binding and negatively regulating the expression of cytokines and granzyme B. Thus, our study for the first time showed that TCF-1 regulates human effector T cell function and its decreased expression could be related to altered T cell function in MS.
Dysregulation of T helper-type cytokines appears prior to and concurrent with appearance of autoantibody specificities during early SLE pathogenesis and contributes to prognosis of transition to classified disease
Authors: Munroe Melissa , Lu Rufei , Guthridge Joel M , Bean Krista M , Fife Dustin A , Chen Hua , Slight-Webb Samantha R , Keith Michael P , Harley John B , James Judith A
Published: January 2023

Autoantibodies and immune dysregulation are associated with systemic lupus erythematosus (SLE), but how these factors influence disease development is unknown. This study evaluates timing and changes in SLE-linked autoantibodies and soluble mediator pathways in the years preceding SLE classification. Serial sera from 84 SLE cases spanning pre- and post-SLE classification (average time = 5.98 years) and matched healthy controls (HC) were obtained from the Department of Defense Serum Repository. Adjusting for multiple comparison, a number of soluble mediators, including IL-5 (q=4.35×10−6), IL-6 (q=8.26×10−6), and IFN-γ (q=0.037), were significantly elevated in cases vs. HC >3.5 years pre-classification, prior to (IL-5 and IL-6) and concurrent with (IFN-γ) the earliest SLE-specific autoantibody specificity, anti-Ro/SSA. Th1-type, Th17-type, and TNF superfamily soluble mediators increased longitudinally in cases approaching SLE classification, but not in HC (q<0.05). In particular, serum levels of BLyS and APRIL were comparable in cases and HC until <10 months pre-classification (q=0.003 and q=0.019, respectively). Random forest models incorporating IL-5, IL-6, and IFN-γ levels (79–82% accuracy) distinguished future SLE patients better than models with ANA alone (58% accuracy) >3.5 years before classification. These data indicate that pre-clinical immune system perturbations allow for the accumulation of autoantibodies preceding clinical symptoms and SLE classification.
A novel paradigm in the clinical context of rheumatoid arthritis: role of Tfh and Th17 Cells in autoimmunity
Authors: Zhang Xin , Budziakowska Magdalena , Shahzad Arif , Zakem Jerald , You Zongbing , Davis William , Quinet Robert
Published: January 2023

Introduction

Rheumatoid Arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovium causing progressive joint destruction. Recently, T follicular helper (Tfh) cells and Th17 cells have emerged as novel T cell subsets controlling autoimmunity. Identifying their roles may break the traditional Th1/Th2 axis dichotomy and bridge knowledge gap of T cell lineages on RA immunopathogenesis. We examined the frequency of circulating Tfh (cTfh) and cTh17 cells in RA patients, and investigated their correlation with disease activity, autoantibody level, and inflammation.

Methods

Peripheral blood was collected from 51 RA patients and healthy donors. RA patients were divided into remission (<2.6) and active groups (>2.6) based on disease activity score (DAS-28). Clinic parameters including Rheumatoid Factor (RF), Anti-CCP, ESR and CRP were obtained. The frequency of cTfh, cTh17 cells, and plasmablasts were measured by flow cytometry. Correlation of the frequency of cTfh and cTh17 cells with DAS-28, plasmablasts, and clinic parameters were statistically determined.

Results

Both cTfh and cTh17 cells were significantly increased in RA patients, expecially in active patients comparing to healthy donors (p<0.05). The frequency of cTfh cells correlated with the percentage of plasmablasts and the level of pathogenic anti-CCP antibody, whereas the frequency of cTh17 cells correlated with serum level of RF and CRP as well as DAS-28.

Conclusions

Tfh cells may contribute to B cell differentiation and autoantibody production, while Th17 cells may involve in the inflamation and pathogenesis of RA. Thus, disrupting the signals provided by Tfh and Th17 cells may offer new therapeutic stategies for active RA patients.
Functional Characterization of the role of Eos (lkzf4) in CD4+ and CD8+ T cells
Authors: Gokhale Ameya Sanjeev , Gangaplara Arunakumar , Rieder Sadiye , Shevach Ethan M
Published: January 2023

Eos is a member of the Ikaros family of transcription factors and is preferentially expressed in T regulatory cells (Treg), and activated T conventional (Tconv) cells. The role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that Eos was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg phenotype and function in vitro and in vivo, but had a deficiency in IL-2 production with an enhanced Th17 response. To further dissect the function of Eos in CD4+ and CD8+Tconv cells, we evaluated the immune responses of Eosfl/flX CD4-Cre mice. IL-2 production in vitro by both CD4+ and CD8+ T cells was diminished upon polyclonal stimulation of cells from Eosfl/flX CD4-Cre mice. EAE was much more severe in the Eosfl/fl X CD4-Cre mice as compared to WT mice. The percentages of CD4+, Foxp3−, IL17+, IFNg+ T cells were significantly higher in spleen, draining lymph nodes, and spinal cord from Eosfl/fl X CD4-cre mice when compared to WT mice. However, the percentages of Treg cells were significantly higher in the WT mice. We also determined if Eos plays any role in the activation of CD8+ cells during LCMV Armstrong infection. On day 9 following infection, no differences between Eos−/− and WT mice were observed in the expansion, expression of activation markers, and cytokine production by tetramer+ LCMV-peptide-specific T cells. Virus was completely cleared on day 7 and anti-viral CTL activity both in vitro and in vivo was similar in the Eos−/− mice and WT mice. Eos appears to play a major role in regulating IL-2 production in vitro in both CD4+ and CD8+ Tconv, but only modulates CD4+, but not CD8+, T effector function in vivo.

This Research was supported by the DIR, NIAID, NIH.
CD8 T cells mediate polyomavirus-associated CNS demyelination
Authors: Mockus Taryn E , Frost Elizabeth L , Shwetank Fnu , Meadowcroft Mark , Lukacher Aron
Published: January 2023

JC polyomavirus (JCV), a common member of the virome in healthy humans, can cause the frequently fatal brain demyelinating disease Progressive Multifocal Leukoencephalopathy (PML) in immunocompromised individuals. PML, a pre-HAART AIDS-defining disease, has reemerged and is increasing in incidence in patients receiving immunodulatory therapies for autoimmune and inflammatory disorders. Lack of an animal model has hampered understanding of PML pathogenesis. We have developed a novel mouse model using intracerebral (i.c.) inoculation of mouse polyomavirus (MPyV), which mimics JCV-associated demyelination and shows similar histological features to PML. By one month after MPyV inoculation, C57BL/6 mice develop diffuse subcortical white matter demyelination, predominantly in the corpus callosum. Diffusion tensor magnetic resonance imaging in infected mice reveals severe peri-ventricular edema and a loss of structural integrity predominately in the corpus callosum and caudate. Virus-specific CD8 T cells robustly infiltrate the CNS and are stably maintained after CNS entry. Antibody-mediated depletion of CD4 and CD8 T cells prior to infection abrogates demyelination in the corpus callosum, despite resulting in a 50–100 fold increase in virus levels. Additionally, CD8 T cell-depletion alone was sufficient to cause higher virus levels in the brain. MPyV was controlled in the absence of CD4 T cells; however, MPyV-specific CD8 T cells in CD4 T cell-deficient mice expressed lower levels of CD103 (aE integrin), which binds epithelial E-cadherin. These data demonstrate that CD8 T cells infiltrating the CNS of MPyV-infected mice play a major role in this demyelinating leukoencephalitis.
Role of GITR and its ligand in sustaining T cell responses against influenza infection
Authors: Chu Kuan Lun , Clouthier Derek L , Watts Tania H
Published: January 2023

Members of the TNFR family play a critical role in determining the magnitude of T cell responses against viral infections. Previous studies using TCR transgenic models showed that GITR, an NF-κB activating TNFR family member, is required intrinsically on CD8 T cells for maximal response against influenza virus. However, it remains unknown how GITR affects the overall endogenous T cell responses during influenza infection. Here, we examined the effect of GITR on endogenous T cell responses against influenza virus in competitive mixed bone marrow chimeras. We found that GITR is intrinsically required for the accumulation of antigen-specific CD4 and CD8 T cells in the lung and secondary lymphoid organs during influenza infection. GITR affected influenza PA224-233-specific CD8 T cells more dramatically than NP366-374-specific CD8 T cells. GITR also had a small positive effect on the number of Tregs. Among different APC subsets in the lung and its dLN, we observed highest GITRL expression on inflammatory DCs and inflammatory macrophages in both organs. Interestingly, we observed peak GITRL expression on these inflammatory subsets on day 3 and day 5 post-influenza infection in the lung but earlier in the dLN. The finding that GITRL is expressed on inflammatory APCs in the lung at the time when T cells enter the lung raises the possibility that GITRL plays a role in the lung tissue and not just in lymphoid organs. We are currently investigating when and where T cells receive signals through GITR, whether early in the dLN or later in the lung during influenza infection. A more thorough understanding of GITR/GITRL axis will provide valuable insights for influenza vaccine development.
T cell help amplifies innate signals in CD8+ DC for optimal CD8+ T cell priming
Authors: Bedoui Sammy
Published: January 2023

DC often require stimulation from CD4+ T cells to propagate CD8+ T cell responses, but precisely how T cell help optimizes the priming capacity of DC and why this appears to differ between varying types of CD8+ T cell immunity remains unclear. We show that CD8+ T cell priming upon HSV-1 skin infection depended on DC receiving stimulation from both IFN-α/β and CD4+ T cells to provide IL-15. This was not an additive effect, but resulted from CD4+ T cells amplifying DC production of IL-15 in response to IFN-α/β. We also observed that increased innate stimulation reversed the helper-dependence of CD8+ T cell priming and that the innate stimulus, rather than the CD4+ T cells themselves, determined how ‘help’ was integrated into the priming response by DC. These findings identify T cell help as a flexible means to amplify varying suboptimal innate signals in DC.
ThCTL expressing NKG2A/C/E represent a unique CD4 cytotoxic effector subset.
Authors: Vong Allen M , Marshall Nikki B , Kuang Yi , Swain Susan L
Published: January 2023

Activated CD4 T cells differentiate into effector subsets tailored to fight widely diverse pathogens. CD4 T cell effectors can help antibody responses, secrete effector cytokines, and lyse infected cells. Cytotoxic CD4 T cells, termed ThCTL, synergize with antibody to provide strong protection against a lethal infection with influenza A virus (IAV) in mice and correlate with better disease outcome in humans. However, their regulation in vivo is not well studied. We find that IAV-generated ThCTL is localized to the lung, the site of viral replication, and express NKG2A/C/E, which is expressed only on a fraction of CD4 effectors. The expression of this marker does not impact cytotoxicity but may play a role in the infiltration or survival of the ThCTL. We used NKG2A/C/E to further characterize the ThCTL subset. We found that ThCTL can secrete effector cytokines, IFNg and TNFa and that they express markers consistent with high levels of activation and cytotoxicity. Microarray analysis indicates these ThCTL down-regulate markers for circulation: Ccr7, S1p1r, and Klf2, while up-regulating cytotoxic molecules: Prf1, Gzmc, and Gzmf. We find the generation of ThCTL in vivo is regulated by the context of late antigen and co-stimulation, such that ThCTL can be induced from effectors at day 6 post infection. Taken together, ThCTL consist of an activated subset of CD4 T cells with unique properties to combat IAV infection.
Overexpression of total STAT1 during lymphopenia-induced proliferation leads to decrease CD4 T cell survival
Authors: Catalfamo Marta , Cecile Le Saout , Luckey Megan A , Villarino Alejandro , Smith Mindy , Myers Timothy G. , Hasley Rebecca B. , Park Jung-Hyun , O’Shea JJ , Lane H. Clifford
Published: January 2023

To preserve the size and diversity of the T cell repertoire throughout life, homeostatic mechanisms regulate both survival and proliferation through IL-7 and TCR signaling. IL-7 signals through a heterodimer receptor composed of the IL-7Rα and the γc chains. Binding of IL-7 to its receptor leads to activation of STAT5 and induction of target genes implicated in survival and proliferation signals. IL-7/IL-7R signaling can also activate STAT1 and STAT3 but the function of IL-7-dependent activation of these transcription factors is largely unknown. Under lymphopenic conditions, IL-7 modulates the expression levels of total STAT1 (t-STAT1) leading to an IL-7-dependent STAT1 signaling in addition to STAT5. We generated a transgenic mouse that overexpress STAT1 in CD4 and CD8 T cells to understand the contribution of IL-7-dependent STAT1 activation on T cell homeostasis in the setting of lymphopenia. We found that under lymphopenia-induced proliferation T cells from the STAT1 transgenic mouse upregulates t-STAT1 leading to diminish CD4 T cell survival. This mechanism could be a physiological mechanism to regulate the expansion of the CD4 T cell pool under lymphopenic conditions.