bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased body temperature induces lymphocyte lipid raft aggregation (35.34)
Authors: Mace Thomas A , Kilpatrick Casey , Zynda Evan , Repasky Elizabeth
Published: January 2023

Previous recent data reveal that mild heating in vitro of CD4+ and CD8+ T cells (purified from murine splenocytes) at 39.5°C induces the aggregation of lipid rafts; approximately 70% of lymphocytes express aggregated lipid rafts compared to only 20% of cells maintained at 37oC as judged by the distribution of the lipid raft marker, fluorescently labeled GM1. Further, we found that the fluidity of the T cell plasma membrane, as measured by fluorescence anisotropy of TMA-DPH, increases when the temperature is raised from 37° to 39.5°C. Based on these in vitro results, we hypothesized that a physiologically relevant increase in body temperature results in an increase in lipid raft aggregation in T cells in situ. We administered whole body hyperthermia (WBH) to raise the temperature of mice to 39.5°C for 6 hours and immediately harvested and fixed cells from spleen and lymph nodes. Utilizing Imagestream flow cytometry, we observed a 3 fold increase in the number of CD8+ T cells with lipid raft aggregates isolated from WBH mice compared to T cells isolated from normothermic control mice. Lymphocytes from spleen and lymph nodes responded similarly to WBH. Overall, these findings may help to understand the role of physiological temperature shifts (e.g., during febrile episodes and inflammation) on costimulation and lymphocyte activation potential.

Supported by NIH P01 CA94045 and RO1 CA71599
Regulation of Tim-1 signaling and localization in T cell activation (35.35)
Authors: Lin Jean Y , Delmastro Meghan M , Kane Lawrence P
Published: January 2023

T cell immunoglobulin and mucin domain 1 (Tim-1) can modulate CD4+ T helper responses. However, the precise mechanisms by which Tim-1 regulates T cell function remain relatively undefined. Work in this study focuses on identifying downstream targets of Tim-1 signaling as well as understanding Tim-1 localization during T cell activation. The consequences of Tim-1 interaction with antibodies or its ligand phosphatidylserine (PS) were explored by luciferase assays to determine levels of NFAT/AP-1 or by Western blotting for Tim-1 induced phosphorylation. Confocal microscopy was used to visualize Tim-1 localization on T cells after transfection of tagged forms of Tim-1 into D10 cells and incubation with antigen loaded antigen presenting cells (APCs). Results/Conclusions: Agonistic Tim-1 antibodies induce a previously unrecognized phosphorylated substrate, which we are currently attempting to identify. No induction of NFAT/AP-1 after interaction with PS expression apoptotic cells was apparent. Tim-1 localizes away from the immunological synapse after T cell activation. Better understanding of Tim-1 interactions with its antibodies, ligands, and APCs will allow for better understanding Tim-1 activation.

NCI and NIAID
Beta-catenin negatively regulates peripheral T cell activation (35.36)
Authors: Driessens Gregory , Gajewski Thomas
Published: January 2023

β-catenin signaling has been shown to play a role in T cell development. However, the function of β-catenin in peripheral T cell regulation is not understood. Activation of T cells via TCR/CD28 ligation resulted in low-level stabilization of β-catenin which was augmented with proteasome inhibition. To determine the consequences of increased β-catenin expression, CAR Tg T cells were transduced with an adenoviral vector encoding a non-degradable β-catenin, which inhibited proliferation and cytokine secretion. Experiments were performed to determine the mechanism of this inhibition. Decreased IL-2 production was confirmed at the mRNA level, and surface expression of TCR and CD28 were not altered. IL-2 production was rescued with PMA+Ionomycin, arguing for a defect in proximal TCR signaling. TCR-induced phosphorylation of Lck, LAT, Akt, and ERK were intact in β-catenin-expressing T cells. However, phosphorylation of PLC-γ1 was significantly reduced, as was Ca++ flux. Conversely, deletion of the β-catenin gene from primary T cells using CAR Tg conditional knockout mice and a Cre-adenovirus caused augmented cytokine production and proliferation. Our results suggest that β-catenin negatively regulates TCR-mediated activation or peripheral T cells via a novel mechanism.
SHP-1 regulation of in vivo CD8 T cell responses (35.43)
Authors: Fowler Carla , Pao Lily , Schietinger Andrea , Tan Xiaoxia , Blattman Joseph , Greenberg Philip
Published: January 2023

OBJECTIVE: Determine the cell-intrinsic effect of SHP-1 deficiency on the primary response and memory formation of CD8 T cells following viral infection.

METHODS: Conditional deletion of SHP-1 in peripheral T cells was achieved by crossing mice transgenic for Cre-recombinase expressed under the distal-LCK promoter to mice bearing a floxed SHP-1 gene. SHP-1+/+, SHP-1+/-, or SHP-1-/- P14 transgenic CD8 T cells specific for LCMVGP33 were adoptively transferred into wild-type (WT) hosts, infected with LCMV, and analyzed.

RESULTS: At the peak of the primary response, SHP-1-/- CD8 T cells showed 2-3 fold greater expansion than WT cells. The increased magnitude of the peak response reflected mostly increased numbers of cells expressing a terminally differentiated surface phenotype. Consistent with this phenotypic data, SHP-1-/- CD8 T cells underwent a larger contraction following infection, and formed long-lived memory populations of similar magnitude to WT CD8 T cells.

CONCLUSIONS: SHP-1 regulates TCR signal strength in vivo and this regulation impacts both the magnitude of peak expansion as well as the number of the peak effector cells that have differentiated towards a short-lived fate. The formation of long-lived memory populations, however, appears to be regulated independently of SHP-1.

SUPPORT: ARCS Foundation, Poncin Fellowship, Bill and Melinda Gates Foundation, NCI, and Leukemia and Lymphoma Society.
Novel function of perforin in regulating CD4+ T cell activation by affecting calcium signaling (35.7)
Authors: sun bing , bi enguang
Published: January 2023

Perforin is a pore forming protein engaged mainly in mediating target-cell death and is employed by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. However, whether it also plays a role in conventional CD4+ T cell function remains unclear. Here we report that in perforin-deficient(PKO) mice, CD4+ T cells are hyperproliferative in response to TCR stimulation. This feature of activated CD4+ T cells is accompanied by an enhancement both in cell division and in IL-2 secretion. The perforin deficiency does not appear to influence T cell development in thymus and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell proliferation and antibody production. Furthermore, PKO mice appear more susceptible to experimental autoimmune uveitis. Examination of the mechanisms involved demonstrate that in PKO CD4+ T cells TCR stimulation increases intracellular calcium flux and subsequently enhances activation of transcription factor NF-AT1. Our results indicate that perforin plays a negative role in regulating CD4+ T cell activation and immune response by affecting TCR-dependent Ca2+ signaling.
GPR34 is not required for mast cell degranulation induced by IgE or lysophosphatidyl-L-serine (36.1)
Authors: Vissinga Christine , Hensley Kelly , Kirchner Jacqueline
Published: January 2023

Mast cells play an important role in allergic inflammation. Upon activation by cross-linking of FcεRI, they rapidly release granule-associated chemical mediators and secrete cytokines, fatty acids and lipids which mediate allergic inflammation.

GPR34 is a GPCR expressed on mast cells and has been proposed to be a receptor for lysophosphatidylserine (lysoPS), a phospholipid derivative which can potentiate antigen-induced mast cell degranulation (BBRC 341: 1078, 2006). To determine if GPR34 is involved in mast cell response, we generated GPR34-/- mice and evaluated mast cell function in vitro and in vivo. Bone marrow-derived mast cells from GPR34+/+ and GPR34-/- mice exhibited similar responses, as measured by beta-hexosaminidase release, to IgE cross-linking or lysoPS stimulation. We evaluated the role of GPR34 in vivo using a passive cutaneous anaphylaxis model induced by crosslinking of FcεRI on mast cells. Mast cells in GPR34+/+ and GPR34-/- mice were activated by SC injection of anti-DNP IgE mAb followed by IV administration of DNP-BSA and Evans blue dye 24 hours later. Activation resulted in vascular permeability which was measured as the amount of dye influx. In this model, the mast cell response was unaffected by GPR34 gene deficiency.

Together, our results indicate that GPR34 is not required for the mast cell response to IgE crosslinking in vitro or in vivo and that GPR34 is not a receptor for lysoPS.
Receptor for advanced glycation end products is required for normal T cell activation and development of T cell induced lung inflammation (36.10)
Authors: Akirav Eitan M , Henegariu Octavian , Clynes Raphael , Schmidt Ann Marie , Herold Kevan C
Published: January 2023

We have shown that the receptor for advanced glycation endproducts (RAGE) is required for normal T cell activation and differentiation into Th1 cells, since RAGE-/- T cells show reduced proliferation and IFNγ expression after activation with alloantigens. Here, we examined T cell activation in RAGE-/- and OT-II.RAGE-/- mice in a model of T cell induced asthma. Intranasal OVA challenge of RAGE-/- mice resulted in reduced T cell activation as measured by increased CD62L expression, decreased CD69 expression, and reduced IL5 production. Cellular infiltrates in the lungs of OVA immunized RAGE-/- mice were reduced when compared with WT mice. Intranasal OVA challenge of OT-II mice induced a robust T cell infiltration of the lung, which was reduced in OT-II.RAGE-/- mice. Moreover, cells from mediastinal lymph nodes of OT-II.RAGE-/- mice showed reduced IL2, IL5, IL10, IL12, and IFN? production. Interestingly, purified RAGE-/- T cells showed lower basal levels of Ca2+ flux when compared with WT cells. These levels were not corrected in response to αCD3 stimulation. Reduced Ca2+ flux was associated with reduced expression of the transcription factor T-bet but with no change in GATA-3 expression in RAGE-/- T cells. In summary, these results reveal an unexpected defect in Ca2+ flux of RAGE deficient T cells under basal and activated conditions and reduced T cell activation in OVA-induced asthma, suggesting a novel role for RAGE in T cell signaling.
Using neurolinguistic-programming as an alternative treatment for allergic rhinitis (36.3)
Authors: Cervantes-Trujano Edgar , Rodriguez-Flores Azucena , Pérez-Perrusquia Marisol , Ramos-Rosales Cintia , Montiel-cortes Miguel , Estrada-Parra Sergio
Published: January 2023

The aim of this study is known as through the conceptual change of health status through neurolinguistics programming (NLP) can modify some mediators such as IgE, cortisol and lymphoid cells.

Material and Methods:

We performed a clinical trial, double-blind, placebo controlled and randomized 18 patients with AR with positive skin tests to aeroallergens. The control group (A) received a placebo of NLP more allergen-specific mixture by sublingual route, by period of 6 months. The experimental group (B) treated with NLP plus placebo mixture of allergens. All patients underwent the following assessments at baseline, three and six months after treatment: seric cortisol and total IgE, determination of lymphocyte populations and subpopulations and eosinophils in peripheral blood by flow cytometry.

Results

The patients were allergic to dust mites, pollens, animals, fungi. All patients had eosinophilia and increased serum IgE, decreased levels of CD4 + CD25 + cells. After 6 months of treatment, levels of IgE and cortisol were not changed, the eosinophilia decreased to normal values in both study groups and levels of CD4+ CD25+ cells increased with respect to its initial values with a statistically significant difference (p = 0.008).

Conclusions

Both treatments changed the status of patients with AR moderate persistent to AR mild intermittent and mild persistent, which shows a better control of the disease.
A Putative Immunoregulatory role for TSLP on CD4+ T cells (36.4)
Authors: de Souza Anjali J , Ziegler Steven
Published: January 2023

TSLP is primarily an epithelial cell derived cytokine that has been implicated in promoting Th-2 cytokine mediated inflammation at mucosal sites. The TSLP receptor is expressed on a variety of cell types including B cells, monocytes, dendritic cells and mast cells.

We have shown earlier that activated CD4+ T cells can also respond to TSLP, express the TSLP receptor and induce IL-4 expression. In this study we have further probed, the effects of TSLP on differentiation and function of other helper T cell lineages. Our data indicate that naive murine CD4+ T cells upon primary activation through the TCR coupled with either TSLP alone or in conjunction with TGF-beta can promote the differentiation and expansion of inducible regulatory T cells (iTregs) indicated by an increased population of FOXP3+ cells and in a manner that requires the TSLP receptor. When added to a cytokine milieu consisting of TGF-β and IL-6 alone or in combination with IL-21 which are known inducers of Th17 differentiation, TSLP downregulates IL-17 production. These data indicate that TSLP can directly influence differentiation of CD4 T cell lineages depending on the cytokine milieu and suggests an immune-regulatory role in disease. Future work is directed at understanding the mechanisms by which TSLP mediates it's effects on T cells and its application in vivo in promoting tolerance and regulating immune responses in allergic and autoimmune diseases.
Cancer-induced activation of CD11b+Gr-1+ cells predispose hosts to antibody-independent anaphylactoid-type reactions (36.5)
Authors: LaFace Drake M , Pande Kalyan , Sathe Manjiri , McClanahan Terrill K , Phillips Joseph H
Published: January 2023

Cancer can induce expansion of CD11b+Gr-1+ myeloid cells, attributed to immune suppression. We noted tumor-activation of these cells further correlated with predisposition for anaphylactoid-type reactions in mice bearing 4T1 mammary carcinomas, which induces strong CD11b+Gr-1+ expansion. To test this hypothesis, 4T1-tumor bearing (4T1+) mice were challenged with recombinant adenoviral vector (rAd), previously shown to activate myeloid lineage cells. When challenged with a single intravenous dose of rAd, 4T1+ mice succumbed to acute anaphylactoid-type reactions. In contrast, minimal responses were seen in rAd treated mice implanted with the related cell line, 66cl4, which do not induce CD11b+Gr-1+ cells. Liposome depletion prior to rAd challenge protected 4T1+ mice, whereas, passive transfer of CD11b+Gr-1+ cells from 4T1+ mice was sufficient to convey susceptibility for anaphylactoid reactions in normal mice. Metabolic pathways were upregulated for nitric oxide and leukotrienes in the 4T1-induced CD11b+Gr-1+ cells. Pre-treating 4T1+ mice with inhibitors of NO-synthetase and leukotrienes inhibited rAd-induced anaphylaxis. Thus, malignant tumor growth can alter CD11b+Gr-1+ myeloid cells, rendering hosts susceptible to anaphylactoid-type reactions. Importantly, there was no requirement for pre-existing Ag-specific IgE or IgG. This reflects clinical reports in which cancer patients undergo treatment-induced anaphylactoid responses in absence of pre-exposure and may provide insight for modeling alternative pathways induced by direct activation of inflammation- or tumor-modified myeloid cells.

* Funded by the Schering-Plough Research Institute
Pattern of skin prick test reactivity to aeroallergens in patients with asthma and rhinitis residing in Riyadh, Saudi Arabia. (36.6)
Authors: Almogren Adel
Published: January 2023

The aim of this study was to determine the pattern of skin prick test reactivity to aeroallergens in patients with asthma and rhinitis (airway allergy) residing in Riyadh region.

During the period between Jan. 2003 and March 2004, allergy skin prick test results of 139 Saudi nationals (53% females and 47% males, mean age 27 + 12 years) with a clinical diagnosis of airway allergy residing in Riyadh region were analyzed in retrospect at King Khalid University Hospital Riyadh. A set of aeroallergens extracts for both indoor and outdoor allergens including fungal spores was used to test the patients.

Of the total 75% (105) patients with airway allergy reacted to one or more allergen extracts. Most frequently reacting indoor allergen was house dust mite (77.8%) followed by the cat (33.6%) and cockroach (19.2%) allergens. Among the outdoor allergens Prosopis juliflora tested positive in 72.1%, Bermuda grass in 53.8%, Chenopodium album in 47.1%, Rye grass in 36.5% and Salsola kali in 36.5%. A significant proportion of patients were also found reacting to Moulds (18.2%) and Aspergillus fumigatus (18.2%) extracts.

Sensitivity to one or more aeroallergens was common in atopic patients, indicating high level of aeroallergen sensitization in patients with airway allergy residing in Riyadh region.
Exploring Stem Cells: the Role of Stem Cells in Immune System Function (Educational Unit) (37.1)
Authors: Thoune Monica
Published: January 2023

Exploring Stem Cells: The Role of Stem Cells in Immune System Function is an educational unit developed for high school life science courses. Students will learn about embryonic and adult stem cells and the role adult stem cells play in the Immune System's function.

Students will learn about the tendency of Systemic Lupus Erythematosus (SLE) and Rheumatoid Arthritis (RA) patients to develop premature cardiovascular disease, specifically atherosclerosis. The unit will explore how the body's immune system, through the production of interferon αmay interfere with the normal development of endothelial progenitor cells (EPCs). Interferons are cytokines that are involved in immune system regulation and the inflammatory response. EPCs are bone marrow-derived stem cells which circulate in the bloodstream and participate in endothelial repair of the lining of blood vessels of the cardiovascular system.

This 5-6 day unit includes pedagogical strategies such as Internet treasure hunts, online interactive tutorials, cooperative group activities, problem solving, and hands-on activities. Students will gain real-world laboratory experience by working with micropipettes during a hands-on activity. They will learn how following proper protocol with minute samples is essential for accuracy and precision.
G-Protein coupled receptor kinases mediate TNF alpha-induced NF kappaB signaling via direct interaction with and phosphorylation of I kappa B alpha (38.15)
Authors: Patial Sonika , Luo Jiansong , Porter Katie Johanna , Benovic Jeffrey L , Parameswaran Narayanan
Published: January 2023

Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibited TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene macrophage inflammatory protein-1β. Consistent with these results, over-expression of catalytically active GRK2 or 5 enhanced TNFα-induced NFκB activity. In addition, we show that GRK2 and 5 interact with IκBα in intact cells as well as in vitro via the N-terminal domain of IκBα and that IκBα is a substrate for GRK2 and 5 in vitro. Taken together, these results demonstrate that GRK2 and 5 are important mediators of TNFα-induced NFκB activity and suggest that GRKs could be potential therapeutic targets for treating TNFα-mediated inflammatory diseases.
KIT Signaling Regulates MITF Expression through microRNAs in Mastocytosis and Mast Cell Proliferation (38.16)
Authors: Takemoto Clifford , Noel Pierre , Mendell Joshua , Wentzel Erik , Carter Melody , Metcalfe Dean D. , Lee Youl-Nam
Published: January 2023

Activating mutations in codon D816 of the tyrosine kinase receptor, c-KIT, are found in the majority of patients with systemic mastocytosis, an aggressive disease characterized by proliferation of abnormal mast cells. Constitutive activation of the KIT signaling pathway is critical to the transformed phenotype. The MITF transcription factor is essential for normal mast cell development. We found that MITF is highly expressed in malignant mast cells from bone marrow biopsies of patients with systemic mastocytosis with the D816V KIT mutation; however, it is not highly expressed in mast cells from bone marrow biopsies of patients with other hematologic conditions. We show that in both primary and transformed mast cell lines, KIT signaling markedly upregulates MITF protein expression. MITF mRNA levels do not change significantly with KIT signaling, suggesting post transcriptional regulation. We performed an array screen for KIT-regulated microRNAs in mast cells and found that miR-539 and miR-381 are downregulated by KIT signals; furthermore, they suppressed MITF expression through conserved sites in the MITF 3' UTR. We demonstrate the requirement of MITF for the transformed phenotype by inhibiting tumor growth in colony forming assays and in a murine xenograft model by knockdown of MITF with a shRNA-expressing lentivirus. This work demonstrates a novel regulatory pathway between two critical mast cell factors, KIT and MITF, mediated by microRNAs; dysregulation of this pathway may contribute to abnormal mast cell proliferation and malignant mast cell disease.
A fundamental role for Interleukin-21 in germinal center antibody responses (38.21)
Authors: Vogelzang Alexis , McGuire Helen , Sprent Jonathan , King Cecile
Published: January 2023

T cell help to B cells is a fundamental property of adaptive immunity, yet only recently have many of the cellular and molecular mechanisms of T cell help emerged. T follicular helper (TFH) cells are the CD4+ T helper cells that provide cognate help to B cells for high affinity antibody production in germinal centers (GC), and have recently been shown to give rise to lymphoid reservoirs of antigen specific memory T cells. TFH cells produce IL-21 and we have shown that IL-21 is necessary for GC formation. However the central role of IL-21 in GC formation reflected its effects on CD4+ T cells rather than on B cells. Furthermore, bone marrow chimeras confirm that IL-21R-deficient B cells are capable of mounting an effective IgG1 response to T dependent antigen. Using an antigen specific model where TCR transgenic CD4 T cells specific for the ovalbumin peptide were introduced into IL-21R-/- mice, we show that IL-21 responsiveness by T cells was important for optimal primary and secondary antibody responses. IL-21 also proved indispensable for affinity maturation of ova-specific antibodies in the GC. This study reveals a previously unappreciated role for TFH cells in the formation of the GC and isotype switching through a CD4+ T cell intrinsic requirement for responsiveness to IL-21.
Lymphotoxin-alpha beta heterotrimers are shed from the surface of activated human lymphocytes, circulate in serum, and are elevated in synovial fluid of rheumatoid arthritis patients (38.22)
Authors: Young Judy C , Yu Xin , Nguyen Allen , Kung Catherine , Wong Wai Lee , DeForge Laura , Grogan Jane
Published: January 2023

Lymphotoxin, a trimeric cytokine in the tumor necrosis family, is expressed by activated T, B, and NK cells and is involved in inflammatory response signaling and secondary lymphoid organ architecture. Using a specific assay for human LTαβ which detects both LTα1β2 and LTα2β1 trimers but not LTα3, we show here that LTαβ is detected in culture supernatants of 293 cells transfected with human LTα and LTβ constructs, and in supernatants of primary activated human T lymphocytes. The levels of soluble LTαβ detected in culture supernatants of human CD4+ Th1 polarized lymphocytes treated with an inhibitor of ADAM17 (TACE) protease, were reduced 3-6 fold compared to supernatants from untreated cells. This suggests that LTαβ cleavage is carried out by TACE. A 26 KDa MW form of LTβ was detected in activated human T cell culture supernatant by Western blot analysis using an anti-LTβ-specific antibody. Soluble LTαβ was detected in serum from normal human donors and rheumatoid arthritis (RA) patients at levels of approximately 100 pg/mL. Soluble LTαβ was also detected in synovial fluid from RA patients at significantly higher levels than in synovial fluid from osteoarthritis patients. Cleavage of LTαβ trimers from the cell membrane and release into circulation has not been previously described. Soluble LTαβ may play a role as an inflammatory mediator of autoimmune disease.

Research supported by Genentech Inc., S. San Francisco, CA
Evaluation of heterophilic antibody blocking agents in the development of sensitive immunoassays for IL-17AA, IL-17FF, and IL-17AF (38.24)
Authors: DeForge Laura E , Loyet Kelly M , Delarosa Donnie , Chinn Jason , Townsend Michael J , Wong Wai Lee T
Published: January 2023

The proinflammatory cytokines interleukin 17 (IL-17) A and F have been implicated in the pathophysiology of autoimmune diseases. In an effort to measure the concentrations of homodimeric IL-17AA, IL-17FF, and the recently identified IL-17AF heterodimer in patient samples, sensitive immunoassays for the three forms of IL-17 were developed in 384 well plates. Samples from autoimmune patients are particularly prone to false readings due to interference from heterophilic antibodies (HA, a term which will be used to encompass true HA as well as rheumatoid factor and human anti-animal antibodies). To evaluate HA interferences in these assays, sera were screened to identify samples that generated false positive signals. These samples were then treated with up to eight different commercial HA blockers. One blocking agent (IIR) was found to consistently eliminate or greatly reduce nonspecific signal and was chosen for use in the final assays. The limits of quantitation of the assays were 0.78 pg/ml, 6.14 pg/ml, and 5.12 pg/ml for IL-17AA, FF, and AF, respectively, with a minimum two-fold sample dilution. IL-17FF and IL-17AF levels were below detection limits for normal, Crohn's disease, and ulcerative colitis (UC) serum as well as osteoarthritis and rheumatoid arthritis (RA) synovial fluid. The greater sensitivity of the IL-17AA assay allowed detection of low levels of IL-17AA in <50% of the UC and RA samples only.
Mercury chloride triggers human mast cells to release vascular endothelial growth factor (VEGF)-relevance to autism (38.26)
Authors: Kempuraj Duraisamy , Hogan Jennifer , Zhang Bodi , Kourelis Taxiarchis , Manola Akrivi , Theoharides Theoharis C
Published: January 2023

Autism Spectrum Disorders (ASD) are pervasive neurodevelopmental disorders of early childhood. There is no pathogenesis or curative therapy. Many ASD patients have "allergic" symptoms and food intolerance; moreover, the prevalence of ASD in patients with mastocytosis is 10-fold higher than the general population, suggesting mast cell involvement. A recent paper with data from the Vaccine Safety Datalink showed significantly increased rate ratios for ASD with mercury exposure from vaccines. We, therefore, investigated the effect of HgCl2 on VEGF secretion, intracellular calcium ion levels and viability of human cultured mast cells. HgCl2 induced statistically significant VEGF release from mast cells at 0.1-10 µM (n=3, p<0.05), but without a dose-dependent relationship; viability was unaffected. To study the mechanism of action, we investigated intracellular calcium ions. HgCl2 increased intracellular calcium ion levels within 15 min. Mast cell stimulation by HgCl2, possibly in combination with other triggers such as viruses, stress hormones or toxins at a vulnerable young age to release VEGF, which is also vasodilatory could disrupt the gut-blood-brain barriers and permit brain inflammation contributing to ASD pathogenesis. This work was supported by Theta Biomedical Consulting and Development Co., Inc. (Brookline, MA).
Ikaros regulates IL-10 expression in CD4+ T cells (38.27)
Authors: Umetsu Sarah E , Winandy Susan
Published: January 2023

IL-10 is a regulatory cytokine critical for controlling inflammatory responses. However, little is known about the molecular mechanisms controlling Il10 gene expression, although GATA-3 is known to be involved in Il10 expression by Th2 cells. Here we show that Ikaros, a zinc finger DNA-binding protein, plays an important role in the regulation of IL-10 in T cells. Upon initial stimulation by T cell receptor, cells deficient in Ikaros (Ikaros null) express significantly lower levels of IL-10 compared to wild type T cells. In addition, under Th2 skewing conditions, which induce high levels of IL-10 production by wild type cells, Ikaros null T cells are unable to properly differentiate and produce only low levels of IL-10. The use of a retroviral vector system to express a dominant negative isoform of Ikaros in wild type Th2 cells represses IL-10 production but does not significantly alter the levels of transcription factors Gata3 and Tbet. Further, expression of Ikaros in Ikaros null T cells restores IL-10 expression. We found that Ikaros binds to conserved regulatory regions of the Il10 gene locus in Th2 cells, supporting a direct role for Ikaros in Il10 expression. In addition, we have evidence to suggest a coordinate role for Ikaros and Gata3 in the regulation of Il10. A direct interaction of these proteins is being examined. Thus, we suggest of novel role of Ikaros in regulating Il10 gene expression.
Analysis of human IL-4 and IL-13 dependent signaling through the IL-4/IL-13Ra1 receptor complex as measured by hTARC production in A-549 cells (38.29)
Authors: Miller Renee , Staples Megan , Febo Ruth , Burns John , Wu Chengbin
Published: January 2023

IL-4 and IL-13 are key TH2 cytokines that contribute to asthma pathogenesis by promoting airway hyper-responsiveness, cellular infiltration and mucus over-production. Both cytokines have similar overlapping functionalities likely the result of having a shared type II receptor complex composed of IL-4R and IL-13Rα1 chains. In the presence of hIL-13Rα1 alone, IL-13 binds to IL-13Rα1 with low affinity but upon IL-4R recruitment a high affinity heterodimeric complex is formed leading to cytokine dependent cellular events. IL-13 can also bind to hIL-13Rα2 with high affinity but the biological mechanism of cell signaling through this receptor remains unclear. Since the A-549 human lung epithelial cell line can respond to both IL-4 and IL-13 stimulation in the presence of recombinant human TNF-alpha (rhTNF-α),

IL-4/IL-13Rα1 expression in A-549 cells was investigated and the molecular basis for IL-13 induced human TARC (Thymus Activation Regulated Chemokine) production was determined. Furthermore, up-regulation of hIL-13Rα2 was observed on A-549 cells following rhTNF-α treatment, therefore, the contribution of IL-13Rα2 signaling as related to TARC production was additionally assessed. Our study indicates that A-549 cells can produce high levels of TARC in response to IL-4 and IL-13 suggesting this response is apparently a result of signaling through the IL-4R/IL-13Rα1 pathway and not the IL-13Rα2 pathway.
Interleukin-7 controls glucose uptake in T-Lymphocytes by regulating Hexokinase II gene expression (38.4)
Authors: Khaled Annette R , Chehtane Mounir
Published: January 2023

Peripheral lymphocytes homeostasis is controlled by cytokine signals of which interleukin-7 (IL-7) figures most prominently. Cells deprived of IL-7 exhibit low levels of Adenosine tri-phosphate (ATP). However, little is known of the mechanism by which IL-7 regulates metabolic activity in dependent cells. We reported that shortly after IL-7 withdrawal, T-lymphocytes decreased their glucose uptake. Here we show that T-cells deprived of IL-7 decreased the gene expression and protein levels of the glycolytic enzyme, Hexokinase II (HXKII), required for generating glucose-6-phosphate. Re-addition of IL-7 to cytokine deprived T-cells restored HXKII gene expression within two hours. This increase in HXKII expression correlated with an increase in glucose uptake. In contrast, gene expression of the glucose transporter GLUT-1 increased after sixty hours of IL-7 readdition to deprived cells, and the gene expression of phospho-fructokinase (PFK) in response to IL-7 showed minimal differences. Inhibition of HXKII activity, using a specific inhibitor, Bromo-pyruvate, decreased glucose uptake in presence, but not the absence, of IL-7 while overexpression of HXKII restored glucose uptake in the absence of IL-7. In summary, we conclude that IL-7 controls glucose uptake in T-lymphocytes by regulating the gene expression of HXKII, suggesting a novel mechanism by which IL-7 supports glucose metabolism in T-cells.
Poly (I:C) induces shedding of soluble TNFR1 from human airway epithelial cells via a TLR3-RIP1-ERK signaling pathway (38.9)
Authors: Yu Man , Levine Stewart  J.
Published: January 2023

Soluble tumor necrosis factor receptors (sTNFR) modulate TNF bioactivity by binding TNF. The type 1 TNF receptor (TNFR1), a key receptor for TNF signaling, is generated by either inducible cleavage and shedding of TNFR1 ectodomains or constitutive release of TNFR1 exosome-like vesicles. The goal of this study was to identify Toll-like receptor (TLR) signaling pathways that induce shedding of sTNFR1. We demonstrated that poly (I:C), a synthetic double-strand RNA (dsRNA) analog which signals via TLR3, induced sTNFR1 shedding from human airway epithelial (NCI-H292) cells in a dose-dependent manner over 2h, whereas ligands for TLR2, TLR4, TLR7, and NOD2 (nucleotide-binding oligomerization domain containing 2) did not. RNA interference experiments showed that poly (I:C)-induced sTNFR1 shedding was mediated via TLR3 and receptor interacting protein1 (RIP1). To further define the TLR3 signaling pathways that mediate sTNFR1 release, we showed that a specific, cell-permeable peptide inhibitor of ERK activation significantly attenuated sTNFR1 release by 55%. In contrast, neither dual oxidase 1 or 2, nor reactive oxygen species (ROS) mediated poly (I:C)-triggered sTNFR1 shedding. Furthermore, the pan-caspase inhibitor z-VAD-fmk attenuated sTNFR1 shedding by 28%, which suggests that caspase-dependent pathways also participate in this process. In conclusion, we show that poly (I:C), a viral dsRNA analog, induces sTNFR1 shedding from human airway epithelial cells primarily via a TLR3-RIP1-ERK signaling pathway, with a minor contribution from caspase-dependent pathways. This identifies a mechanism by which airway epithelial cells can modulate viral-induced airway inflammation.

This work was funded by the Division of Intramural Research in NHLBI/NIH.
A2A adenosine receptor mediates suppression in a mouse model of colitis (39.1)
Authors: Kurtz Courtney C , Naganuma Makoto , Alam Mohammad S , Feldman Sanford H , Linden Joel M , Ernst Peter B
Published: January 2023

Background: Adenosine accumulates in inflamed tissue and can suppress proliferation and cytokine production in helper T cells (Th). Th cells from mice lacking the A2A Adenosine Receptor (A2AAR-/-) do not function properly in the CD45RB transfer model of colitis. Methods: To examine the role of A2AAR in regulating colitis, we tested colonic myeloperoxidase (MPO) activity after adoptive transfer of Th cell susbsets from wildtype or A2AAR-/- mice. Th cells from mesenteric lymph nodes (MLN) of colitic mice were treated with an A2AAR agonist to assess effects on cytokine production. To investigate the role of A2AAR on myeloid cells in the CD45RB model of colitis, we adoptively transferred wildtype Th cells into RAG1/A2AAR double knockout (DKO) mice. Results: Colons of mice that received A2AAR-/- CD45RBhi cells with wildtype or A2AAR-/- CD45RBlo Treg had significantly greater MPO activity than mice that received wildtype CD45RBhi and CD45RBlo cells. Cytokine production by MLN Th cells from colitic mice was suppressed by an A2AAR agonist. Adoptive transfer of CD45RBhi Th cells into either RAG1-/- or DKO mice induced colitis but co-transfer of CD45RBlo Tregs attenuated colitis only in RAG1-/- mice. Conclusions: These data indicate that adenosine signaling through A2AAR plays an important suppressive role in colitis and that this is mediated through effects on both T cells and myeloid cells. CK supported by a CCFA Fellowship.
Melanocortin 5 receptor expressed by APC mediates activation of Treg cells during experimental autoimmune uveoretinitis (166.21)
Authors: Lee Darren , Taylor Andrew
Published: January 2023

We previously showed that IRBP-specific memory Treg cells (Tregs) emerge in the spleen of experimental autoimmune uveoretinitis (EAU) recovered mice and are necessary to protect from a memory immune response to IRBP. Induction of these memory Tregs require melanocortin 5 receptor (MC5r), and APC also from EAU recovered mice. We investigated if the T cell or the APC requires MC5r in the induction of memory Tregs that emerge during EAU resolution. EAU was induced in C57BL/6 wild type and MC5r(-/-) mice with IRBP, the spleen cells were collected througout EAU to assay for IRBP stimulated T cell cytokine production of IFN-γ, IL-17, and TGF-β. We observed a MC5r dependent shift in the cytokine profile from effector cytokines, Th1 and Th17, in the beginning of EAU to regulatory. MC5r was required for a consistent increase in TGF-β production through the course of EAU, continuing through resolution. Isolated APC from the spleens of EAU-recovering mice were able to convert IRBP-specific MC5r(-/-) Th1 cells into Tregs, that are capable of suppressing EAU when adoptively transferred into mice with EAU. We have found that during the course of EAU a nonconventional APC emerges in the spleen. This APC requires expression of MC5r to promote induction of memory Tregs. Therefore, stimulation of the melanocortin pathway during EAU is required for the reestablishment of immune tolerance to ocular autoantigens.
Fever regulates inflammation through microRNAs and SOCS in pulmonary epithelial cells (166.23)
Authors: Pickering Edward , Luzina Irina , Hasday Jeffrey , Atamas Sergei
Published: January 2023

Inflammation is often accompanied by fever, which may affect patients' survival in severe acute diseases. Pulmonary epithelium is involved in lung inflammation, and a major role for microRNAs has been suggested. To determine whether febrile temperature (FT) affects the response to inflammation at the level of microRNAs, primary small airway epithelial cells were incubated at either 37 deg C or 39.5 deg C with or without 5 ng/ml TNF-α for three hours. Total RNA was isolated, labeled, and hybridized to Affymetrix microRNA arrays. After data normalization and using a 1.5 fold change as a cutoff value, we identified 59 microRNAs levels of which changed up to 8 fold in response to FT. Also, levels of 46 microRNAs changed up to 5 fold in the presence of FT plus TNF-α. Putative gene targets for these microRNAs were identified using microRNA.org, Targetscan, and Pictar databases. Many of the identified targets are involved in inflammatory pathways, including members of the suppressor of cytokine signaling (SOCS) family SOCS1 (miR-19), SOCS2 (miR-214, -139, -194), SOCS3 (miR-19, -30), SOCS4 (miR-27, -34, -125), SOCS5 (miR-18, -24, -25, -92, -130), and SOCS6 (miR-15, -17, -19, -20, -23, -27, -30, -106, -128, -130). Levels of all 10 microRNAs potentially targeting SOCS6 were decreased by both FT and TNF-α activation, with an additive effect of these two activators on miR-15, -30, and -130. Thus, innate inflammatory mechanisms involve broad microRNA-mediated regulation of SOCS by fever.
Host immunological conditions that determine status of Helicobacter pylori as pathogenic or commensal bacterium (166.26)
Authors: Wendelsdorf Katherine , Cabo Adria , Hoops Stefan , Hontecillas Raquel , Bassaganya-Riera Josep
Published: January 2023

H. pylori is a leading cause of peptic ulcer, gastric lymphoma and adenocarcinoma. In spite of being a highly prevalent bacterium in the gastric mucosa, only 15 % of carriers develop overt disease. Bacterial pathogenesis is believed to arise from an interaction of strain-specific immunity-mediating capabilities and the host susceptibility. To investigate the mechanisms of immune modulation by H. pylori we have created a mathematical model of immune cells of the gastric mucosa, gastric lymph nodes and their response to H. pylori. A local sensitivity analysis suggests that, in a healthy host, the H. pylori strains that are asymptomatically tolerated are those with the ability to invade the mucous layer as well as those that inhibit the ability of epithelial cells to secrete defensin and chemoattractants. Interestingly, our model also indicates that persistence of H. pylori in the gastric mucosa is facilitated by a strain’s ability to induce secretion of inflammatory mediators in the gastric epithelium. However, H. pylori strains that induce a tolerogenic phenotype in APCs or induce cytoskeletal changes in epithelial cells are likely to elicit a pathological inflammatory response. A global sensitivity analysis revealed novel context-dependent functional interactions occurring between the host and H. pylori.
Normal development of regulatory T cells in the post natal mouse liver requires both MyD88 and TGF-β1 (166.3)
Authors: Maria Ann , Epstein Samantha , English Kathryn , Gorham James
Published: January 2023

Regulatory T cells (Treg) play a major role in the maintenance of immune tolerance and homeostasis. The liver microenvironment promotes immune tolerance, but very little is known about the development of the Treg compartment in liver. We examined the kinetics of Treg (FoxP3+CD4+ T cells) development in the post-natal mouse liver. Liver Tregs were first detected in liver at post-natal day 2 (P2). The Treg frequency in liver rapidly surged thereafter, peaking to 20-25% of liver CD4+ T cells at P8, over twice as frequent as in spleen (10%), and declined thereafter to reach an adult frequency of ~3% CD4+ T cells. The liver receives a large supply of its blood through the portal vein, which drains the intestine. We hypothesized that the early “surge” in liver Treg frequency occurs in response to colonization of the gut by commensal bacteria. In support of this hypothesis, the surge was partially abrogated in Myd88+/- mice and completely abrogated in Myd88-/- mice. The cytokine TGF-β1 has been previously shown to be important for several aspects of the Treg response pathway; the liver Treg surge was abrogated in Tgfb1-/- mice. We conclude that the early post-natal surge of Treg in liver is dependent on both MyD88 and TGF-β1. We suggest the following model: colonization of the newborn gut drives a surge in the liver Treg compartment, likely mediated by one or more TLR pathways, and TGF-β1 is an important effector cytokine of this response.
Generation of colitogenic Th17 CD4 T cells and experimental colitis is enhanced by IL-17+ γδ T cells (166.5)
Authors: Do Jeongsu , Visperas Anabelle , Baldwin III William , Min Booki
Published: January 2023

Adoptive transfer of naïve CD4 T cells into T cell deficient mice induces severe colitis, a widely used animal model to study inflammatory bowel disease (IBD) including ulcerative colitis and Crohn’s disease. Th17 cells have been implicated in the pathogenesis of T cell-mediated colitis, although cellular mechanism(s) that supports in vivo Th17 differentiation and colitis induction remains unclear. We report here that γδ T cells play critical roles in amplifying Th17 differentiation and subsequent development of colitis in mice. Following naïve CD4 T cell transfer, TCRβ-/- mice developed severe colitis with enhanced Th17 differentiation, while TCRβ/δ-/- mice showed no sign of weight loss with very mild colitis. Th17 differentiation in TCRβ/δ-/- recipients was significantly diminished compared to that in TCRβ-/- recipients. Adoptive transfer of CD4 T cells with γδ T cells fully restored colitis in TCRβ/δ-/- mice and Th17 differentiation was accordingly elevated. Surprisingly, IL-17 production by γδ T cells was found to be very important for colitogenic effect because cotransfer of CCR6+ (IL-17+) γδ T cells was sufficient to promote Th17 differentiation and colitis yet transfer of CCR6- (IL-17-) γδ T cells failed to do so. Collectively, our results demonstrate that IL-17 production by γδ T cells play a critical role in fostering in vivo Th17 differentiation and inducing colitis, implying that targeting these cells might be beneficial to treat the disease.
Epidermal ADAM17 provides barrier immunity and prevents myeloproliferative disease by regulating Notch-AP1 interaction (166.7)
Authors: Murthy Aditya , Shao Yang Washington , Guinea-Viniegra Juan , Narala Swami , Zúñiga-Pflücker Juan Carlos , Wagner Erwin , Khokha Rama
Published: January 2023

Epithelial cells are the major component of mucosal tissues such as the skin, gut and lungs. They are critical barriers against environmental stress factors which induce significant immunopathology. Keratinocytes in the skin may act as key decision-makers of immune cell function by directing local adaptive immune responses and hematopoietic development, however mechanisms driving epithelial:immune crosstalk are incompletely understood. The cell surface sheddase A Disintegrin and Metalloproteinase 17 (ADAM17) cleaves numerous proteins with key roles in development, stress response and inflammation. Here we show that loss of epidermal Adam17 leads to severe atopic dermatitis and myeloproliferative disease. ADAM17 deficiency in keratinocytes enhanced AP1 signaling, increasing the production of epithelial cytokines (e.g. TSLP, IL-33) to drive Th2-polarized lymphocyte activation. We also observed the release of myeloid growth factors (e.g. G-CSF), causal to myeloproliferative disease in these mice. Next, we identified that ADAM17 promotes ligand-independent Notch activation in keratinocytes, revealing a novel mechanism of Notch-AP1 interaction that abrogates stress signaling. Notably, ectopic Notch activation in the epidermis of ADAM17 deficient mice rescued local inflammation and myeloproliferation. Thus Notch activation by ADAM17 in keratinocytes antagonizes AP1-induced inflammation, thereby establishing a mechanism of epithelial:immune crosstalk.
Synovial fluid cells in proteoglycan-induced arthritis contain a myeloid-derived suppressor population (167.21)
Authors: Egelston Colt , Besenyei Timea , Tryniszewska Beata , Rauch Tibor , Glant Tibor , Mikecz Katalin
Published: January 2023

Joint damage in rheumatoid arthritis (RA) is largely mediated by synovial fluid (SF) neutrophils. In proteoglycan (PG)-induced arthritis (PGIA), an autoimmune mouse model of RA, SF cells show high expression of the myeloid lineage markers CD11b and Gr-1. The effects of this neutrophil-like SF cell population on the activation of dendritic cells (DCs) or T cells have not been studied in RA or its animal models. We co-cultured SF cells of PGIA mice with bone marrow-derived DCs, and found that levels of MHC II and CD86 expression by DCs decreased significantly in the presences of SF cells. To determine if SF cells also reduced the antigen (Ag)-presenting capacity of the DCs, we added naïve PG-specific TCR transgenic T cells and cognate Ag to the co-cultures. T cells cultured in the presence of SF cells and Ag-loaded DCs showed significant decreases in CD69 expression and proliferation compared to T cells cultured with Ag-loaded DCs only. Arginase-1, a marker of myeloid-derived suppressor cells (MDSCs) was upregulated at both transcriptional and protein levels in SF cells. Expression and activity of arginase-1 were also elevated in spleen cells of arthritic mice as compared to spleen cells of naïve non-arthritic mice. Our results suggest that SF cells in the arthritic joints and spleens of mice with PGIA contain a population of MDSCs, which might have the capacity to suppress autoreactive T cell responses and thus accelerate the resolution of joint inflammation.