bluuebunny/crossref_metadata_2025 huggingface dataset:
BCR-induced actin rearrangement provides a driving force for the formation of surface BCR signalosomes (84.1)
Authors: Song Wenxia , Liu Chaohong , Miller Heather , Orlowski Gregory , Sharma Shruti , Lam Hui-King , Grooman Brian , Upadhyaya Arpita
Published: January 2023

In response to antigens, the BCR forms surface signalosomes where it initiates signaling cascades and antigen internalization. This study examines the role of the actin cytoskeleton in BCR signalosome formation. Both multi-valent soluble and membrane-associated antigens induce BCR clustering and actin reorganization. Actin is actively polymerized at BCR microclusters upon their formation and surrounding BCR clusters as they are merged into central clusters. Actin regulators, WASP, cofilin and gelsolin, are activated and recruited to BCR clusters. Latrunculin or jasplakinolide treatment, which blocks BCR-induced actin reorganization by depolymerizing or stabilizing F-actin, inhibits surface BCR clustering and B cell spreading. Jasplakinolide blocks antigen-triggered tyrosine phosphorylation. In contract, latrunculin alone, in the absence of antigenic stimulation, induces BCR microclustering and tyrosine phosphorylation, however, in a much slower kinetics than those induced by antigens. Btk deficiency inhibits not only antigen-induced actin polymerization, but also antigen-induced B cell spreading and BCR clustering. These results indicate that BCR-induced, Btk-dependent actin reorganization, consisting of temporospatially regulated actin polymerization and depolymerization, promotes the formation of surface BCR signalosomes by increasing the speed and the extend of BCR engagement and clustering. Our study reveals an actin-mediated positive feedback mechanism for BCR signaling.
Mice lacking IgA exhibit defective IgG antibody responses to polysaccharide conjugate vaccine (84.14)
Authors: Wilson-Welder Jennifer , Kirimanjeswara Girish , Metzger Dennis
Published: January 2023

Streptococcus pneumoniae is a leading cause of global disease. While capsular polysaccharide conjugate vaccines have decreased disease burden; a portion of the population remains unresponsive to vaccination. In order to understand the mechanisms responsible vaccine non-responsiveness, studies have been performed using mice with a targeted gene deletion that causes lack of IgA but few alterations in expression of other Ig isotypes. Surprisingly, IgA-/- mice were found nonetheless to exhibit specific defects in both IgA and IgG antibody responses to the polysaccharide (PS) component of pneumococcal conjugate vaccine. Neither IgM anti-PS responses nor IgG antibody responses to the protein component of the conjugate vaccine were affected. Furthermore, IgA-/- mice expressed fewer peritoneal B1a cells. As B1a and marginal zone B cells are considered to be the primary cell types that respond to polysaccharide antigens, this might to some extent explain the decreased anti-PS serum antibody titers seen in IgA-/- mice. Studies using bone marrow chimeras showed that reconstitution of IgA-/- mice with IgA+/+ B cells failed to restore antibody responsiveness to the PS component of the conjugate vaccine. Taken together, these results indicate that a defect in IgA expression affects B cell homeostasis, which is partially influenced by the environment that is present during B cell activation. (Supported by NIH grants RO1 41715 and R21 83878).
Anti-CD20 therapy alters immune tolerance by disrupting crosstalk between B cells and FoxP3+ Tregs (84.20)
Authors: Ray Avijit , Dittel Bonnie
Published: January 2023

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) thought to be due to dysregulation in immune responses. To study the regulatory role of B cells in CNS autoimmunity, we used the animal model of MS, experimental autoimmune encephalomyelitis (EAE), in which we demonstrated a regulatory role for B cells in the resolution of EAE using B cell deficient (μMT) mice. FoxP3+ T regulatory (Treg) cells have also been shown to suppress EAE. We have demonstrated a link between B cells expressing B7 and the presence of FoxP3+ Tregs in the CNS of mice with EAE, suggesting crosstalk between B cells and Tregs. To further study this crosstalk, we performed anti-CD20 depletion of B cells. Following B cell depletion, mice exhibited chronic disease as in μMT, but partial depletion of B cells resulted in resolution of EAE. We then investigated the role of B cells in the development, peripheral maintenance and function of Tregs. B cell depletion had no effect on Treg development or function. However, a significant reduction in the number of Tregs paralleled total B cell loss, whereas with partial B cell depletion Tregs were unaffected. Resolution of EAE and partial restoration of Treg numbers following B cell reconstitution provided further evidence of the importance of crosstalk between B cells and Tregs in immune regulation. These data suggest a role for B cells in regulating immune tolerance and controlling CNS autoimmunity by maintaining the number of Tregs.
GL7 antigen is expressed spatio-temporally selectively in rodent and human lymphocytes (84.21)
Authors: Laszlo Gloria , Balogh Andrea , Adori Monika , Matko Janos
Published: January 2023

GL7 was first described as a late activation antigen in mouse, expressed by pre- and immature B-cells, certain thymocytes, and used since as marker of germinal centers. Later GL7 was also found on some human cell lines and sialic acid residues (Sia) were identified as the critical part of the epitope. Enzymes involved in glycosylation show considerable differences between human and mouse and the specificity of some Sia-binding siglecs also vary between species. Here we investigated GL7 expression and membrane localization in rodent and human immunocytes and lymphoid tissues. Mouse and human cell lines, primary (resting and activated) mouse, rat and human lymphocytes were assayed with flow cytometry and confocal microscopy. Our results show that the GL7 epitope expression pattern is similar in rats and mice. In contrast, human lymphocytes selectively and constitutively express GL7, and its expression level can further increase or decrease upon activation of T and B lymphocytes, respectively. The correlation pattern between GL7 expression and binding of various lectins to the surface immunocytes further confirms that the α2,6-linked Neu5Ac is involved in the GL7 epitope in human, as well. The expressed GL7 epitope (possibly attached to protein or lipid molecules) is highly raft-associated in lymphoid cells. This and its spatio-temporally selective expression suggest possible regulatory or adhesion functions for the GL7 epitope-linked molecule(s) in human.
Inhibition of cyclooxygenase-2 activity impairs antibody production in human B cells and in mice infected with vaccinia virus (84.8)
Authors: Phipps Richard , Bancos Simona , Topham David , Chapman Timothy , Bernard Matthew
Published: January 2023

Generation of protective humoral immune responses against vaccines depends on optimal production of antibodies by B lymphocytes. Non-steroidal anti-inflammatory drugs (NSAIDs) are used to curb undesirable symptoms of vaccination, including pain, fever and swelling. We previously demonstrated in B cells that cyclooxygenase-2 (Cox-2) is induced in response to both T-dependent and T-independent stimuli, including the TLR9 ligand CpG DNA, and that Cox-2 is crucial for antibody production. Expression of plasma cell transcriptional regulatory factors, including Blimp-1 and Xbp-1, were dependent on Cox-2 activity. Significantly fewer antibody secreting plasma cell precursors were generated in the presence of Cox-2 selective inhibitors, indicating that Cox-2 is essential for human B cell terminal differentiation. Cox-2 selective inhibitors were administered in vivo to determine the role of Cox-2 in a humoral immune response to live infection with vaccinia virus. Mice chronically treated with Cox-2 selective inhibitors produced less viral neutralizing IgG following live virus infection. Inhibition of Cox-2 resulted in a reduced frequency of IFN-γ producing T cells, indicating that T cell priming of B cells was attenuated. These data support the idea that impaired humoral immunity in the absence of Cox-2 activity can be dependent on both B cells and T cells and that the use of Cox-2 selective inhibitors have the potential to dampen humoral immunity to live virus infection and vaccines.
Loss of miR-181a-mediated DUSP6 repression increases the TCR threshold of naive CD4 T cells in the elderly (85.11)
Authors: Li Guangjin , Yu Mingcan , Lee Won-Woo , Weyand Cornelia , Goronzy Jorg
Published: January 2023

With increasing age, the ability of the immune system to respond to vaccination with a CD4 T cell response and the production of antibodies declines. The decline is only partially explained by a loss of naïve and central memory CD4 T cells due to thymic involution. Naïve CD4 T cells are present in sufficient, albeit compared to young adults reduced frequencies in most elderly suggesting that additional functional defects are important. In these studies, we compared naïve CD45RO- CD4+ T cells from 20 to 35 (n=117) and 70 to 85 (n=80) year-old healthy adults. Elderly T cells were less responsive to superantigen/dendritic cell (p<0.001) stimulation. Signaling studies by PhosFlow showed reduced ERK (p<0.0001), but normal ZAP70 phosphorylation in the first 5 minutes after stimulation, suggesting a selective age-related defect in the ERK pathway. In murine systems, DUSP6 has been implicated in differentiation-dependent TCR threshold calibration. In Western blot studies, DUSP6 protein was increased in elderly CD4 T cells (p=0.02), however, DUSP6 transcription was not different suggesting a defect in posttranscriptional regulation. Expression of miR-181a that binds to the DUSP6 3′UTR was decreased in elderly naïve CD4 T cells. Overexpression of miR-181a downregulated DUSP6 expression and restored ERK responses in elderly CD4 T cells. Our data suggest that loss of miR181a in naïve CD4 T cells increases the TCR threshold and contributes the immune defect in the elderly.
Selective regulation of glucocorticoid receptor translational isoforms in T cells and B cells (85.7)
Authors: Shin Soon Cheon , Schleimer Robert , Lu Nick
Published: January 2023

The glucocorticoid receptor (GR) has eight translational isoforms, each of which has a distinct ability to regulate gene expression and to mediate apoptosis. Little is known, however, about whether cell activation or differentiation alters the GR isoforms. We determined the pattern of GR isoform expression in purified human lymphocytes at rest and after activation with mitogens. Stimulation of T cells with ConA led to a slight increase in GR-A and induced a 3-5-fold increase in GR-C. In contrast, activation of B cells with CpG containing DNA did not alter the expression of GR isoforms. To determine the functional consequences of elevated GR-C in activated T cells, we screened the results of microarray studies of Jurkat cells expressing only single GR isoforms to search for genes that were selectively induced by glucocorticoids in cells expressing GR-C but not the other isoforms. We found seven genes that were selectively activated by dexamethasone (DEX) in GR-C-expressing cells and tested the influence of T cell activation on the ability of DEX to activate the expression of these genes. Two of the tested genes, AIM1 and TUBA3E, were significantly increased by DEX only in activated T cells, suggesting that induction of the GR-C isoform had enabled the glucocorticoid to alter the expression of these genes. These results suggest that activation of immune cells can lead to alterations in the expression of GR isoforms that modify the glucocorticoid-induced gene expression.
Promiscuous processing of human α/β-protryptases (86.13)
Authors: Le Quang , Gomez Gregorio , Zhao Wei , Min Hae-Ki , Fukuoka Yoshihiro , Katunuma Nobuhiko , Schwartz Lawrence
Published: January 2023

Human β-tryptase is stored in secretory granules of human mast cells as a heparin-stabilized homotetramer. Although β-protryptase is sequentially processed in vitro by autocatalysis at Arg-3 followed by cathepsin (CTS)-C proteolysis to the mature enzyme, CTSC-deficient mice effectively process the murine pro form of mast cell protease-6, a mouse tryptase. Tryptase processing activity(ies) distinct from CTSC were recognized in and partially purified from human HMC-1 cell extracts by ion exchange and gel filtration chromatography, and identified by LC-tandem mass spectroscopy to include CTSB and CTSL. Importantly, CTSB and CTSL could process α-protryptase (Q-3) and mutated β-protryptase (R-3Q) as well as β-protryptase, indicating no need for autocatalytic cleavage at Arg-3, whereas CTSC-dependent processing required the autocatalytic step. Heparin was required for processing and for tryptase tetramer formation, and also protected tryptase from degradation by CTSB and CTSL. Processed protryptases were tetrameric by gel filtration and mature by N-terminal sequence analysis. CTSL, CTSB and β-tryptase were co-localized to skin mast cell secretory granules based on their co-release upon degranulation, indicating that they traffic together within the mast cells. Thus, CTSL and CTSB may be central to the processing of both α and β protryptases in mast cells of human and perhaps other species, and are potential targets for attenuating production of mature tryptase.
TRPM7 kinase domain confers magnesium sensitivity to G-protein regulated mast cell degranulation (86.14)
Authors: Zierler Susanna , Ryazanova Lillia , Ryazanov Alexey , Penner Reinhold , Fleig Andrea
Published: January 2023

TRPM7 is a bi-functional protein where an ion channel domain is fused with an atypical α-kinase domain. Impairment of the kinase domain leads to hypomagnesemia and sensitization to allergic reactions. We here investigate whether TRPM7 is involved in receptor-stimulated peritoneal mast cell degranulation and histamine release. Using the whole-cell patch clamp technique, we find that mimicking hypomagnesemic conditions by removal of extracellular Mg2+ shifts the degranulation response to intracellular Ca2+ from a graded to an all-or-none process independent of functional TRPM7 activity. In contrast, G-protein-induced mast cell degranulation requires higher intracellular Ca2+ levels in cells isolated from a mouse heterozygotic for a TRPM7 kinase domain knock-out (dKhet). Removal of extracellular Mg2+ compensates for this effect by both significantly increasing the degranulation amplitude and accelerating the rate of secretion in dKhet but not wild type (wt) cells. These observations are confirmed in a histamine release assay, where G-protein induced histamine secretion using MIP-1α is greatly enhanced in dKhet mast cells under hypomagensemic conditions, whereas Ca2+-induced degranulation by FcϵRI crosslinking is similar compared to wt. We conclude that Ca2+-induced mast cell activation is sensitive to extracellular Mg2+ concentrations independent of TRPM7 and that the TRPM7 kinase domain, but not the channel domain, is involved in G-protein-regulated degranulation.
Basophils are transiently recruited into the draining lymph nodes during helminth infection via IL-3 but infection-induced Th2 immunity can develop without basophil lymph node recruitment or IL-3 (86.19)
Authors: Min Booki , Kim Sohee , Prout Melanie , Ramshaw Hayley , Lopez Angel , Le Gros Graham
Published: January 2023

Basophils are recognized as immune modulators through their ability to produce IL-4, a key cytokine required for Th2 immunity. It has also recently been reported that basophils are transiently recruited into the draining LN after allergen immunization and that the recruited basophils promote the differentiation of naïve CD4 T cells into Th2 effector cells. Likewise, basophils were recruited into the draining mediastinal and mesenteric LN at 3 and 10 days after infection with intestinal nematode Nippostrongylus brasiliensis, respectively. However, the recruitment was completely abolished in mice deficient in IL-3 or IL-3Rβ, suggesting that the IL-3/IL-3R system is absolutely required. Activated CD4 T cells appeared to be the primary source of IL-3. Experiments using bone marrow chimera strategy further revealed that the cell types expressing IL-3Rβ are of bone marrow origin. Unexpectedly, the lack of IL-3 or of basophil LN recruitment played little role in N. brasiliensis-induced Th2 immune responses as unaltered Th2 immune responses were observed in N. brasiliensis infected IL-3-/- mice. Consistent with these findings, basophil depletion in infected mice did not diminish the development of IL-4-producing CD4 T cells. Therefore, our results reveal a previously unknown role of IL-3 in recruiting basophils to the LN and demonstrate that basophils are not necessarily associated with the development of Th2 immunity during parasite infection.
Mast cells are initiators of inflammatory pain in thermal pain in mice. (86.2)
Authors: Chatterjea Devavani , Engblom Camilla , Mora-Solano Carolina , Maitland-McKinley Paul , Allen Juliann
Published: January 2023

Pain is a cardinal component of inflammation and a significant public health issue. Cytokines such as TNF-alpha, IL-1beta, IL-15 and CXCL1/IL-8, as well as neutrophil influx have been implicated in the potentiation of peripheral inflammatory pain in models of thermal and mechanical hindpaw hypernociception in rodents. These lines of evidence point to sentinel mast cells in the tissues as possible regulators of these pain responses. Degranulation of mast cells has been shown to be important for phosphorylation of Erk in nociceptive neurons in a rat model of migraine-like stimulation. Mechanical bladder pain in a model of interstitial cystitis was abrogated in mast cell deficient mice. We show that c4880-mediated degranulation of mast cells causes pain as well as significant edema and upregulation of local TNF-alpha and IL-1beta levels in the hindpaws of mice in a model of thermal pain. Mast cell-deficient Wsh mice show abrogation of this pain response. IgE/Ag mediated degranulation of mast cells in the paws of WT mice also causes pain and edema but it is not accompanied by detectable upregulation of TNF-alpha in the paws. This suggests that mast cells may be necessary for the initiation of inflammatory pain but that the underlying mechanisms of potentially protective inflammatory pain vs. potentially pathological allergic pain may be different.
Biomarkers of IgE- and IgG-dependent anaphylaxis (86.20)
Authors: Khodoun Marat , Strait Richard , Armstrong Laura , Yanase Noriko , Perkins Charles , Potter Crystal , Herbert De’Broski , Finkelman Fred
Published: January 2023

Anaphylaxis is classically induced by IgE-dependent FceRI crosslinking that triggers mast cell and basophil mediator, enzyme and cytokine secretion. Anaphylaxis can also be induced in mice, however, by IgG- mediated crosslinking of macrophage and basophil FcgRIII. This alternative pathway may be responsible for some cases of human anaphylaxis, particularly those associated with protein-based drugs, including humanized mAbs. We used mouse passive anaphylaxis models to attempt to identify markers that could distinguish between IgE- and IgG-mediated anaphylaxis. Mice sensitized by i.v. injection of TNP-specific IgE or IgG mAbs were challenged i.v. 24h later with TNP-OVA; changes in cells and serum were then evaluated. Changes 2h post-challenge in both IgE- and IgG-mediated anaphylaxis included: 1) up to 90% decreases in percentages of basophils and monocytes in blood; 2) doubling of the percentage of neutrophils in blood; and 3) ~25% increases in the ability of neutrophils to generate reactive oxygen intermediates and mobilize Ca++. IgE-, but not IgG-mediated anaphylaxis induced greatly increased serum levels of IL-4 and MMCP1 and an ~30% increase in T cell membrane IL-4Ralpha 4 h post-challenge, while IgG-, but not IgE-dependent anaphylaxis decreased neutrophil membrane FcgRIIb/RIII expression by ~30% 2-12 h post-challenge. Studies will be performed to determine if these cellular markers can distinguish between IgE-dependent and putative IgG-dependent anaphylaxis in humans.
Novel G-Protein Coupled Receptors in the Activation of Human Mast Cells by C3a-Related Peptides (86.23)
Authors: Kashem Sakeen , Subramanian Hariharan , Magotti Paola , Lambris John , Ali Hydar
Published: January 2023

Studies with animal models have shown that complement components C3a and C5a play an important role in the effector phase of allergic asthma. We have previously shown that C3a activates human CD34+-derived mast cells endogenously expressing G protein coupled receptors (GPCR) for C3a (C3aR) as well as RBL-2H3 cells stably expressing C3aR. The purpose of this study was to utilize peptide C3a receptor agonists to further characterize the role of C3aR on mast cell activation. We found that C3a (a 74 amino acid peptide) and a C3aR peptide superagonist (H-WWGKKYRASKLGLAR-OH) activated human mast cells with similar potency. Surprisingly, a scrambled C3aR superagonist peptide also caused mast cell degranulation. RT-PCR analysis demonstrated that all four known human Mas-related genes, MrgX1, MrgX2, MrgX3, and MrgX4 are expressed in human mast cells. RBL-2H3 cells stably expressing MrgX2 responded to its known ligand, cortistatin-14 for sustained Ca2+ mobilization and degranulation. Furthermore, C3aR superagonist peptide, scrambled C3aR superagonist peptide and substance P induced degranulation in RBL-2H3 cells expressing MrgX2 but not in untransfected cells. These findings suggest that Mrg receptors may play a novel role in activation of mast cell by complement-derived and other positively charged peptides and could serve as therapeutic targets for mast cell-dependent allergic and inflammatory diseases.
TGFb1 and IL-4 Provide Antagonistic Control of the Mast Cell Response (86.5)
Authors: Ryan John , Morales Johanna , Macey Matthew
Published: January 2023

Cytokine networking is an important concept for understanding how the immune system is controlled. We hypothesized that the initial presence of pro-inflammatory cytokines could desensitize mast cells to anti-inflammatory signals, rendering them more likely to be activated in allergic reactions. Conversely, the initial presence of anti-inflammatory cytokines might suppress the effects of pro-inflammatory cytokines in allergic type responses. In this study, we show that IL-4 and TGFβ1 act as an antagonistic pair in controlling the mast cell response. Mast cell pre-culture with IL-4 decreased TGFβR1 and TGFβR2 RNA and protein levels through a STAT6-dependent mechanism. Further, IL-4 diminished TGFβ1 signaling, blocking SMAD2 phosphorylation. IL-4 also blocked TGFβ1-mediated mast cell death and suppressed TGFβ1-mediated mast cell migration. Pre-culture with TGFβ1 mirrored this antagonistic effect. TGFβ1 pre-culture reduced IL-4R protein levels, suppressed IL-4-mediated Stat6 phosphorylation, and diminished mast cell migration toward IL-4. When both are present simultaneously, TGFβ1 appears to have a dominating inhibitory effect over IL-4 by blocking proliferation in vitro and macrophage recruitment in vivo. We obtained similar results using human mast cells. These data are important to understanding how cytokine networks can impact mast-cell mediated diseases like asthma. Supported by NIH grants 1R01 AI59638 and U19A1077435.
Eosinophils regulate immunity during chronic infection by Trichinella spiralis (87.14)
Authors: Gebreselassie Nebiat , Fabre Valeria , Bliss Susan , Gagliardo Lucille , Lee Nancy , Lee James , Appleton Judith
Published: January 2023

Eosinophilia is a hallmark of worm infection and allergy. The parasitic nematode Trichinella spiralis establishes chronic infection in skeletal muscle. We have shown previously that in eosinophil-ablated, transgenic mice, T. spiralis larvae die in large numbers in the muscle. Death is promoted by inducible nitric oxide synthase (iNOS). We report here that transcription of iNOS was upregulated in the context of an established alternative activation phenotype in local leukocytes. Infiltrating macrophages, dendritic cells, and neutrophils produced iNOS. These changes were preceded by a reduced T cell recruitment to infected muscle that was coincident with a lag in larval growth, suggesting compromise of the parasite’s habitat. The results further define the cellular interactions that occur at a site of chronic nematode infection in which the eosinophil functions as a pivotal regulator of immunity.
Parthenolide, an anti-inflammatory NFκB inhibitor, stimulates platelet production and blunts platelet activation (87.17)
Authors: Sahler Julie , Bernard Jamie , Spinelli Sherry , Casey Ann , Blumberg Neil , Phipps Richard
Published: January 2023

Platelets play a significant role in hemostasis, and are critically involved in common medical complications including thrombocytopenia, ischemia, and atherosclerosis. Platelets can also directly induce inflammation and influence immune cells by releasing inflammatory mediators such as CD40 Ligand, TGF-β, PGE2, IL-1β, and MIP-1α. Despite these recent findings, platelet signaling pathways remain unclear and are under current investigation. Recently, our lab identified the presence of NFκB transcription factors in platelets. Because platelets are anucleate, we hypothesize an alternative role for NFκB in platelet function. To further study the role of NFκB in platelets, we tested the effects of an NFκB inhibitor, Parthenolide, on human platelets and Meg-01 cells, a human megakaryoblastic cell line. Parthenolide is an extract of the Feverfew plant used in herbal therapies to dampen inflammation. We found that Parthenolide directly influenced human platelets by diminishing clot retraction and spreading. In addition, we found that Parthenolide increased platelet production, increased heme oxygenase-1 (up to 2000-fold), and increased glutathione levels over time (p=0.02) in Meg-01 cells. These data suggest that the anti-inflammatory properties of Parthenolide may be partially due to its ability to suppress platelet activation and up-regulate anti-oxidant pathways.
Ptaquiloside-induced immunotoxic effects are reversed by selenium supplementation (87.21)
Authors: Latorre Andreia , Caniceiro Beatriz , Wysocki Harry , Haraguchi Mitsue , Gardner Dale , Górniak Silvana
Published: January 2023

Pteridium aquilinum (Pa) is a poisonous plant and its ingestion has been closely associated with human and animal cancers. Our earlier studies have shown that Pa reduces white pulp of spleen and NK cell cytotoxicity in mice. In addition, we observed that selenium (Se) reversed Pa-induced spleen damage. In this study the aim was evaluate if ptaquiloside (Ptq), the main toxic principle found in Pa, is responsible for its immunotoxic effects and if Se would reverse these effects. For in vivo study, mice were separated into 4 groups: control, Ptq, Ptq+Se, and Se. Mice were treated by gavage for 14 days with Ptq (5.3mg/kg BW) and/or Se (1.3mg/kg BW). Mice were killed and it was prepared a non-adherent splenic cells suspensions to perform NK cell activity assay against YAC cell line. For in vitro study, cultures of non-adherent splenic cells were performed for each mouse as follows: untreated, Se 0.1mM, RPMI extract of Pa (RE) 4mg/ml, RE 4mg/ml+Se 0.1mM (co-incubation), and RE 4mg/ml+Se 0.1mM (RE/1h wash and then Se/1h). After treatment, the cells were washed to proceed both NK cell activity assay and MTT assay. We observed that Ptq reduced NK cytotoxicity, which permits to suppose that Ptq is the principle responsible by immunotoxic effects. Moreover, NK cell activity was completely recovered after Se supplementation in vivo and in vitro. These results showed for the first time that Pa-induced immunotoxic effects are caused by Ptq and that these effects are fully recovered by Se.
Involvement of estrogen receptor alpha on MCP1 production in C57BL/6 kidney mesangial cells (87.29)
Authors: Dasgupta Subhajit , Eudaly Jackie , Molano Ivan , Baker DeAnna , Mollenhauer Meagan , Gilkeson Gary
Published: January 2023

Abstract We demonstrate an estrogen independent involvement of ERα in TLR2 mediated expression of MCP1 in mesangial cells. In vitro treatment with the TLR2 ligands Pam3CysSK4, LTA and PGN-SA induced production of MCP1 in mesangial cells isolated from kidneys of female C57BL/6 mice in a dose dependent manner. Treatment with LTA to C57BL/6 mesangial cells was found to activate NF kB and induced its nuclear localization. Interestingly enough, we found LTA treatment induced phosphorylation of ERα in primary mesangial cells and also in SV40 virus transformed B6 mesangial cell line. Nuclear translocation of pERα (Ser118) was found in the LTA treated B6 mesangial cells. ERα selective antagonist MPP and NF kB antagonist TPCA1 attenuated MCP1 production in TLR2 ligand treated mesangial cells suggesting influence of pERα in NF kB dependent MCP1 gene expression. The LTA was found to induce DNA binding activity of NF kB in B6 mesangial cell line in a time dependent manner. Transient transfection with ERα siRNA was also found to reduce expression of MCP1 in Pam3CysSK4 treated mesangial cells. Taken together, our study indicates an unique role of ERα in modulation of TLR2-mediated signals in mesangial cells. Acknowledgement: We are thankful to the Department of Medicine, Division of Rheumatology/Immunology and Ralph V Johnson VA research service, MUSC. The research support was provided by VA research service. We do not have any conflict of interests.
IL-17 plays differential role in primary verse memory immune responses and recognition of cellular verse soluble antigens (87.30)
Authors: Guan Hongbing , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

IL-17 is a proinflammatory cytokine that has been linked to many autoimmune related diseases such as rheumatoid arthritis, asthma, lupus, and multiple sclerosis. Recent studies have indicated that IL-17 may also play central role in driving the development of germinal center-derived autoantibody and Th response in an autoimmune disease model setting. In addition, activated and memory T cells can both produce IL-17. In this report, we found that IL-17 differentially affected primary versus memory immune response to cellular versus soluble antigen. Briefly, we initiated primary or memory immune response with SRBC or OVA and blocked IL-17 using anti-IL-17 antibody (Amgen). We found that blockade of IL-17 did not affect OVA-induced primary or memory response, nor did it affect SRBC-induced memory response; however, the blockade inhibited SRBC-induced primary response. SRBC-induced hemagglutination, DTH and specific T cell proliferation were inhibited; serum IFN-gamma, IL-12 and SRBC-specific IgG1 were decreased; numbers of germinal centers and IgG1-producing B cells in the germinal center were decreased; IL-17R knockout-CD4 T cells showed a decreased expression of BTLA and ICOS; however, percentages of CD4, CD8, CD3 and CD19 subsets in the spleen did not change with the antibody blockade. Our report suggests a differential role of IL-17 on T-B, T-DC cell interaction or Ig class switch in response to cellular versus soluble antigens, and primary versus memory immune response.
Complement Factor H regulation in the central nervous system (87.5)
Authors: Fraczek Laura , Martin Carol , Martin Brian
Published: January 2023

The complement system is a critical component of innate immunity that requires regulation to avoid inappropriate activation. This regulation is provided by many proteins including complement Factor H (CFH), a secreted protein of the alternative pathway. Despite its regulatory function, gene and promoter polymorphisms of CFH have been implicated in patients with age-related macular degeneration and atypical hemolytic uremic syndrome, indicating that a better understanding of this gene’s regulation is valuable. Presently, little work has been done to address the role of CFH transcriptional control in the central nervous system (CNS). We have cloned the murine CFH promoter and have made truncation constructs to examine CFH gene regulation in cells of the CNS. Our studies show that CFH mRNA is present in several CNS cell types, and that specific regions throughout the promoter contain enhancers and repressors that are positively and negatively regulated by inflammatory cytokines. Database mining of these regions indicates that there are potential transcription factor binding sites conserved between different species, which led us to investigate specific transcription factor binding interactions in these regions. Through these studies we hope to elucidate the transcriptional regulation of CFH in the CNS.
Regulation of CD20 in rituximab-resistant cell lines (RRCL) and B-cell non-Hodgkin’s lymphomas (NHL) (88.18)
Authors: Tsai Ping-Chiao , Naveen Bangia , Olejniczak Scott , Hernandez-Ilizaliturri Francisco , Czuczman Myron
Published: January 2023

Rituximab(R) resistance is associated with low CD20 expression in relapsed/refractory (r/r) lymphomas. Studies had shown a direct correlation between CD20 levels and R-induced complement cell lysis. CD20 up-regulation by IL-4 serves as a useful tool to study regulatory mechanisms of CD20 expression. We demonstrated that chronic exposure of B-cell to R leads to decrease CD20 at mRNA and protein levels. Our current work defines regulatory mechanisms of CD20 expression in RRCL and evaluates the role of CD20 in R activity. Differences in CD20 mRNA stability and promoter activity were determined between RSCL and RRCL by Northern blot and luciferase reporter assays. Levels of CD20 regulatory factors were determined by Western Blot. IL-4 effects in CD20 promoter activity, CD20 surface expression and R activity was studied in RSCL, RRCL and in tumor cells derived from r/r NHL. No difference in CD20 mRNA stability was noted between RSCL and RRCL. CD20 promoter activity was lower in RRCL. Detailed analysis of different CD20 promoter fragments indicated that low CD20 promoter activity occurred within the first 300 base pairs, suggesting lack of positive regulatory factors. Exposure to IL-4 increased CD20 promoter activity, CD20 expression and R activity. Our data suggest that CD20 promoter activity plays a role in CD20 down-regulation in RRCL. We are currently studying differences in PU.1 or Oct-1 binding to CD20 promoter in an attempt to define their role, if any, in R-resistance
Innate production of Th2 cytokines by adipose tissue-associated c-Kit+Sca-1+ lymphoid cells (89.11)
Authors: Moro Kazuyo , Koyasu Shigeo
Published: January 2023

Innate immune responses are important in combating various microbes during the early phases of infection. NK cells are innate lymphocytes that, unlike T and B lymphocytes, do not express antigen receptors but rapidly exhibit cytotoxic activities against virus-infected cells and produce various cytokines. Here we report a new type of innate lymphocyte present in a novel lymphoid structure associated with adipose tissues in the peritoneal cavity. These cells do not express lineage (Lin) markers but do express c-Kit, Sca-1, IL7R and IL33R. Similar lymphoid clusters were found in both human and mouse mesentery and we term this tissue ‘FALC’ (fat-associated lymphoid cluster). FALC Lin-c-Kit+Sca-1+ cells are distinct from lymphoid progenitors and lymphoid tissue inducer cells. These cells proliferate in response to IL2 and produce large amounts of Th2 cytokines such as IL5, IL6 and IL13. IL5 and IL6 regulate B-cell antibody production and self-renewal of B1 cells. Indeed, FALC Lin-c-Kit+Sca-1+ cells support the self-renewal of B1 cells and enhance IgA production. After helminth infection and in response to IL33, FALC Lin-c-Kit+Sca-1+ cells produce large amounts of IL13, which leads to goblet cell hyperplasia—a critical step for helminth expulsion. In mice devoid of FALC Lin-c-Kit+Sca-1+ cells, such goblet cell hyperplasia was not induced. Thus, FALC Lin-c-Kit+Sca-1+ cells are Th2-type innate lymphocytes, and we propose that these cells be called ‘natural helper cells’.
Synergistic Signals for Natural Cytotoxicity Are Required to Overcome Inhibition by c-Cbl (89.16)
Authors: Kim Hun Sik , Das Asmita , Gross Catharina , Bryceson Yenan , Long Eric
Published: January 2023

Natural killer (NK) cells do not express variable antigen-specific receptors but are activated by combinations of germ-line encoded receptors. How activation is achieved and controlled during encounter of NK cells with target cells is unclear. We showed here that co-engagement of receptors NKG2D and 2B4 triggered synergistic activation of PLC-γ2, degranulation and cytotoxicity, which were controlled positively by Vav1 and negatively by the E3 ubiquitin ligase c-Cbl. Knockdown of c-Cbl blocked ubiquitylation of Vav1 and bypassed the requirement for synergy, as NKG2D and 2B4 could induce degranulation on their own. The enhanced degranulation obtained after c-Cbl knockdown was still Vav1-dependent. Inhibition of cytotoxicity through binding of CD94-NKG2A to HLA-E on target cells was independent of inhibition by c-Cbl. Therefore, natural cytotoxicity is controlled not only by dominant inhibitory receptors, but also by an activation threshold set by c-Cbl, which is overcome by Vav1-dependent synergy of coactivation receptors.
Calcium hydroxide attenuates the inflammatory potential of lipoteichoic acid (LTA) from Enterococcus faecalis by deacylating the LTA (89.22)
Authors: Baik JungEun , Yun Cheol-Heui , Han SeungHyun
Published: January 2023

Enterococcus faecalis is a pathogenic gram-positive bacterium closely related with refractory apical periodontitis. Since lipoteichoic acid (LTA) is a major virulence factor of gram-positive bacteria, we prepared highly pure LTA from E. faecalis and investigated its inflammatory responses in a murine macrophage-like cell line, RAW 264.7. Upon exposure to the E. faecalis LTA, the cells substantially produced tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in a dose- and time-dependent manner. Notably, the action of the LTA was through Toll-like receptor 2 (TLR2), but not TLR4. The LTA enhanced the DNA-binding activity of a transcription factor, NF-κB, which plays an important role in the transcriptional activation of genes encoding various inflammatory mediators. However, induction of TNF-α and NO production was not observed in the cells exposed to LTA pre-treated with calcium hydroxide, which is a widely used as intracanal medicament for sterilizing root canals of teeth. Furthermore, calcium hydroxide-treated LTA was not able to stimulate TLR2. Structural analysis using MALDI-TOF mass spectrometry showed that calcium hydroxide deacylated the glycolipid moiety in the LTA structure. Conclusively, we suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA structure.
Crucial role of calcium signaling in TCR-dependent functions of Vgamma2Vdelta2 T cell (89.23)
Authors: Li Haishan , Pauza C. David
Published: January 2023

Vgamma2Vdelta2 T cells in human peripheral blood recognize phosphoantigen and play important roles in host defense and immunoregulation. However, the molecular mechanisms responsible for phophogantigen-mediated activation of human gammadelta T-cells remain unclear. We have previously reported that phosphoantigen isopentyl pyrophosphate (IPP) activated MEK/Erk and PI-3K/Akt pathways rapidly. Specific inhibition of Erk and Akt significantly impaired the functional response to IPP. In the present study, we found that IL2 can also activate MEK/Erk and PI-3K/Akt pathways without inducing functional responses. When given together with IPP, the effect of IL2 was to enhance cytokines production, degranulation and cytotoxicity. These data implicate the activation of MEK/Erk and PI-3K/Akt pathways as necessary but not sufficient to induce functional responses in Vgamma2Vdelta2 T cells. Further, we found that FK506 or cyclosporine A (CsA) inhibitors of calcineurin, completely blocked IPP-induced cytokines production, and partly inhibited TCR-dependent degranulation and cytotoxicity functions of Vgamma2Vdelta2 T cells. IPP but not IL2 induced the calcium-dependent nuclear translocation of NFAT. Our results indicate that calcium signaling pathway plays a crucial role in TCR-dependent functions in Vgamma2Vdelta2 T cells that can be distinguished from the effect of IL2.
A predominance of M2b macrophages in mesenteric lymph nodes of mice exposed to whole body 137Cs γ-irradiation (89.32)
Authors: Kobayashi Makiko , Cornforth Michael , Kinoshita Manabu , Shinomiya Nariyoshi , Suzuki Fujio
Published: January 2023

In accompanying studies, we have demonstrated that mice exposed to 5 Gy whole body 137Cs γ-irradiation (5 GY-mice) are greatly susceptible to bacterial translocation. M1Mϕ (IL-12+IL-10− Mϕ), host defense effector cells against bacterial translocation, were not demonstrated in mesenteric lymph nodes (MLN) of these mice. M2Mϕ (IL-12−IL-10+ Mϕ) were predominated in MLN of 5 GY-mice. M2Mϕ are inhibitory on Mϕ conversion from resident Mϕ to M1Mϕ. In this study, MLN-M2Mϕ isolated from mice 14 days after 5 Gy γ-irradiation were characterized biophysically. F4/80+ cells (1 x 106 cells/ml) isolated from MLN were cultured with 106 heat-killed Enterococcus faecalis for 48 hrs. Culture fluids harvested were assayed for IL-10, CCL17, CCL1 and CXCL13 by ELISA. Also, cells harvested from the cultures were assayed for mannose receptor and LIGHT mRNA expression by RT-PCR. In the results, CCL1 and IL-10 were detected in the culture fluids of MLN F4/80+ cells isolated from 5 GY-mice. Also, these Mϕ expressed LIGHT mRNA. CCL17 and CXCL13 were not produced and mannose receptor mRNA was not expressed by these Mϕ. An 86% of antigen-stimulated Mϕ conversion from resident Mϕ to M1Mϕ was inhibited by MLN F4/80+ cells isolated from 5 GY-mice. CCL17 and mannose receptor are biomarkers of M2aMϕ, CCL1 and LIGHT are biomarkers of M2bMϕ, and CXCL13 and mannose receptor are biomarkers of M2cMϕ. These results indicate that F4/80+ Mϕ isolated from MLN of 5 GY-mice are M2bMϕ.
M1 macrophages as a major host defense effector cell in bacterial translocation induced by whole body 137Cs γ-irradiation (89.34)
Authors: Suzuki Fujio , Kinoshita Manabu , Shinomiya Nariyoshi , Cornforth Michael , Kobayashi Makiko
Published: January 2023

In thermally injured hosts, mesenteric lymph node macrophages (MLNMϕ) are identified as major effector cells against bacterial translocation. In this study, the antibacterial function of MLNMϕ from mice exposed to whole body γ-irradiation was investigated. BALB/c mice, 14 days after 0.5 (0.5 GY-mice) or 5 Gy (5 GY-mice) whole body γ-irradiation, were tested for their resistance against oral infection with 106 CFU/mouse of Enterococcus faecalis. Also, the resistance of decontaminated NOD-SCID IL-2rγ-/- mice (NOD-SCID mice), inoculated with MLNMϕ from 0.5 GY-mice or 5 GY-mice, to E. faecalis oral infection was examined. Furthermore, MLNMϕ isolated from 0.5 GY-mice and 5 GY-mice were biophysically characterized. In the results, 100% of 5 GY-mice died after E. faecalis oral infection, while 0.5 GY-mice similarly infected with the pathogen were shown to be resistant. All NOD-SCID mice inoculated with 5 GY-mouse MLNMϕ died after the infection, while 100% of these mice inoculated with 0.5 GY-mouse MLNMϕ survived. MLNMϕ from 0.5 GY-mice produced IL-12 (but not IL-10) and expressed iNOS mRNA. MLNMϕ from 5 GY-mice produced IL-10 (but not IL-12) and did not express iNOS mRNA. These results indicate that 0.5 GY-mouse MLNMϕ are M1Mϕ, and 5 GY-mouse MLNMϕ are M2Mϕ. Bacterial translocation in whole body γ-irradiated mice may be controlled by immunological intervention of M1Mϕ distributed in mesenteric lymph nodes.
Role of microRNA-214-targeting PTEN in advanced glycation endproducts-induced apoptosis delay of monocytes (89.35)
Authors: Zen Ke , Li Li-Min , Liu Dan-Qing , Guo Ya-Lan , Liu Yuan , Zhang Chen-Yu
Published: January 2023

Advanced glycation endproduct(AGE) delays spontaneous apoptosis of human monocytes and contributes to the development of a local inflammatory response. The mechanism by which AGEs effect monocyte apoptosis, however, is unclear. Here we report that AGE-modified BSA and β2-microglobulin(β2m) can increase miR-214 expression in monocytes, which in turn, enhances cell survival by reducing the expression of phosphatase and tensin homolog(PTEN). Employing miRNA microarray, we obtains a genome-wide miRNA expression profiling of THP-1 cells treated with/without AGEs. Among 470 miRNAs screened, 6 miRNAs including miR-214 are upregulated while 5 miRNAs were down-regulated (fold-change>2) in AGE-treated cells. AGE-induced miRNA differential expression is validated by miRNA qRT-PCR. Significant upregulation of miR-214 is confirmed in normal human monocytes treated with AGEs and monocytes isolated from patients with chronic renal failure. Both AGE-treated monocytes and patient monocytes have a delayed apoptosis. Luciferase reporter assay shows that miR-214 targets PTEN mRNA 3’-untranslated region. Furthermore, PTEN expression is inversely correlated with miR-214 expression in monocytes and the forced expression of miR-214 leads to a delayed apoptosis of THP-1. In contrast, decrease of miR-214 expression via anti-miR-214 largely abolishes AGE-induced cell survival. In summary, our findings define a new regulation role of miR-214 on AGE-induced monocyte survival by impeding PTEN expression.
Impairment of human dendritic cell function by a food-borne carcinogenic heterocyclic aromatic amine, 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (89.38)
Authors: Kim SunKyung , Jeon Jun Ho , Im Jintaek , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Carcinogens frequently provoke immunosuppressive effects and thereby cancer cells are not easily removed by host immune system having carcinogen(s). Heterocyclic amines (HCA) such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine (PhIP), and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), are dietary carcinogens that are abundantly produced by overcooking meat and fish. Here, we investigated the effect of HCA on the activation of dendritic cells (DCs) which play an important role in the initiation of immune responses. When human monocyte-derived DCs were stimulated with lipopolysaccharide (LPS), DCs became mature with an increase in the expression of co-stimulatory receptors such as CD80 and CD86, and MHC molecules but a decrease in the phagocytic capacity. However, Trp-P-1 but not PhIP or IQ alleviated all of these phenomena under the same condition. In addition, Trp-P-1 inhibited the production of cytokines, TNF-α and IL-12, by LPS-stimulated DCs. Furthermore, DCs pre-exposed to Trp-P-1 were less efficient in the activation and proliferation of autologous T cells than the control DCs. The suppressive effect of Trp-P-1 on the LPS-induced DC activation appears to be due to interference with the interaction between LPS and DCs as determined by a binding assay using FITC-labeled LPS. Conclusively, these results suggest that Trp-P-1 impairs the function of DCs for the appropriate induction of immune responses.