bluuebunny/crossref_metadata_2025 huggingface dataset:
ADP-Ribosylation of Rho by C3 Ribosyltransferase Inhibits IL-2 Production and Sustained Calcium Influx in Activated T Cells
Authors: Angkachatchai Vachras , Finkel Terri H.
Published: January 2023

Activation of the T lymphocyte induces dramatic cytoskeletal changes, and there is increasing evidence that disruption of the cytoskeleton inhibits early and late events of T cell signal transduction. However, relatively little is known about the signaling molecules involved in activation-induced cytoskeletal rearrangement. The rho family of small GTP-binding proteins, which include rho, rac, and cdc42, regulates the cytoskeleton and coordinates various cellular functions via their many effector targets. In prior studies, the Clostridium botulinum toxin C3 exoenzyme has been used to ADP-ribosylate and inactivate rho. In this study, we demonstrate that treatment of T cells with C3 exoenzyme inhibits IL-2 transcription following ligation of the TCR. Inhibition of IL-2 expression correlated with loss of sustained increase in [Ca+2]i and mitogen activated protein kinase (MAPK/Erk) activity, but not with activation of the tyrosine kinase, lck. These findings are the first to show that ADP-ribosylation of rho by C3 ribosyltransferase (exoenzyme) inhibits IL-2 production due, in part, to the requirement for sustained calcium influx and MAPK activation after Ag receptor ligation.
IFN-α Activates Stat6 and Leads to the Formation of Stat2:Stat6 Complexes in B Cells
Authors: Gupta Sanjay , Jiang Man , Pernis Alessandra B.
Published: January 2023

IFN-α consists of a family of highly homologous proteins, which exert pleiotropic effects on a wide variety of cell types. The biologic activities of IFN-α are mediated by its binding to a multicomponent receptor complex resulting in the activation of the Janus kinase-STAT signaling pathway. In most cell types, activation of Stat1 and Stat2 by IFN-α leads to the formation of either STAT homo-/heterodimers or of the IFN-stimulated gene factor 3 complex composed of Stat1, Stat2, and p48, a non-STAT protein. These distinct transcriptional complexes then target two different sets of cis-elements, γ-activated sites and IFN-stimulated response elements. Here, we report that IFN-α can activate complexes containing Stat6, which, until now, has been primarily associated with signaling by two cytokines with biologic overlap, IL-4 and IL-13. Induction of Stat6 complexes by IFN-α appears to be cell type specific, given that tyrosine phosphorylation of Stat6 in response to IFN-α is predominantly detected in B cells. Activation of Stat6 by IFN-α in B cells is accompanied by the formation of novel Stat2:Stat6 complexes, including an IFN-stimulated gene factor 3-like complex containing Stat2, Stat6, and p48. B cell lines resistant to the antiproliferative effects of IFN-α display a decrease in the IFN-α-mediated activation of Stat6. Activation of Stat6 as well as of Stat2:Stat6 complexes by IFN-α in B cells may allow modulation of target genes in a cell type-specific manner.
Mapping the Major Interaction Between Binding Protein and Ig Light Chains to Sites Within the Variable Domain
Authors: Davis David P. , Khurana Ritu , Meredith Stephen , Stevens Fred J. , Argon Yair
Published: January 2023

Newly synthesized Ig chains are known to interact in vivo with the binding protein (BiP), a major peptide-binding chaperone in the endoplasmic reticulum. The predominant interactions between the light chain and BiP are observed early in the folding pathway, when the light chain is either completely reduced, or has only one disulfide bond. In this study, we describe the in vitro reconstitution of BiP binding to the variable domain of light chains (VL). Binding of deliberately unfolded VL was dramatically more avid than that of folded VL, mimicking the interaction in vivo. Furthermore, VL binding was inhibited by addition of ATP, was competed with excess unlabeled VL, and was demonstrated with several different VL proteins. Using this assay, peptides derived from the VL sequence were tested experimentally for their ability to bind BiP. Four peptides from both β sheets of VL were shown to bind BiP specifically, two with significantly higher affinity. As few as these two peptide sites, one from each β sheet of VL, are sufficient to explain the association of BiP with the entire light chain. These results suggest how BiP directs the folding of Ig in vivo and how it may be used in shaping the B cell repertoire.
Characterization of an Avian (Gallus gallus domesticus) TCR αδ Gene Locus
Authors: Kubota Takayuki , Wang Jin-yi , Göbel Thomas W. F. , Hockett Richard D. , Cooper Max D. , Chen Chen-lo H.
Published: January 2023

Mammalian TCRδ genes are located in the midst of the TCRα gene locus. In the chicken, one large Vδ gene family, two Dδ gene segments, two Jδ gene segments, and one Cδ gene have been identified. The TCRδ genes were deleted on both alleles in αβ T cell lines, thereby indicating conservation of the combined TCRαδ locus in birds. Vα and Vδ gene segments were found to rearrange with one, both or neither of the Dδ segments and either of the two Jδ segments. Exonuclease activity, P-addition, and N-addition during VDJδ rearrangement contributed to TCRδ repertoire diversification in the first embryonic wave of T cells. An unbiased Vδ1 repertoire was observed at all ages, but an acquired Jδ1 usage bias occurred in the TCRδ repertoire. The unrestricted combinatorial diversity of relatively complex TCRγ and δ loci may contribute to the remarkable abundance of γδ T cells in this avian representative.
TNF Receptor p55 Is Required for Elimination of Inflammatory Cells Following Control of Intracellular Pathogens
Authors: Kanaly Suzanne T. , Nashleanas Michelle , Hondowicz Brian , Scott Phillip
Published: January 2023

The elimination of lymphocytes within inflammatory lesions is a critical component in the resolution of disease once pathogens have been cleared. We report here that signaling through the TNF receptor p55 (TNFRp55) is required to eliminate lymphocytes from lesions associated with intracellular pathogens. Thus, TNFRp55−/− mice, but not Fas-deficient mice, maintained inflammatory lesions associated with either Leishmania major or Rhodococcus equi infection, although they developed a Th1 response and controlled the pathogens. Inflammatory cells from either L. major- or R. equi-infected C57BL/6 mice were sensitive to TNF-induced apoptosis, and conversely the number of apoptotic cells in the lesions from TNFRp55−/− mice was dramatically reduced compared with wild-type mice. Furthermore, in vivo depletion of TNF in wild-type mice blocked lesion regression following R. equi infection. Taken together, our results suggest that signaling through the TNFRp55, but not Fas, is required to induce apoptosis of T cells within inflammatory lesions once pathogens are eliminated, and that in its absence lesions fail to regress.
Monocyte-Driven Activation-Induced Apoptotic Cell Death of Human T-Lymphotropic Virus Type I-Infected T Cells
Authors: Wakamatsu Shin-ichi , Makino Masahiko , Tei Chuuwa , Baba Masanori
Published: January 2023

We attempted apoptotic cell death induction of T cells infected with human T lymphotropic virus type I (HTLV-I) which induces HTLV-I-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. T cells acutely infected and expressing HTLV-Igag Ags were killed by cross-linking their TCR with anti-CD3 mAb. Cells in apoptotic process were found by staining with annexin V. The apoptosis was not affected by costimulation through CD28 molecules and was resistant to ligation of Fas molecules. Whereas the virus-infected T cells expressed higher levels of HLA-DR, CD25, CD80, and CD86 Ags than apoptosis-resistant PHA-blasts, the T cell apoptosis was enhanced by addition of exogenous IL-2. Furthermore, in this apoptosis, monocytes played an important role because T cells infected in the absence of monocytes were resistant to the death signals. The apoptosis-sensitive T cells responded to TCR signaling more strongly by proliferating than those apoptosis-resistant cells. Monocytes weakly affected the expression levels of viral Ags on T cells. However, HTLV-I-infected monocytes primed T cells to die by subsequent TCR signaling. T cells primed with the monocytes, subsequently infected in the absence of monocytes, were killed by TCR signaling. These observations suggest that primed and infected T cells could be killed by activation-induced cell death.
The Inhibitory Action of Mycobacterium ulcerans Soluble Factor on Monocyte/T Cell Cytokine Production and NF-κB Function
Authors: Pahlevan Ali A. , Wright David J. M. , Andrews Caroline , George Kathleen M. , Small Pamela L. C. , Foxwell Brian M.
Published: January 2023

Buruli ulcer is a chronic and progressive necrotizing ulcer for which there is no medical treatment. Historically, a soluble toxin (factor) derived from the causative Mycobacterium ulcerans was found to induce the massive necrosis of skin and s.c. tissue seen in this condition. However, the persistence of the disease is thought to be caused by a lack of any immune response. We therefore investigated whether the factor was related to immunosuppression. A protocol to partially purify the factor was developed, and its effects on immune competent cells were tested. The factor produced >95% inhibition of LPS-induced release of TNF and IL-10 from human monocytes and caused a loss of adherence of these cells without cell death. The factor also blocked the production of IL-2 from activated T lymphocytes. The factor had no effect on TNF-induced cytotoxicity, but abrogated TNF-induced NF-κB activation. Surprisingly, a synergy was observed between the factor and phorbol ester-directed NF-κB activation. The factor had no effect on IL-1- or LPS-induced NF-κB activity, indicating selective activity of the factor. The factor did not inhibit the degradation of IκBα induced by TNF, indicating that the target for its activity lies within an undefined part of the TNF signaling mechanism. The data indicate that the localized immunosuppression associated with Buruli ulcer relates to the activity of the released factor, and this may provide a target for future therapeutic strategies for this intractable disease.
Pulmonary Epithelial Cells are a Source of IL-8 in the Response to Mycobacterium tuberculosis: Essential Role of IL-1 from Infected Monocytes in a NF-κB-Dependent Network
Authors: Wickremasinghe Melissa I. , Thomas Lynette H. , Friedland Jon S.
Published: January 2023

Pulmonary epithelial cells, covering a 70-m2 surface area, have not previously been considered an important source of chemokines in pulmonary tuberculosis. We analyzed IL-8 secretion from A549 cells and primary normal human bronchial epithelial cells (NHBE) infected by Mycobacterium tuberculosis. Direct infection of A549 cells by M. tuberculosis caused IL-8 secretion of 7720 ± 1610 pg/106 cells, but no IL-8 secretion from NHBE after 24 h. In contrast, conditioned media from M. tuberculosis-infected human monocytes (CoMTB) induced a much greater IL-8 secretion of 92,635 ± 13,180 pg/106 A549 cells and 13,416 ± 3,529 pg/106 NHBE after 24 h. CoMTB induced rapid IL-8 mRNA accumulation, which was stable over 24 h, compared with TNF-α-induced transcripts. CoMTB stimulated nuclear binding of p65, p50, and c-Rel subunits of NF-κB to IL-8 promoter sequences. Transient transfections with IL-8 promoter reporter constructs showed NF-κB binding-site mutations abolished IL-8 promoter activity while NF-IL-6 binding-site mutations decreased promoter activity to 50.2 ± 6.3% of wild-type activity. IL-1R antagonist but not neutralizing anti-TNF-α inhibited epithelial cell IL-8 secretion, mRNA accumulation, and NF-κB binding. Recombinant IL-1β (2 ng/ml) induced similar levels of IL-8 secretion to CoMTB in both A549 cells and NHBE. Pulmonary epithelial cells are a major source of IL-8 in the initial host response to pulmonary tuberculosis. Such IL-8 secretion is NF-κB dependent, NF-IL-6 requiring, and activated by an IL-1-mediated pathway as a consequence of phagocytosis of M. tuberculosis by monocytes.
Regulation of Complement Activation by C-Reactive Protein: Targeting the Complement Inhibitory Activity of Factor H by an Interaction with Short Consensus Repeat Domains 7 and 8–11
Authors: Jarva Hanna , Jokiranta T. Sakari , Hellwage Jens , Zipfel Peter F. , Meri Seppo
Published: January 2023

C-reactive protein (CRP) is a major acute phase protein whose functions are not totally clear. In this study, we examined the interaction of CRP with factor H (FH), a key regulator of the alternative pathway (AP) of complement. Using the surface plasmon resonance technique and a panel of recombinantly expressed FH constructs, we observed that CRP binds to two closely located regions on short consensus repeat (SCR) domains 7 and 8–11 of FH. Also FH-like protein 1 (FHL-1), an alternatively spliced product of the FH gene, bound to CRP with its most C-terminal domain (SCR 7). The binding reactions were calcium-dependent and partially inhibited by heparin. In accordance with the finding that CRP binding sites on FH were distinct from the C3b binding sites, CRP preserved the ability of FH to promote factor I-mediated cleavage of C3b. We propose that the function of CRP is to target functionally active FH and FHL-1 to injured self tissues. Thereby, CRP could restrict excessive complement attack in tissues while allowing a temporarily enhanced AP activity against invading microbes in blood.
TNF-α and IL-1 Sequentially Induce Endothelial ICAM-1 and VCAM-1 Expression in MRL/lprLupus-Prone Mice
Authors: McHale Julie F. , Harari Olivier A. , Marshall Diane , Haskard Dorian O.
Published: January 2023

Dysfunctional leukocyte-endothelial interactions are thought to play a key role in systemic lupus erythematosus pathogenesis. We questioned the importance of TNF-α and IL-1 for endothelial activation in MRL/lpr lupus-prone mice. Endothelial ICAM-1 and VCAM-1 expression increased significantly with disease evolution in kidney, heart, and brain, as shown by i.v. injected radiolabeled Ab uptake. Lung endothelial VCAM-1 also increased, while lung endothelial ICAM-1 did not rise above a high basal level. Immunoassays showed a significantly raised circulating level of TNF-α by 14 wk, with levels of circulating IL-1α and IL-1β being additionally raised by 20 wk. With 14-wk-old MRL/lpr, anti-TNF-α antiserum inhibited expression of ICAM-1 and VCAM-1 by endothelial cells cultured with sera in vitro, and uptake of anti-ICAM-1 and anti-VCAM-1 mAb in lung, kidney, brain, and heart in vivo. In contrast, both anti-TNF-α and anti-IL-1 antisera were required for maximal inhibition in vitro and in vivo at 20 wk. These data indicate that TNF-α is largely responsible for the early up-regulation of endothelial ICAM-1 and VCAM-1, but that IL-1 enhances expression in late disease. Our observations provide novel insights of possible relevance to understanding endothelial activation in systemic lupus erythematosus, and highlight an approach that can be extended to dissecting other chronic inflammatory diseases.
Inhibition of Bacterial Cell Wall-Induced Leukocyte Recruitment and Hepatic Granuloma Formation by TGF-β Gene Transfer
Authors: Song Xiao-yu , Zeng Li , Pilo Carey M. , Zagorski John , Wahl Sharon M.
Published: January 2023

Intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats results in dissemination of SCW to the liver, spleen, bone marrow, and peripheral joints. The uptake of SCW by Kupffer cells in the liver initiates a chain of events largely mediated by T lymphocytes and macrophages. Local synthesis and secretion of cytokines and growth factors in response to the persistent SCW lead to the evolution and maintenance of a chronic T cell-dependent granulomatous response and result in granuloma formation and irreversible hepatic fibrosis. In an attempt to impede the development of the chronic granulomatous lesions in the liver, we injected a plasmid DNA encoding TGF-β1 i.m. to the SCW animals to determine the effect of TGF-β1 gene transfer on the course of liver inflammation and fibrosis. A single injection of plasmid DNA encoding TGF-β1 resulted in virtual abolition of the development of the SCW-induced hepatic granuloma formation and matrix expansion. TGF-β1 DNA not only reduced key proinflammatory cytokines including TNF-α, IL-1β, IFN-γ, and IL-18, but also inhibited both CXC and CC chemokine production, thereby blocking inflammatory cell recruitment and accumulation in the liver. Moreover, TGF-β1 gene delivery inhibited its own expression in the liver tissue, which is otherwise up-regulated in SCW-injected animals. Our study suggests that TGF-β1 gene transfer suppresses hepatic granuloma formation by blocking the recruitment of inflammatory cells to the liver, and thus may provide a new approach to the control of hepatic granulomatous and fibrotic diseases.
Protection from Radiation-Induced Colitis Requires MHC Class II Antigen Expression by Cells of Hemopoietic Origin
Authors: Marguerat Samuel , MacDonald H. Robson , Kraehenbuhl Jean-Pierre , van Meerwijk Joost P. M.
Published: January 2023

Ulcerative colitis, an inflammatory bowel disease, is believed to result from a breakdown of dominant tolerance mechanisms that normally control intestinal immunity. Although CD4+ T lymphocyte subpopulations and expression of MHC class II molecules have been shown to play a role in the pathogenesis of the disease, the nature of the responsible mechanisms remains unclear. In this paper we describe a novel mouse model for inflammatory bowel disease, radiation-induced colitis, that occurs with complete penetrance 6–8 wk postinduction. A combination of high dose gamma-irradiation and lack of MHC class II expression on cells of hemopoietic origin results in development of colitis in C57BL/6 mice. Because of its versatility (due to susceptibility of mice of the widely genetically manipulated C57BL/6 background), high reproducibility, and 100% penetrance, radiation-induced colitis will be a useful mouse model for colitis and a significant tool to study dominant immunological tolerance mechanisms. Moreover, our data imply that tolerization to enteric Ags requires MHC class II mediated presentation by APC of hemopoietic origin.
IL-2 Receptor-Targeted Cytolytic IL-2/Fc Fusion Protein Treatment Blocks Diabetogenic Autoimmunity in Nonobese Diabetic Mice
Authors: Zheng Xin Xiao , Steele Alan W. , Hancock Wayne W. , Kawamoto Kensaku , Li Xian Chang , Nickerson Peter W. , Li Yongsheng , Tian Yan , Strom Terry B.
Published: January 2023

High affinity IL-2R5 is present on recently activated but not on resting or memory T cells. Selective targeting of T cells bearing high affinity IL-2R is an attractive therapy for many T cell-dependent cytopathic disease processes. A variety of rodent mAbs directed against the α-chain of the IL-2R, as well as IL-2 fusion toxins, have been used in animals and humans to achieve selective immunosuppression. Here we report on the development of a novel IL-2R targeting agent, a cytolytic chimeric IL-2/Fc fusion protein. This immunoligand binds specifically and with high affinity to IL-2R and is structurally capable of recruiting host Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activities. The Ig component ensures an extended circulating t1/2 of 25 h following systemic administration. To subsequently explore the mechanisms of the antidiabetogenic effects of IL-2/Fc, we have mutated the FcR binding and complement C1q binding (Fc−/−) domains of the Fc fragment to render the Fc unable to direct Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity activities. In a model of passive transfer of diabetes in nonobese diabetic mice, lytic IL-2/Fc, but not nonlytic IL-2/Fc−/−, exhibited striking antidiabetogenic effects. Together with the negligible potential of IL-2/Fc for immunogenicity, this finding forecasts that cytolytic IL-2/Fc may offer a new therapeutic approach for selective targeting of auto and alloimmune T cells.
Hyperexpression of CD40 Ligand (CD154) in Inflammatory Bowel Disease and Its Contribution to Pathogenic Cytokine Production
Authors: Liu Zhanju , Colpaert Stefaan , D’Haens Geert R. , Kasran Ahmad , Boer Mark de , Rutgeerts Paul , Geboes Karel , Ceuppens Jan L.
Published: January 2023

CD40 ligand (CD40L or CD154), a type II membrane protein with homology to TNF, is transiently expressed on activated T cells and known to be important for B cell Ig production and for activation and differentiation of monocytes and dendritic cells. Both Crohn’s disease and ulcerative colitis are characterized by local production of cytokines such as TNF and by an influx of activated lymphocytes into inflamed mucosa. Herein, we investigated whether CD40L signaling participates in immune responses in these diseases. Our results demonstrated that CD40L was expressed on freshly isolated lamina propria T cells from these patients and was functional to induce IL-12 and TNF production by normal monocytes, especially after IFN-γ priming. The inclusion of a blocking mAb to CD40L or CD40 in such cocultures significantly decreased monocyte IL-12 and TNF production. Moreover, lamina propria and peripheral blood T cells from these patients, after in vitro activation with anti-CD3, showed increased and prolonged expression of CD40L as compared with controls. Immunohistochemical analyses indicated that the number of CD40+ and CD40L+ cells was significantly increased in inflamed mucosa, being B cells/macrophages and CD4+ T cells, respectively. These findings suggest that CD40L up-regulation is involved in pathogenic cytokine production in inflammatory bowel disease and that blockade of CD40-CD40L interactions may have therapeutic effects for these patients.
Sepsis-Induced T Cell Immunoparalysis: The Ins and Outs of Impaired T Cell Immunity
Authors: Jensen Isaac J. , Sjaastad Frances V. , Griffith Thomas S. , Badovinac Vladimir P.
Published: January 2023

Sepsis results in a deluge of pro- and anti-inflammatory cytokines, leading to lymphopenia and chronic immunoparalysis. Sepsis-induced long-lasting immunoparalysis is defined, in part, by impaired CD4 and CD8 αβ T cell responses in the postseptic environment. The dysfunction in T cell immunity affects naive, effector, and memory T cells and is not restricted to classical αβ T cells. Although sepsis-induced severe and transient lymphopenia is a contributory factor to diminished T cell immunity, T cell–intrinsic and -extrinsic factors/mechanisms also contribute to impaired T cell function. In this review, we summarize the current knowledge of how sepsis quantitatively and qualitatively impairs CD4 and CD8 T cell immunity of classical and nonclassical T cell subsets and discuss current therapeutic approaches being developed to boost the recovery of T cell immunity postsepsis induction.
Morphine and TAT: Additive modulation of wound healing events (100.15)
Authors: Martin Josephine L. , Roy Sabita
Published: January 2023

Interactions between chronic morphine and the HIV-1 protein TAT are believed to play a major role in accelerating wound pathogenesis of HIV positive patients. Chronic morphine users infected with HIV often display infected and improper wound closure events compared to non-drug abusive HIV populations. Although it is well documented that HIV-1 proteins such as TAT up-regulate chemokine secretion, our laboratory is the first to show morphine suppression of TAT-induced leukocyte migration and chemokine secretion. Simultaneous treatment of morphine and lipopolysaccharide results in altered cytokine profiles of monocyte chemotactic protein-1 and macrophage inflammatory protein-2. Such alterations lead towards altered immune cell recruitment profiles of neutrophils and macrophages resulting in inadequate bacterial clearance and ultimately, delayed closure of wounds.

We have recently found further exacerbation of murine wound beds following co-administration of Tat proteins and morphine using ELISA, flow cytometry and histological assays.

Taken together, our data implicates a mu opioid receptor-mediated immune suppression of wound healing events following Tat and chronic morphine treatment and shows the potentially detrimental effects chronic morphine administration may have in the wound healing processes of HIV positive patients.

Supported by: NIDA R01-DA12104, K02-DA015349 and P50-DA11806.
Wide Expression and Cellular Localization of Inflammation-associated TSG-6 Protein in Atherosclerotic Lesions (100.17)
Authors: Mindrescu Catalin , Coppola John , Doty Stephen , Antonescu Cristina , Staniloae Cezar
Published: January 2023

TNF-stimulated gene 6 (TSG-6) is induced by TNF-α during inflammation and its secreted product TSG-6 glycoprotein is involved in immune-mediated inflammatory diseases and fertility. It regulates COX-2 and prostaglandin synthesis, and participates in extracellular matrix remodeling. Considering the chronic inflammatory nature of atherosclerosis we hypothesized that TSG-6 is expressed in atherosclerotic plaques and investigated TSG-6 protein expression and cellular distribution on 12 superficial femoral artery endarterectomy specimens from 6 diabetic and 6 non-diabetic patients with peripheral vascular disease. Six histologically normal radial artery specimens were analyzed as control. Paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-TSG-6 antibody. TSG-6 expression was consistently present in all atherectomy specimens but not in control specimens. A distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. Cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. Patchy TSG-6 expression was noted in the extracellular matrix. Within the inflammatory plaques from diabetic patients, TSG-6 stained the foamy macrophages. Our study identifies for the first time the preferential expression of TSG-6 in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. TSG-6 is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation.
High levels of ROS block the differentiation of human and mouse osteoclasts (100.5)
Authors: Moreno Jose Luis , Celluzzi Christina M , Keegan Achsah D , Williams Mark S
Published: January 2023

During chronic inflammation at skeletal sites, production of inflammatory cytokines by cells from the joint tissues and recruited activated T cells generate a pro-inflammatory environment with high levels of Reactive Oxygen Species (ROS). A critical cytokine expressed by the joint tissue and by the activated T cells is RANKL, which exerts its activity by inducing the differentiation of osteoclasts (OC) from cells of the monocyte-macrophage lineage. The effect of RANKL on the OC is mediated by a transient generation of ROS which are critical in regulating RANKL signaling. Therefore, we investigated the effect of increased ROS levels on OC differentiation from human and mouse progenitors. We found that addition of the glutathione depleting drug BSO to the cultures blocked OC generation from CD14+ human PBMC and from mouse bone marrow cells. This blockage was reversible; withdrawal of BSO allowed for the recovery of OC formation. The level of recovery diminished with increasing concentrations of BSO. The inhibition was time dependent, and it had a more profound effect if monocytes were the precursor cells rather than macrophages. Thus, while RANKL-induced OC development requires ROS, excess ROS can inhibit OC development.

(Supported by PHS AI59775 (ADK) AI49807 (MSW))
Toll-Like Receptor Expression is Necessary for Synovial Type II Cells to Respond to LPS: a Potential Mechanism of Reactive Arthritis (101.14)
Authors: Hao Hsiao-Nan , Yang Shang-Yu , Wooley Paul H
Published: January 2023

Reactive arthritis is predominantly associated with abnormal immune reactivity to gram-negative bacterial infections in children and young adults, but the precise molecular mechanism of the pathology remains unknown. Our previous results indicated fibroblast-like synovial cells (FLSC) express inducible Toll-like receptors (TLR). In this study we investigated the potential relationship of synovial cell TLR-dependent responses to the pathologen of reactive arthritis. To define TLR positive synovial cell subtypes, cell specific surface markers and flow cytometry were used both to isolate and to characterize the cells from mouse synovial tissues. Using synovial cell cultures and anti-TLR oligonucleotides, we examined whether the synovial immunological responses to LPS is dependent on TLR expression. Our data shows partial of FLSC were consecutive positive for TLR4, and inducible by LPS or cytokines. These cells were different from typical fibroblasts which are TLR4 negative. The TLR4+ FLSCs contribute approximately thirty percent of the isolated FLSC population, and sensitive to gram-negative bacterial endotoxin to produce inflammatory cytokines. Suppression of TLR4 expression on the cell surface significantly decreased the production of LPS-induced cytokines. The findings suggest synovial cells may orchestrate the immunological responses to bacterial components via TLR-based mechanisms.
MAP kinase phosphatase-1 plays a pivotal role in the protective effects of dexamethasone against endotoxic shock (101.15)
Authors: Wang Xianxi , Nelin Leif D , Kuhlman Joshua R , Meng Xiaomei , Welty Stephen E , Liu Yusen
Published: January 2023

Although glucocorticoids potently inhibit the biosynthesis of pro-inflammatory cytokines, the underlying mechanism is still not fully understood. We have previously shown that glucocorticoids induce the expression of MAP kinase phosphatase (MKP)a-1 in innate immune cells. Since MKP-1 is a critical negative regulator in innate immune response, we hypothesize that induction of MKP-1 plays an important role in the inhibition of pro-inflammatory cytokine biosynthesis by glucocorticoids. To test this hypothesis, we compared the anti-inflammatory effects of dexamethasone between wild-type and Mkp-1-deficient mice. We found that prophylactic administration of dexamethasone completely protected the wild-type mice, but not the Mkp-1−/− mice, from mortality caused by high-dose of lipopolysaccharides (LPS). Dexamethasone attenuated TNF-α production to lower levels in LPS-challenged wild-type mice than in similarly challenged Mkp-1−/− mice. Likewise, TNF-α and IL-6 production in vitro by wild-type macrophages upon LPS stimulation was decreased to lower levels by dexamethasone than did cytokine production by Mkp-1−/− macrophages. Dexamethasone pretreatment shortened the duration of p38 and JNK activities in LPS-stimulated wild-type macrophages, but had little effect in similarly treated Mkp-1−/− macrophages. Our results indicate that the inhibition of p38 and JNK activities by glucocorticoids is mediated by enhancing MKP-1 expression. These results demonstrate that induction of MKP-1 constitutes an important part of the anti-inflammatory mechanism of glucocorticoids.
Gene delivery strategies for targeting microglia cells (101.17)
Authors: Puzis Leopold , Odashima Kay , Pearse Damien D.
Published: January 2023

Microglia (MG) are the resident immune cells of the CNS and are implicated as both important sensors for biological defense as well as mediators of various pathological conditions through the various inflammatory and oxidative molecules they produce. In the current study we compared different techniques for gene targeting of microglia using non-viral and viral methods in vitro for eventual application to the in vivo setting. For this investigation we employed the EOC2 MG cell line and three methods of gene introduction for pmax plasmid-GFP:

electroporation,cationic lipid co-delivery andlentiviral vector infection.

Electroporation when employed with various buffers and at different voltages led to low transfection efficacies (>1%). Transfection with cationic lipids (LipofectAmine, NovaFector, GeneFector) showed higher transfection rates (%), but these were still relatively low. In contrast, lentiviral vector delivery produced robust infection (>95%) and GFP expression. In addition, the activation of the cells, so that they underwent morphological (rounded) and immunophenotypical (ED1 immunoreactivity) changes, prior to or during transfection with the lower efficiency methods, did dramatically enhance efficiency (>95%). We conclude, that in CNS pathologies, where MG cells are activated, both non-viral and viral methods of gene therapy will likely be highly successful for the introduction of specific anti-inflammatory or immune suppressive genes.

FUNDING: The Miami Project to Cure Paralysis, The Buoniconti Fund, The US Army Medical Research and Material Command
Biliary obstruction expands TREG which suppress liver T cell function (101.2)
Authors: Katz Steven C , Chaudhry Umer , Kingham T. Peter , Raab Jesse , DeMatteo Ronald P
Published: January 2023

INTRODUCTION: The mechanism by which obstructive jaundice predisposes to infection has not been clearly defined. We hypothesized that extrahepatic biliary obstruction would directly impair the function of T cells within the liver.

METHODS: Bile duct ligation (BDL) or sham laparotomy (SL) was performed in mice. Liver or spleen T cell function was assessed by proliferation in response to dendritic cells or anti-CD3 stimulation. TREG (CD4+CD25+FoxP3+) were measured by flow cytometry. CD4+C25+ (70% FoxP3+) liver T cells were purified by FACS and used as suppressors.

RESULTS: Following BDL, liver T cells were markedly less responsive to allogeneic or antigen-loaded syngeneic dendritic cells. In contrast, spleen T cell function was preserved. Even in the presence of direct anti-CD3 stimulation, liver T cells from BDL mice were functionally deficient and produced less IL-2 relative to spleen T cells. BDL expanded the number of liver TREG which suppressed the response of CD4+C25− T cells to DC in vitro.

CONCLUSION: BDL expands TREG, which may contribute to the increased risk of infection in patients with obstructive jaundice.
The effects of Immunosuppressive agents on NK cell function (102.18)
Authors: Wai Lu-En , Fujiki Masato , Takeda Saori , Hsieh Christine , Martinez Olivia M , Krams Sheri M
Published: January 2023

Natural killer (NK) cells are potent effector cells in allogeneic bone marrow and xeno transplantation. NK cells infiltrate solid organ allografts and a role for NK cells in acute and chronic rejection has been suggested. Little is known about the effects of immunosuppressive agents on NK cells

Primary NK cell lines were analyzed for proliferation in the absence or presence of increasing doses (0–100 ng/ml) of cyclosporine (CSA), FK506 (FK) or Rapamycin, (RAPA). NK cells demonstrated robust proliferation both in the absence and presence of CSA and FK. In contrast, RAPA significantly (p< 0.05) inhibited the proliferation of NK cells in a dose-dependent manner. The effects of immunosuppressive agents on NK cells were determined in a rat liver transplant model. Peripheral NK cell levels in graft recipients treated with CSA and FK remained stable whereas there was a marked decrease in the levels of NK cells in recipients treated with RAPA. The immunosuppressive agents did not alter IFNγ production by NK cells however NK cell–mediated cytotoxicity was significantly (p< 0.05) inhibited in the presence of RAPA (but not CSA or FK).

These data indicate that CSA and FK have little effect on NK cell function. In contrast, NK cell proliferation and function are significantly decreased in the presence of RAPA. Understanding the differential effects of immunosuppressive drugs on NK cell function is important in clinical transplantation.
Diversity and fate of pre-immune anti-tumor T cells after umbilical cord blood transplantation (102.28)
Authors: Merindol Natacha , Caty Martine , Gimmig Sylvain , Duval Michel , Champagne Martin A. , Soudeyns Hugo
Published: January 2023

Umbilical cord blood (UCB) has been used successfully as a source of progenitor cells to treat a variety of haematological and immune disorders in children. Donor-derived T cells play a key role in antitumoral and antiviral immunity and contribute to the survival of the graft recipient. Here we studied the diversity, persistence, fate and antitumoral function of UCB-derived T cells and characterized their persistence and function in the graft recipient. Naïve CD8+ T cells specific for the Melan-A26-35 peptide were isolated from UCB and UCB recipients at 1, 3, and 6 months post-transplant using Melan-A26-35-HLA-A2 tetramers and cell sorting, and were cultured in the presence of IL-2, IL-7, PHA and peptide. Melan-A-specific cytotoxicity was tested in 51Cr-release assays, and the clonal diversity of the pre-immune Melan-A-specific repertoire was determined by sequencing of the T cell receptor β chain CDR3 region. 14 UCB recipients were enrolled. Melan-A-specific CD8+ T cells were detected at a median frequency of 2.28% (range=0.0–8.18%) in 4 of 6 UCB samples initially analyzed. These cells proliferated in the presence of Melan-A peptide in 4 of 6 cases, and showed cognate cytotoxicity in 2 of 6 cases. Finally, tetramer-positive cells were detected in UCB recipients as early as 1 month post-transplant. This study will lead to a better understanding of the fate of the pre-immune T cell repertoire in UCB recipients.
A Novel In Vitro Murine Epithelial Culture System To Study Cutaneous GVHD (102.6)
Authors: Arditti Fabian David , Friedman Thea M , Murphy George , Korngold Robert
Published: January 2023

Graft-versus-host disease (GVHD), following allogeneic hematopoietic stem cell transplantation, involves a donor T cell attack against a number of tissue systems, including the skin, resulting in high levels of morbidity and mortality. The development of an in vitro murine model system for examining the underlying processes of specific target cell injury would be invaluable for increasing the understanding of the immunobiology of GVHD. In an effort to mimic the in situ nature of the target tissue, primary skin epithelial cell cultures were generated from ear skin of adult mice, after aggressive enzymatic digestion, without addition of growth factors or chemokines. The cultured cytokeratin-positive epithelial cells can be expanded and cryo-preserved for several passages. To test the ability of alloreactive T cells to mount responses against cultured epithelial cells, T lymphocytes from C57Bl/6 (B6) mice were added to the epithelial cells grown from minor histocompatibility antigen-mismatched BALB.B or syngeneic B6 mice. In vitro responses were characterized by day 6 supernatant interferon gamma levels (anti-BALB.B, 336.5 ± 32 pg/ml; anti-B6, 16.7 ± 12.2 pg/ml). Up-regulation of H2Kb was also observed on the surface of BALB.B epithelial cells (3.5 ± 2.2 and 90.5 ± 25 MFI on days 1 and 7, respectively). In addition, there was an increased amount of epithelial cell death, as denoted by 7-AAD staining.
Reduced sensitivity of effectors to regulation, and not reduced Treg function, may lead to autoimmune diabetes in NOD (128.1)
Authors: Nettles Adam , Honkanen-Scott Minna , Bansal-Pakala Pratima
Published: January 2023

T regulatory cells [Tregs] play an important role in controlling autoimmunity. The NOD mouse provides an excellent model to delineate the role of Tregs. Transfer experiments have shown that Tregs can prevent the progression of T1D. Similar results were obtained from depletion of Tregs in vivo. However, quantitative and qualitative changes in Tregs with the progression of T1D have not been examined in detail. In this study we studied changes in number and function of Tregs during progression of T1D in NOD. Since NOD males and females are very similar, yet the incidence of diabetes in males is much lower than the females, we examined sex based differences in Tregs and effectors.

We examined the expression of CD4+CD25+FoxP3+ to define the quantitative differences in Tregs. Time-course analysis showed that Treg numbers remained constant or slightly increased from 6 week to 16 weeks of age. Also, both female and male NOD had similar numbers of Tregs, suggesting that T1D progression is not due to a quantitative change in Tregs. Functional assays for purified Tregs from both male and female NOD showed marginal differences between their activities in vitro and in vivo. We found that islet-reactive T cells from female NOD were more auto-aggressive and refractory to regulation by Tregs than male effectors. These data suggest, for the first time, that refractory effector T cells, and not the number or function of Tregs, may account for T1D.
Humoral and cellular immune responses to IGF-2 in type 1 diabetes (128.21)
Authors: Louis Céline , Renard Chantal , Bosseloir Alain , Weets Ilse , Gorus Frans , Geenen Vincent
Published: January 2023

Background

IGF-2 is the dominant member of insulin family expressed in the thymus, Igf2 expression contributes to complete tolerance of insulin, and Igf2 transcription is defective in the thymus of diabetes-prone BB rats. Objectives

To evaluate the immunological tolerance to IGF-2 in T1D patients; andTo analyse the type of cellular response induced by IGF-2 B11-25, the homologous sequence of Insulin B9-23, a

major T1D auto-antigen.

Results

Using a sensitive and specific radio-binding assay, the presence of autoantibodies against IGF-2 was investigated in 100 newly diagnosed T1D patients and 100 healthy controls. No significant difference appeared between the two groups.We also investigated the profile of IL-10/IFN-γ secretion following presentation of Insulin B9-23 and IGF-2 B11-25

to PBMCs from T1D patients and healthy controls. Compared to Insulin B9-23 in ELISpot, ELISA and real-time RT-PCR, the presentation of IGF-2 B11-25 induced a regulatory/tolerogenic profile with a significantly higher increase of IL10 transcription and IL-10 secretion.

Conclusions

The very strong tolerance to IGF-2 is associated to active regulatory properties that might be exploited for development of a tolerogenic self-vaccine against T1D.

(Supported by the Belgian NFSR, Walloon Region TOLEDIAB project, and FP6 Euro-Thymaide Integrated Project)
Regulatory interactions between two innate immune cells - iNKT and MZB cells - suppress autoimmunity (128.24)
Authors: Wen Xiangshu , Yang Jun-Qi , Singh Ram Raj
Published: January 2023

In vivo activation of iNKT cells prevents autoimmune diseases such as lupus at least during early stages of disease development. Conversely, loss of iNKT cells in CD1d-KO mice worsens nephritis in lupus-prone BWF1 mice. Mechanisms of regulation of autoimmunity by iNKT cells remain poorly understood. Here, using a variety of in vitro and in vivo experimental systems, including adoptive transfers in SCID and iNKT cell-deficient (Ja18–/–) mice, we demonstrate that although iNKT cells trans-activate B cells to increase expression of activation markers and production of normal Ig, they selectively suppress IgG autoantibody production. Whereas increased expression of B cell activation markers occurs via cytokines secreted by iNKT cells, iNKT cell-mediated suppression of IgG autoantibody production requires a direct contact between iNKT cells and B cells. Furthermore, although iNKT cells increase production of cytokines by other immune cells, they specifically inhibit IL-10 production by B cells in a contact-dependent manner. Furthermore, whereas iNKT cells increase activation markers on all B cell subsets including B1, B2 and marginal zone B (MZB) cells, they significantly reduce the MZB cell population in lupus-prone BWF1 mice. Further support for the role of iNKT cells in regulating MZB cells came from iNKT cell transgenic (Va14Tg) and deficient (Ja18–/–) mice. Whereas the proportion of MZ-B cells are increased in Ja18–/– mice as compared to wild type BALB/c mice, these cells are reduced in Va14Tg mice. Thus, iNKT cells suppress autoreactive B cells and prevent systemic autoimmunity. They do so via regulating MZ-B cells. Ongoing investigations will elucidate how activated iNKT cells activate MZB cells but reduce their IL-10 and autoantibody production.
Harnessing the Fas pathway for type 1 diabetes therapy (128.29)
Authors: Mohamood Abdiaziz Sheikh , Xiao Zuoxiang , Schneck Jonathan P , Hamad Abdel Rahim A
Published: January 2023

The Fas pathway is not required for T cell activation yet loss-of-function mutation that inactivates Fas (lpr) or FasL (gld) prevents autoimmune diabetes in NOD mice without interfering with normal immune responses. However, the Fas pathway has not been considered a viable therapeutic option because these mutations also cause double negative (DN) T cell lymphoproliferation and lupus-like syndrome. Using bone marrow chimeras and adoptive transfer, we show that gld mutation on either hemopoietic or nonhemopoietic compartment is equally protective from autoimmune diabetes; however, gld mutation on hemopoietic cells is responsible for most of DN T cell lymphoproliferation. Therefore, limiting gld mutation to the nonhemopoietic tissue completely controls diabetogenicity of wild type T cells without causing lymphoproliferation. The protection is recapitulated therapeutically by treating NOD-wt mice with FasL neutralizing antibody. Anti-FasL treatment curtailed insulitis and did not lead to lupus-like disease or DN T cell lymphoproliferation. These results suggest the potential of generating a new class of immunotherapy that is based on targeting T cell apoptosis inducing ligand rather than costimulatory pathways.
Role of Fas in Autoimmune Diabetes (128.30)
Authors: Mora Conchi , Wen Li , Gomis Ramon , Green E. Allison , Chervonsky Alexander V. , Wong F. Susan , García Ainhoa , Flavell Ricahard A.
Published: January 2023

We have studied the role of Fas in the induction of autoimmune diabetes in NOD mice and found that although Fas is required for the development of diabetes, early overexpression of this molecule on pancreatic β cells delays diabetes onset and reduces diabetes incidence in NOD mice. Moreover, Fas overexpression on β cells is not sufficient to restore the diabetic phenotype in Fas-deficient NOD mice. On the other hand we show that induction of β cell death by CD4 T cells is dependent on the expression of Fas on β cells. IL-1β and IFNγ are key cytokines involved in Fas up-regulation on mouse β cells. However, here we demonstrate that IL-1β is not required for either spontaneous t or adoptively transferred diabetes. Interestingly we have also observed that FasL overexpression on β cells seems to confer immune-privilege to islet cells against diabetogenic CD4 T cells.

We hypothesize that early, immune-independent, Fas expression on β cells protects from diabetes by promoting immune tolerance to islet antigens, while late, cytokine-mediated induction of Fas has the opposite effect by promoting massive β cell death and diabetes. There is redundancy regarding the cytokines involved in Fas up-regulation on β cells in vivo.
A critical role of cyclon, a novel cytokine-inducible gene, in the regulation of immune homeostasis (128.31)
Authors: Fleur Shella Saint , Hoshino Akemi , Fujii Hodaka
Published: January 2023

Autoimmunity, which results from hyperreactivity and hypersensitivity of lymphocytes against self antigens, can be caused by various defects in lymphoid cells, including impaired activation induced cell death (AICD) in effector T and B cells. Elimination of activated self-reactive T and B cells may effectively cure autoimmune diseases. We recently identify a novel cytokine-inducible gene, cyclon, by DNA microarray analysis. Cyclon is induced in both CD4+ and CD8+ T cells by T cell receptor ligation. Our data show that transgenic expression of Cyclon has no significant effect on T cell development and functions in wild-type mice. However, transgenic expression of Cyclon normalizes the number of activated T cells and suppresses enlargement of peripheral lymphoid organs in some autoimmune mice such as mice lacking the α subunit or the common γ chain of Interleukin-2 receptor. in vitro assays also suggest that the transgenic expression of Cyclon decreases viability of activated T cells and enhances AICD. Collectively, these results indicate that Cyclon may play an important role in immunosuppression by enhancing AICD.

This work is supported by NIH grant R01 AI059315 to H. F.