bluuebunny/crossref_metadata_2025 huggingface dataset:
Toll-like receptor3 overexpression alters responses to toll-like receptor7 ligand (111.6)
Authors: Zheng Wenjie , Crampton Steve , Bolland Silvia
Published: January 2023

Macrophages are key components of the inflammatory response. PolyIC induces type I IFN and inflammatory responses through TLR3 or MDA5 pathway, depending on the activating conditions and cell types. In our hands, bone marrow-derived macrophages (BMM) in vitro preferentially engage TLR3 in response to stimulation with naked poly IC. In contrast, the same macrophages activate TLR3-independent pathways in response to Fugene-complexed poly IC.These results suggest that lipid-complexed poly IC is more likely to induce inflammatory cytoplasmic pathways. To further investigate the role of TLR3 in macrophage activation, we have generated TLR3-overexpressing transgenic mice (TLR3 Tg). TLR3 Tg mice contain 20-30 copies of a BAC clone which spans the entire mouse tlr3 gene. As a consequence, TLR3 mRNA expression is greatly enhanced in BMM and splenoctes of TLR3 Tg mice. These mice had normal life span and no obvious changes in immunological characteristics or spontaneous pathology. When we stimulated TLR3 Tg BMM with naked poly IC, we detected a small increase in their hyperresponsiveness to poly IC compared to WT cells. Interestingly, TLR3 Tg BMM greatly enhanced the synergistic effect of TLR7 agonist pretreatment with naked poly IC in the inflammatory cytokine secretion, but did not enhance the TLR9 agonist pretreatment. In the future, we will further investigate the mechanisms involved in these effects and explore in vivo consequences of TLR receptor cross-regulation.
Inhibition of Th17 cells and promotion of Tregs in Fcγ chain-deficient mice contributes to the attenuated atherosclerotic lesions (112.1)
Authors: Ng Hangpong , Burris Ramona , Nagarajan Shanmugam
Published: January 2023

The presence of anti-oxLDL IgG is well documented in clinical and animal studies. However, the role for FcγRs to the progression of atherosclerosis has not been studied in detail. In the present study, we investigated the role for activating FcγR in the progression of atherosclerosis using apoE-Fcγ-chain double knockout (DKO) mice. Relative to apoE KO mice, arterial lesion formation was significantly decreased in apoE-Fcγ-chain DKO mice. Bone marrow chimera studies showed reduced lesions in apoE KO mice receiving the bone marrow of apoE-Fcγ-chain DKO mice. Cytokine responses by activated CD4+ cells showed higher levels of both IL-10 and IFN-γ secretion. Moreover, compared to apoE KO mice, anti-oxLDL IgG1 (Th2) and IgG2a (Th1) were increased in apoE-Fcγ-chain DKO mice. Interestingly, the number of Th17 cells and the secretion of IL-17 by activated CD4+ cells were decreased in apoE-Fcγ-chain DKO mice. Notably, the number of T-regulatory cells, expression of mRNA, and secretion of TGF-β and IL-10 were increased in apoE-Fc-chain DKO mice. Furthermore, secretions of IL-6 and STAT-3 phosphorylation essential for Th17 cell genesis were reduced in apoE-Fcγ-chain DKO mice. Collectively, our data suggest that activating FcγR promotes atherosclerosis by inducing Th17 response in the hyperlipidemic apoE KO mouse model. Our findings indicate that blocking activating FcγR may be a possible therapeutic approach to inhibit T cell responses contributing to the progression of atherosclerosis.
CD8 effector cell immunological synapse and cell division in the virally infected CNS (112.20)
Authors: Kim Jiyun , Stewart-Hutchinson Phillip , Kang Silvia , McGavern Dorian , Dustin Michael
Published: January 2023

Two-photon intravital imaging through thinned skull was used to visualize the dynamics of LCMV specific P14 effector T cells on day 6 LCMV meningitis. Most P14 were motile without forming stable synapses with infected stroma before the mice succumb to immunopathology. To determine if a subset of effector T cells can form more stable interactions with professional antigen presenting cells (APCs), we visualized the dynamics of CFP+ P14 T cells in CD11c-YFP transgenic or CX3CR1GFP/+ mice. p14 formed synapses with CD11c+ dendritic cells, found mostly on the meninges and perivascular spaces in the layer 1 cortex, and with activated CX3CR1+ microglia in the brain parenchyma and perivascular areas. Long contacts were rare with ramified CX3CR1+ microglia and small CD11c+ cells. P14 motility increased upon infusion of anti-MHC class I and anti-LFA-1 blocking antibodies. Fluo-Red dye infusion revealed Ca2+ flux in both P14 and tissue cells. Unexpectedly, during extensive imaging, we noted division of P14. P14 underwent divisions after contacting CD11c+ and away from these cells during the imaging period. We conclude that while the dominant behavior of P14 effector T cells during LCMV meningitis is continuous motility, a subset arrests with professional APCs. These prolonged interactions were sometimes terminated by cell division. Other instances of cell division suggest that there is significant division of anti-viral effector cells at the site of infection.
The transcription factor Klf2 regulates B cell trafficking in a lineage-specific manner (112.21)
Authors: Sebzda Eric , Hoek Kristen , Cendron Delphine , Gordy Laura , Collins Patrick , Parekh Vrajesh , Aune Thomas , Joyce Sebastian , Thomas James
Published: January 2023

Manipulation of homing receptors on effector lymphocytes may provide therapeutic relief from autoimmune disorders; however, few treatments are available to effectively limit B lymphocyte trafficking and prevent effector cell access to peripheral tissues. Understanding the molecular networks that regulate lymphocyte trafficking will be required to develop stable, efficacious drugs that work in a lineage-specific manner. In this regard, our preliminary findings indicate a regulatory link between Kruppel-like factor 2 (Klf2) and B cell homing receptors. Conditional excision of Klf2 leads to lineage-specific defects in mature B cell compartmentalization including displacement of marginal zone (MZ) B cells in the spleen, aberrant trafficking of follicular (FO) B cells, and an absence of peritoneal B1 lymphocytes. These phenotypes are quickly replicated in vivo following tamoxifen-induced Klf2 excision, consistent with a post-developmental defect in mature B cell homeostasis. Moreover, we demonstrate that Klf2 differentially regulates homing receptor expression in a B cell lineage-specific manner, resulting in functionally altered migration patterns. These results highlight Klf2 as a non-redundant transcription factor involved in naïve B cell homing. In turn this study may provide impetus for the rational design of therapeutics that target the molecular mechanisms governing B cell migration.
The role of β2 integrins in host immune responses to bacterial infection (112.26)
Authors: Bose Tina , Lefrancois Leo
Published: January 2023

β2 (CD18) integrins (CD11a,b,c,d) are important adhesion molecules necessary for leukocyte migration, homing, and cell to cell interactions. CD18-deficiency in humans is characterized by recurrent bacterial infections and poor wound healing, due to defects in leukocyte activation and migration. However, the in vivo role of each of the individual β2 family members is poorly understood. CD11a, CD11b and CD11c are all upregulated on activated CD8 T cells. In order to study the potential role of β2 integrins in host defense, CD11a, CD11b and CD11c deficient and wildtype mice were infected with Listeria monocytogenes (LM). Our results indicate that the absence of CD11b and CD11c had no effect on antigen specific CD8 T cells in response to infection with LM. On the contrary, after infection, antigen specific CD8 T cells in CD11a -/- mice exhibited diminished proliferation and activation resulting in significantly reduced numbers at the peak of the response. Moreover, CD11a KO mice exhibited a reduced differentiation of short lived effectors subset (KLRG1 hi CD127 lo) although cytokine and granzyme B production was unaffected. Interestingly, upon secondary infection with LM, antigen specific memory CD8 T cells in CD11a KO responded robustly. Overall, these results suggest that CD11a plays a crucial role in regulation of primary CD8 T cell effector differentiation, but may be dispensable for secondary responses to Listeria infection.
Thrombin mediates kinesis in human resting CD8 T cells from healthy controls and HIV infected individuals through specific interaction with thrombin receptor (PAR-1). (112.7)
Authors: Hurley Amanda , Smith Mindy , Karpova Tatiana , Nikel Erin , Packard Beverly , Imamichi HIromi , MacNally James , Higgins Jeanette , Sneller Michael , Lane Clifford , Catalfamo Marta
Published: January 2023

Chronic infections, such as HIV, are complicated with coagulation abnormalities and increased risk of cardiovascular events, however the mechanisms that link infection/inflammation and coagulation are not clearly defined. Thrombin is the main protease of the coagulation cascade and also participates in the host inflammatory response to tissue insult through activation of protease activated receptor-1 (PAR-1). PAR-1 is expressed in cells of the innate immune system. In this study we characterized the expression of PAR-1 on T cells from healthy controls. PAR-1 was expressed on CD4 and CD8 T cells with effector memory phenotype (23%±1.9 and 48%±3.5 respectively). Compared to healthy controls, patients with HIV infection had 1.6 times increased (p=0.0001) expression of PAR-1 on CD8 but not CD4 T cells. We hypothesized that thrombin could be involved in the recruitment of T cells to the place of tissue insult. Therefore, we imaged the effect of thrombin in individual cells over time using time-lapse microscopy. Thrombin was able to induce rapid kinesis after the addition of thrombin to human resting CD8 T cells in a PAR-1 dependent fashion. In addition, CD8 T cells from HIV infected patients were able to migrate in response to thrombin suggesting that during HIV infection CD8 T cells could respond to burst of thrombin leading to increased inflammation. This study provides new evidence of cross-talk between infection/coagulation adding a new player: T cells.
T cell expression of NOX2 regulates T helper differentiation (113.10)
Authors: Shatynski Kristen , Chen Haiyan , Williams Mark
Published: January 2023

Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In seeming contradiction, immune responses to agents such as H. pylori and influenza imply an altered or augmented adaptive immune response in CGD. We have shown NOX2 expression in T cells, and oxidase deficient T cells demonstrate Th1 skewed cytokine secretion upon in vitro stimulation. The goal of this study was to determine if these differences are T cell inherent, or if other cells promote T helper polarization. Activation of purified naive CD4+ T cells from NOX2-deficient mice led to augmented IFN-γ and diminished IL-4 production, consistent with a Th1 skewing of naïve T cells. This was supported by enhanced expression of the transcription factor T-bet and lower levels of GATA-3. Similar to NOX2-deficient cells, Th2-biased BALB/c T cells treated with the antioxidant N-acetyl cysteine produced increased IFN-γ and decreased IL-4 as compared to untreated controls. Inhibition of TCR-induced STAT5 phosphorylation was identified as a potential mechanism for skewed T helper differentiation. Stimulation of TCR-transgenic, OT-II T cells in vitro with NOX2-deficient splenic APC produced similar amounts of IL-4, IL-17, and IFN-γ as compared to those incubated with wild type APC, supporting the conclusion that NOX2 can regulate the adaptive immune response in a T cell inherent fashion.
Twist1 regulates Th1 cell development by interfering with its transcriptional network (113.11)
Authors: Pham Duy , Kaplan Mark
Published: January 2023

Although T-bet and STAT4 are required for Th1 programming, detailed transcriptional networks in Th1 cells have not been clearly defined. Twist1 is a basic helix-loop-helix (bHLH) transcription factor that is regulated by STAT4 in Th1 cells. We demonstrated that knocking down Twist1 in Th1 cells using Twist1-targeting small hairpin RNA vector increased IFN-γ and TNF-α and that overexpression of Twist1 in Th1 cells results in the decrease in IFN-γ and TNF-α production, which correlates with a reduction of STAT4 activation, and the expression of number of Th1 genes including T-bet, Runx3, and Hlx1. However, ectopic expression of Runx3 but not IL-12Rβ2 or T-bet can rescue the inhibitory effect of Twist1. Twist1 functions through several mechanisms including the direct binding of Twist1 to the E-box containing regions of the Ifng, Il12rb2, Tbx21 and Runx3 promoters in activated Th1 cells. Moreover, there is a physical interaction between Twist1 and Runx3 in both cell line and primary T cell culture. This study provides new insight on how Twist1 negatively regulates the Th1 transcriptional network, and how modulating Twist1 expression could be an approach for gene-based therapy in autoimmune diseases mediated by Th1 cells.
Deficiency of epidermal fatty acid-binding protein promotes Foxp3 expression in adaptive T regulatory cells (113.22)
Authors: Li Bing , Zhang Lihua , Hansen Richard , Stout Robert , Suttles Jill
Published: January 2023

Fatty acid-binding proteins (FABPs) are cytosolic proteins that serve as intracellular lipid chaperones. In previous work we demonstrated that CD4+ T cells deficient for expression of epidermal fatty acid-binding protein (E-FABP) exhibit elevated activity of peroxisome proliferator-activating receptor γ (PPARγ) which suppressed IL-6-induced STAT3 activity, resulting in suppression of Th17 differentiation. E-FABP-deficiency was accompanied by enhanced levels of FoxP3+ CD4+ T cells in vivo. Herein, we demonstrate that E-FABP deficiency promotes TGF-β-induced Foxp3 expression in adaptive T regulatory cells. These cells are characterized by enhanced PPARγ and AMP-activated protein kinase (AMPK) activities with elevated TGF-β and IL-10 production, but normal Smad2 and Smad3 phosphorylation. AMPK activators, but not PPARγ agonists, increased TGF-β-driven Foxp3 expression, yet inhibition of either AMPK or PPARγ downregulated Foxp3 expression. AMPKα1 deficiency results in impaired Foxp3 induction upon TGF-β stimulation. Taken together, our data reveal that E-FABP deficiency in CD4+ T cells enhances Foxp3 expression via PPARγ-AMPK-mediated pathway(s).
miRNA regulates the Glycoprotein A repetitions predominant expression in Tregs (113.23)
Authors: Zhou Qihui , Schnabel Christine , Schulze-Koops Hendrik , Skapenko Alla
Published: January 2023

Regulatory T cells (Tregs) are important for maintaining peripheral tolerance. A novel surface protein, Glycoprotein A repetitions predominant (GARP) was recently identified as a specific marker of human Tregs. Several studies suggested that GARP expression in Tregs is post-transcriptionally regulated. To test the hypothesis that miRNAs are involved in the regulation of GARP expression, we performed an in sillico analysis to predict putative miRNAs binding sites in the 3’UTR of GARP mRNA. Six overlapping fragments of GARP 3’UTR were cloned into pEGFP-C1 and pmirGlo vectors downstream of their reporter genes. Expression of the reporter proteins after nucleofection of T cells was analyzed by flow cytometry or luciferase assay. In fact, one of the six analyzed fragments of the GARP 3’UTR was capable of down-modulating the reporter protein expression. Within this fragment, three putative binding sites were recognized by their respective miRNAs when analyzed individually. In the context of the full-length 3’UTR, however, only two of those were recognized by their miRNAs. Mutation of these sites abrogated the respective miRNA recognition confirming the specificity of the binding to GARP 3’UTR. Together, we identified and characterized an miRNA regulation locus in the GARP 3’UTR, confirming the hypothesis of post-transcriptional regulation of GARP expression. The involvement of this site into regulation of GARP expression in Tregs still needs to be investigated.
Immune dysregulation by psychological distress (113.26)
Authors: Mathews Herbert , Janusek Linda
Published: January 2023

The molecular basis for the immune dysfunction that accompanies psychological distress is unknown. The purpose of this study was to determine whether epigenetic modifications underlie this immune dysregulation. Women newly diagnosed with early stage breast cancer were enrolled into the study and psychological, immunological and epigenetic assessments were made at diagnosis and four months later, after completion of cancer treatment. At diagnosis women reported increased perceived stress, anxiety, and mood disturbance and the peripheral blood mononuclear cells (PBMC) of these women exhibited reduced natural killer cell activity and reduced production of interferon gamma, which contrasted with results, obtained after completion of treatment. At the epigenetic level, the PBMC of these women at diagnosis exhibited distinct epigenetic patterns, with reduced nuclear acetylation of histone residues H4-K8 and H4-K12, as well as reduced phosphorylation of H3-S10, when compared to PBMC derived after the completion of treatment. PBMC function (natural killer cell activity and interferon-gamma production) associated with nuclear acetylation and phosphorylation status of these histone residues. These findings demonstrate associations among PBMC nuclear epigenetic patterns and the immune dysregulation that accompanies psychosocial distress.
Transcription regulation of human NCR1/NKp46, an activating NK cell receptor (113.4)
Authors: Lai Chiehmin Benjamin , Cheng Grace , Moradian Annie , Morin Gregg , Mager Dixie
Published: January 2023

Natural Killer (NK) lymphocytes are innate immune cells, mainly known to eliminate cancerous and virus infected cells. The NCR1/NKp46 receptor is expressed specifically in the NK lineage. Activation of NK effector functions results when the receptor binds viral haemagglutinin or unidentified tumor ligands. Knockout studies have shown that the molecule is indispensible in the control of influenza infection, lymphoma growth, and the development of type I diabetes. Yet the molecular basis behind the specificity of NCR1 expression is unknown. We seek to uncover the genetic and epigenetic mechanisms that regulate human NCR1 transcription. Comparative genomics and transient luciferase assays suggest that crucial cis-regulatory elements are found within 300bp upstream of the gene. Within this proximal region, luciferase experiments also indicate the presence of a basal, non-specific promoter as well as a tissue-specific regulatory switch. Through bioinformatics and transcription factor RNA expression screens, RUNX3 was identified as a probable regulator of the switch. Chromatin immunoprecipitation (ChIP) studies were conducted to confirm RUNX3 binding in NK cells. A proteomics approach is currently underway to identify additional factors. Finally, preliminary ChIP data hints at transcriptional control by histone 3 lysine 4 methylation in blood lineages.
IFN-γ protects from IL-17 mediated lethal encephalomyelitis independent of neutrophils (114.4)
Authors: Savarin Carine , Stohlman Stephen , Cua Daniel , Bergmann Cornelia
Published: January 2023

Immunodeficient SCID recipients of virus-primed memory CD4 T cells survive CNS infection by neurotropic coronavirus (JHMV), whereas IFN-γ deficient (GKO) donor CD4 T cells substantially increase morbidity. Control of virus replication and demyelination are similar in both recipients. However, distinct from wild type (wt) CD4 T cell recipients, GKO recipients were characterized by massive CNS neutrophil infiltration and detection of IL-17 mRNA. To determine if the lack of IFN-γ is directly correlated to rapid mortality, wt recipients were depleted of IFN-γ. Surprisingly, anti-IFN-γ treatment did not accelerate mortality despite substantially increasing CNS neutrophils. Accelerated mortality was only observed in recipients which were deficient in IFN-γ and expressed IL-17 mRNA in the CNS. Confirming IL-17 as the pathogenic mediator, inhibition of IL-17 in GKO recipients significantly decreased mortality. Moreover, co-transfer of wt with GKO donor CD4 T cells overcame the lethal phenotype, albeit without reducing IL-17 transcription. Survival of co-transferred SCID recipients was IFN-γ dependent, suggesting that IFN-γ afforded protection by inhibiting detrimental IL-17 protein responses, rather than IL-17 mRNA. Together these data demonstrate that IL-17, in the absence of IFN-γ, can accelerate mortality during viral encephalomyelitis by a mechanism independent of the magnitude of CNS neutrophil infiltration and reversible by IFN-γ.
IL-17A, IL-17C and IL-17 receptor A are involved in atherogenesis in apolipoprotein E-deficient mice. (114.7)
Authors: Galkina Elena , Phillips Tracy , Butcher Matthew
Published: January 2023

Atherosclerosis is a disease of large arteries that is characterized by the formation of atherosclerotic plaques in the arterial wall and the immune response accompanies this process. The pro-inflammatory conditions of atherogenesis promote Th17 and IL-17A+γδ+ T cell generation in atherosclerosis-prone Apolipoprotein E (Apoe-/-) deficient mice, however, the role of these cells are controversial. To examine the role of IL-17A in atherogenesis, we have generated IL-17A-deficient Apoe-/- (Il17a-/-Apoe-/-) and IL-17 receptor A-deficient Apoe-/- (Il17ra-/-Apoe-/-) mice. Western diet (WD)-fed Il17a-/-Apoe-/- and Il17ra-/-Apoe-/- mice had reduced plaques compared to diet matched Apoe-/- controls. The absolute number of aortic Th1, Th17, macrophages, and neutrophils were reduced in Il17a-/-Apoe-/-, Il17ra-/-Apoe-/- aortas, and corresponded with diminished expression of multiple aortic chemokines and cytokines. In contrast, the expression of IL17C mRNA was elevated in Il17a-/-Apoe-/- and Il17ra-/-Apoe-/- mice. Flow cytometric analysis of Il17a-/-Apoe-/-, Il17ra-/-Apoe-/- spleens and aortas revealed elevated levels of IL17C+ T cells compared to Apoe-/- mice. Ex vivo treatment of Apoe-/- and Il17ra-/-Apoe-/- aortas with IL-17C strongly promoted monocyte adhesion and aortic CXCL1 expression. These results suggest that IL17A, IL17RA and IL17C play a pro-atherogenic role through the induction of aortic chemokines and the recruitment of leukocytes to atherosclerotic lesions.
Innate interleukin-22 confers protection during acute liver infection (114.8)
Authors: Zenewicz Lauren , Pedra Joao , Booth Carmen , Fikrig Erol , Flavell Richard
Published: January 2023

Interleukin-22 (IL-22) is an important cytokine in the regulation of tissue responses during inflammation. This molecule is highly expressed by lymphoid tissue inducer (LTi)-like cells, which are found in lymphoid tissues such as those of the tonsils or gastrointestinal tract. The functional significance of these innate immune cells and whether these cells are present in other tissues, such as the liver, has not been well characterized. In this study, we have identified a population of RORγt-dependent LTi-like cells in the liver that secrete IL-22 upon stimulation with IL-23. To examine the role of innate derived IL-22 in liver immune responses, we utilized the obligate intracellular bacterium Anaplasma phagocytophilum. This important emerging tick-borne pathogen provokes a host innate immune response-driven hepatitis. Innate IL-22 provided protection to this liver injury as well as from bacterial infection. Additionally, A. phagocytophilum infection led to hepatocyte upregulation of the IL-22 receptor in an IL-22 dependent manner. Modulation of the IL-22 signaling pathway during infection may allow for optimal tissue responses to protect the host. Thus, IL-22 derived from LTi-like cells plays an important protective role in acute liver infection.
Regulatory T cells induced by GM-CSF suppress disease severity and selectively inhibit acetylcholine receptor-stimulated T cell responses in experimental myasthenia gravis (115.1)
Authors: Sheng Jianrong , Prabahakar Bellur , Meriggioli Matthew
Published: January 2023

We and others have demonstrated the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce and maintain semi-mature, tolerogenic DCs in vivo, and have shown that treatment could suppress autoimmunity by inducing CD4+CD25+ regulatory T cells (Tregs). In the current study, we have confirmed the critical role of Tregs in the therapeutic effects of GM-CSF both in vitro and in vivo utilizing extensive adoptive transfer experiments. Specifically, we show that Tregs from GM-CSF treated EAMG mice transferred to animals with EAMG suppress clinical disease more potently than equal numbers of Tregs from untreated mice. In addition, GM-CSF-induced-Tregs (GM-CSF iTregs) from EAMG mice potently and specifically suppress T cell proliferation induced by AChR, but do not alter T cell proliferation in response to irrelevant antigens to any greater extent than Tregs obtained from naive mice (so called natural Tregs [nTregs]). These results are consistent with the hypothesized mechanism of action of GM-CSF involving the mobilization of “semimature” dendritic cell precursors which, upon antigen (AChR) capture, initiate a suppression of the anti-AChR immune response, predominantly through the activation of AChR-specific iTregs. These preclinical findings provide important knowledge that may allow for clinical application of GM-CSF or related molecules to the treatment of human MG in the future.
Protective Tnfrsf9 haplotype mediates increased survival of functionally superior CD137+ T regulatory cells in type 1 diabetes (115.16)
Authors: Kachapati Kritika , Adams David , Wu Yuehong , Mittler Robert , Ridgway William
Published: January 2023

CD137 is a T cell costimulatory molecule that increases cell survival and promotes cytokine production, and is a candidate gene (Tnfrsf9) in NOD.B10 Idd9.3 congenic mice. We have shown that Tregs constitutively expressing CD137 are the target of anti-CD137 antibody that protects NOD from diabetes. Here, we show that NOD.B10 Idd9.3 CD137pos, but not CD137neg or NOD, Tregs have increased in vivo survival. Mixed bone marrow chimera mice made with NOD and NOD.B10 Idd9.3 bone marrow showed that increased survival in Tregs expressing the B10, but not NOD, Tnfrsf9 allele was an intrinsic cellular effect. NOD.B10 Idd9.3 CD137pos Tregs also express more anti-apoptotic Bcl-xl than NOD Tregs. To understand the impact of increased survival of CD137pos Tregs on disease, we performed functional studies that showed that CD137pos Tregs are significantly more suppressive than CD137neg Tregs in-vitro. We show that CD137pos Tregs, not CD137neg Tregs or T effectors, are the primary producers of the alternatively spliced CD137 isoform, soluble CD137. Soluble CD137 directly suppressed effector T cells by binding to CD137L on T cells in an APC independent assay. The increased suppression of CD137pos Tregs was associated with increased production of soluble CD137 and these studies show the importance of CD137pos Tregs in T1D.
Sex hormones and gender influence the expression of Foxp3 and regulatory T cells in SLE patients (115.18)
Authors: Dinesh Ravi , Hahn Bevra , Singh Ram
Published: January 2023

Regulatory T cells have been implicated in the maintenance of immune tolerance. Whether sex hormones and gender influence the expression of FoxP3 and regulatory T cells remain obscure. We provide the first evidence in this study that numbers and percent expression of regulatory T cells are significantly reduced in healthy females compared to healthy males. We found that both CD4+CD25+hi and CD8+CD25hi+ subsets of healthy males had 3-4-fold higher Foxp3 mRNA compared to healthy females. Estrogen increases proinflammatory cytokines and plasma estradiol positively correlated with IL-21 in SLE pts. Levels of estradiol positively correlated with SLEDAI score. Testosterone level was decreased in SLE women compared to healthy women. Furthermore, testosterone significantly increased FoxP3 expression in CD4+CD25hi cells from women with SLE. Plasma concentrations of testosterone positively correlated with the expression of FoxP3 in Treg. These data demonstrate that women may be more susceptible than men to SLE and other autoimmune diseases in part because many healthy women have fewer regulatory T cells and less FoxP3 expression. In addition, women with SLE, compared to healthy women, seem to have less ability to generate CD4+ Treg in response to physiologic concentrations of 17b-estradiol, whereas testosterone metabolite increases the generation of Tregs. These data suggest that gender and sex hormones influence susceptibility to SLE via their effects on Treg and Foxp3 expression.
Auto-reactive exTreg cells are generated during the initiation of autoimmune inflammation. (115.23)
Authors: Bailey-Bucktrout Samantha , Zhou Xuyu , Rosenthal Wendy , Bluestone Jeffrey
Published: January 2023

The onset of autoimmunity is associated with a reduced frequency of Foxp3+ T regulatory (Treg) cells, or reduced Foxp3 expression. We have observed that Tregs can lose Foxp3 under homeostatic conditions to form exTreg cells, and that islet-specific exTreg cells induced autoimmune type 1 diabetes (T1D). ExTreg generation in the islets correlates with low IL-2Rα (CD25) expression, and IL-2 complexes directed at Treg cells can rescue T1D. Unstable Foxp3 expression may weaken the ability of the Treg compartment to suppress effectors, and, exTregs can themselves be pathogenic. In this study, we used class II tetramers conjugated with myelin oligodendrocyte peptide 38-49 (MOG38-49) to analyze the myelin-reactive potentially pathogenic CD4+ conventional effector T cells and Treg cells during experimental autoimmune encephalomyelitis (EAE). We observed exTreg generation from Tregs enriched for MOG38-49-specificity, and this correlated with EAE progression. CD25 expression was high on Tregs and exTregs in the inflamed central nervous system, so low CD25 cannot explain Foxp3 instability in this setting. This is the first description of auto-reactive Treg cells within a normal T cell repertoire losing Foxp3 expression during the onset of autoimmunity. It supports the hypothesis that Tregs are enriched for self-reactivity, and that unstable Tregs are associated with progressive autoimmune disease.
Blockade of Programmed death-1 induces functional CD4+ regulatory T cells to resist apoptosis in (New Zealand Black x New Zealand White)F1 lupus-prone mice (115.25)
Authors: Wong Maida , La Cava Antonio , Hahn Bevra
Published: January 2023

PD-1 is a negative regulatory signal in T cells. We hypothesize that regulation of PD-1 signaling is required for maintenance of functional regulatory T cells (Treg) and subsequent control of autoimmunity in BWF1 mice, and that regulatory capacity of the cells is sustained in part by resistance to apoptosis, resulting in Treg survival. By toleration with an artificial peptide pCons, we have shown induction of CD4+ and CD8+ Foxp3+Treg with reduced of PD-1 expression decrease anti-dsDNA and nephritis and prolong survival. The same outcomes are observed when PD-1 expression is blocked by a neutralizing antibody. Here, anti-PD1 or pCons treatments maintained total numbers of functional suppressive Foxp3+CD4+CD25+ T cells between 10 and 40 weeks of age. The decline in IL-2 was significantly delayed by anti-PD1, and TGF-β production was increased. These CD4+Treg were resistant to spontaneous apoptosis, suppressed CD4+CD25- T cell proliferation, and induced apoptosis in B cells. In particular, Fas and FasL are two candidate molecules in the apoptotic pathways that were upregulated in splenic CD4+Treg upon pCons-tolerization or PD-1 blockade. To conclude, PD-1 is central in control of some regulatory T cells that suppress autoimmunity; their PD-1 expression may need fine tuning to permit the cells to survive and retain suppressive capacities. A mechanism by which PD-1 sustains these Treg is by reducing their susceptibility to apoptosis, which may relate to altering Fas/FasL pathway.
Host-derived CD4+ T cells protect lethally irradiated B6.g7 mice from stem cell mediated transfer of autoimmunity. (115.26)
Authors: Rajasekaran Narendiran , Wang Nan , Truong Phi , Rinderknecht Cornelia , Beilhack Georg , Shizuru Judith , Mellins Elizabeth
Published: January 2023

In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic TCR initiate disease by helping B cells to produce arthritogenic anti-GPI (glucose-6- phosphate isomerase) autoantibodies. We found that lethally-irradiated lymphocyte-deficient B6.g7 (I-Ag7+) Rag-/- mice reconstituted with K/BxN HSC (ckit+Lin-Sca-1hi) exhibit arthritis by week 4 and have anti-GPI Ab levels comparable to K/BxN mice. The normal B6.g7 mouse recipients of KBN HSC, however, showed only a mild arthritis with limited extent and duration. Titers of anti-GPI Abs in reconstituted B6.g7 mice were ~60 fold lower than in K/BxN mice. To investigate the basis of this attenuated autoimmunity, we examined the chimerism in the reconstituted B6.g7 mice. Although the B cell and myeloid compartments in blood and spleen had 98%±0.1 donor-derived cells, the CD4+ T cells were only 12%± 2.5 donor-derived. Further, host-derived CD4+ T cells in the reconstituted B6.g7 mice were 18%±3.5 CD25+FoxP3+ Treg cells compared to 8%±1.76 Tregs in donor-derived CD4+ T cells (P<0.024). Notably, attenuation of autoimmune diabetes and high percentage of host-derived Tregs (hTreg) were also observed when B6.g7 mice were reconstituted with HSCs from BDC2.5 transgenic mice. The hTreg were functional in vitro in standard assays. Our findings show that despite myeloablation, hTreg emerge in these mice and attenuate autoimmunity.
ITAM signaling negatively regulates dendritic cell function via SOCS-1 (116.19)
Authors: Ito Hiroaki , Lau Laura , Hamerman Jessica
Published: January 2023

Dendritic cells (DCs) play a crucial role in both the innate and adaptive immune system. DCs use several receptor systems to recognize pathogens, including Toll-like receptors (TLR). We have found that two immunoreceptor tyrosine-based activation motif (ITAM) adaptors (DAP12 and FcRγ) negatively regulate TLR responses in DCs, however the precise mechanism by which ITAM signaling suppresses TLR responses remains to be elucidated. Here, we show that aberrant SOCS-1 expression was found in ITAM signaling-deficient DCs. SOCS-1 is a well-described suppressor for TLR signaling as well as cytokine signaling, including signaling from the IFN-γ and IFN-α/β receptors. We found decreased SOCS-1 expression in DAP12/FcRγ-deficient BM-derived DCs (BMDCs) in comparison with WT BMDCs before and after TLR stimulation at both mRNA and protein levels. MHC class II expression on DAP12/FcRγ-deficient BMDCs was higher than that of WT BMDCs even under steady state conditions, similar to SOCS-1 deficient DCs. Consistent with the decrease of SOCS-1 expression in DAP12/FcRγ-deficient BMDCs, STAT-1 phosphorylation was increased in DAP12/FcRγ-deficient BMDCs upon IFN-γ stimulation in comparison with WT BMDCs. These results suggest that ITAM signaling positively regulates SOCS-1 expression to inhibit DC activation.
A negative regulatory role for Traf3 in DNA-induced type I interferon production (116.21)
Authors: Dev Anurupa , Wang Yaya , Ghaffari Amirali , Cheng Genhong
Published: January 2023

While the adaptor protein TRAF3 is known to be a positive regulator of the type I interferon (IFN) response to RNA-mediated TLR and RLR activation, its role in DNA-induced IFN induction is not clear. Here we provide evidence of a negative regulatory role for TRAF3 in the IFN response to intracellular DNA. Traf3-/- MEFs infected with HSV-1 or stimulated with B-form DNA produce elevated levels of type I IFNs, and subsequently have a significant reduction in HSV-1 viral replication and production as compared to wildtype cells. Interestingly, cells lacking TRAF2 or cIAP1/2, components of the TRAF3-containing complex required for suppression of noncanonical NF-κB activation, were also found to hyperinduce IFN upon stimulation with B-DNA. Further studies indicate that components of the noncanonical NF-κB signaling pathway interact and also synergize with TBK1 in IFN production, and activating this pathway provides protection against HSV-1 infection both in vitro and in vivo. Thus, our studies suggest that TRAF3 plays opposing roles in regulating pathogenic DNA versus RNA-induced IFN responses.
Kamebakaurin inhibits lipopolysaccharide-induced dendritic cell maturation by blocking transforming growth factor-beta activated kinase 1 (116.30)
Authors: Kim Jee Youn , Kim Yeon Jin , Ryu Hwa Sun , Kim Hyung Sook , Roh Eunmiri , Kim Hwan Mook , Kang Jong Soon , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Transforming growth factor-beta activated kinase-1 (TAK-1) is a serine/threonine kinase in the mitogen-activated protein kinase kinase kinase family. Once activated, TAK-1 will activate p38, JNK, and IKK, followed by activation of transcription factors AP-1 and NF-κB. Due to its role in the regulation of cell survival, differentiation, and inflammation, TAK-1 becomes one of the important drug targets in many human diseases. In this study, we show that TAK-1 can be directly inhibited by natural compound, kamebakaurin. Kamebakaurin inhibited the production of nitric oxide, IL-12, TNF-α, and IL-1β by dendritic cells, but did not affect their maturation process. We also showed that kamebakaurin inhibited the phosphorylation of p38 and JNK, but not ERK, in LPS-treated DCs. Kamebakarin also inhibited the phosphorylation of MEK3/6, upstream kinase of p38, and MKK4/7, upstream kinase of JNK, but not MEK1/2, upstream kinase of ERK. In addition, kamebakaurin inhibited NF-κB pathway, sequentially IKK phosphorylation, IκB-α degradation and nuclear translocation of NF-κB p65. Finally, we proved that kamebakaurin directly blocked autophosphorylation and kinase activity of TAK-1. Taken together, we show that kamebakaurin is a direct inhibitor of TAK-1 and suggest that it might be candidate for the treatment of inflammatory diseases.
Complement alternative pathway is activated in preeclampsia (116.35)
Authors: Zhang Ming , Velickovic Ivan , Dalloul Mudar , Alam Amit , Lai Chih Yuan , Medranda Giorgio , Abulafia Ovadia , Sherer David
Published: January 2023

In a previous study of a cohort of pregnant women (mostly whites, with 4.6% preeclampsia), complement factor B, a central molecule of alternative complement pathway, is observed as a predictor of preeclampsia in early pregnancy. Whether this observation could be applied to other populations is unknown. Further, the status of factor B in the fetal circulation of preeclampsia is unclear. We investigated these important questions by examining a cohort of pregnant women with high risk of preeclampsia. Fifty pregnant women (mostly African Americans, with 14% preeclampsia) were enrolled. Preeclampsia were diagnosed before delivery. Blood samples were collected from maternal and fetal umbilical cord blood immediately after delivery. Plasma Bb levels, the active fragment of factor B, were quantified by ELISA. We found that maternal plasma Bb levels were significantly higher in women with preeclampsia than those of normal pregnancy (increased 72%, P=0.02). In addition, Bb levels in the fetal cord blood of preeclampsia group were also significantly higher than those of normal (increased 92%, P=0.03). In summary, in a cohort of minority pregnant women with high risk of preeclampsia, complement factor B is activated in both maternal and fetal circulations during preeclamptic pregnancy. Activation of alternative complements may contribute to the systemic inflammatory response in both mothers and fetus.
DAP12 negatively regulates toll-like receptor responses in macrophages through SHIP1 and DOK3 (116.5)
Authors: Humphrey Mary Beth , Malhotra Shikha
Published: January 2023

DAP12, an ITAM- adaptor, associates with TREM2 and together they limit cytokine responses to LPS in macrophages. The aim of this study was to identify molecular mechanisms needed for the negative regulation of TLR signals by TREM2 and DAP12. LPS stimulation of macrophages induces the co- immunoprecipitation of DAP12 with src-homology containing inositol phosphatase-1 (SHIP1). LPS induced SHIP1 phosphorylation in wild type but not DAP12- deficient macrophages. LPS stimulated DAP12 or SHIP1-deficient macrophages produced significantly more TNFa than wild type macrophages. Further analysis showed that SHIP1 co-immunoprecipitates with another adaptor protein DOK3 in response to LPS. Phosphorylation of DOK3 was dependent on SHIP1 as SHIP1- deficient macrophages had a defect in DOK3 phosphorylation upon LPS stimulation. Knock down of DOK3 expression in RAW264.7 cells lead to increases in TNF-alpha production in response to LPS. These data indicate that DOK3 has a negative function in regulating LPS response. Thus, our study suggests a novel pathway that negatively regulates TLR4 responses. Upon LPS-stimulation of macrophages, DAP12 recruits SHIP1 which in turn regulates the activation and localization of DOK3. DAP12, SHIP1, and DOK3 are required to control LPS-induced cytokine production possibly via the Erk pathway. Studies are in progress further define the key proteins required for this negative pathway and to validate our findings in mouse models of shock.
EAT-2 links SLAM-family receptors with PLCγ1 to activate human NK cells (116.8)
Authors: Wilson Timothy , Brown Marion
Published: January 2023

Cell surface receptors are important mediators of pathogen recognition and intercellular communication. The SLAM-family receptors are a group of Immunoglobulin Superfamily receptors that signal through the small SH2 domain-containing adapters SAP and EAT-2. These adapters promote cellular activation through interactions with Src-family kinases and PKC-θ, though putative inhibitory signaling can be found under some conditions. Our laboratory previously reported a novel interaction between the adapter protein EAT-2 and PLCγ1, suggesting a potent activating potential for EAT-2 in NK cells. Subsequent experiments have demonstrated that EAT-2 phosphorylated on Tyr-127 specifically recruits PLCγ1 but not PLCγ2. By shRNA-mediated knockdown of human NK cell gene expression, we determined that activation of cytotoxicity through SLAM-family receptors was critically dependent on PLCγ1. Importantly, cytotoxicity through other receptors such as CD2 or NKp30 was unaffected by the absence of PLCγ1. While mouse studies have implicated PLCγ2 as a primary mediator of NK cell activation, our study indicates parallel pathways for induction of calcium flux, and demonstrates the importance of the EAT-2/PLCγ1 pathway in human NK cell activity. Further experiments to measure the individual contributions of SAP and EAT-2 have revealed important roles for these proteins in NK cell cytotoxicity.
Stimulating effect of alternatively spliced Interleukin-4 on cultured human T lymphocytes (117.15)
Authors: Luzina Irina , Lockatell Virginia , Atamas Sergei
Published: January 2023

Interleukin-4 is naturally produced in two splice isoforms, one encoded by exons 1-4 (IL-4), and the other encoded by exons 1, 3, and 4 (IL-4δ2). Our recent studies have suggested that both variants are biologically active in vivo, with partially overlapping, yet distinct effects on immune inflammation. In contrast, experiments in cell culture revealed minimal, if any, independent activity of recombinant IL-4δ2. We hypothesized that the reason for this difference between the in vivo system and cell culture may be the instability of purified recombinant IL-4δ2. To address this hypothesis, we co-cultured primary human T cells with normal human fibroblasts that have been engineered to secrete either IL-4δ2 or IL-4. In this system, IL-4δ2 can act on T cells immediately upon release, and it may be also stabilized by other factors generated by cells. Lymphocytes were labeled with CFSE, and their proliferation was analyzed by flow cytometry after 7 days of co-culture. One to three successive generations of cells were observed. Multiplex ELISA assays revealed a 2.5-fold increase in IFN-γ, a 10-fold increase in TNF-α, and more than a 100-fold increase in IL-1β in the presence of IL-4δ2, compared to control co-cultures or those expressing IL-4. There was no difference in the expression of IL-6 or MCP-1 between the IL-4δ2- and IL-4-expressing co-cultures. Thus, IL-4δ2 is biologically active in vivo and in vitro, and its effects are different from those of IL-4.
Human CD4 and CD8 T cells distinctly upregulate Suppressors of Cytokine Signaling 1 and 3 following Interferon-β treatment. (117.24)
Authors: Schneider Raphael , El-Khoury Lama , Beauseigle Diane , Duquette Pierre , Prat Alexandre , Arbour Nathalie
Published: January 2023

Suppressor of cytokine signaling (SOCS) proteins have been identified as a negative feedback loop to cytokine signaling. Emerging evidence supports the potential role of SOCS in controlling immune disorders, but data regarding their expression in human T cells is still sparse. Interferon-β (IFN-β) is a broadly used and effective treatment for multiple sclerosis (MS), although mechanisms of actions are still incompletely resolved. We postulate that IFN-β mediates beneficial effects through the induction of SOCS in peripheral T cells. We first evaluated the expression of SOCS-1 and SOCS-3 at the mRNA (qRT-PCR) and protein levels (FACS, Western blot, immunocytochemistry) by human CD4 and CD8 T cells following IFN-β treatment. A rapid and significant increase of SOCS-1 and SOCS-3 was observed upon cytokine addition, especially in the CD8 T cell compartment. To mimic patients under IFN-β treatment, both T cell subsets were chronically exposed to physiological doses of IFN-β measured in treated patients. Only SOCS-1 but not SOCS-3 levels were elevated after each IFN-β re-exposure. These effects were more pronounced in CD8 T cells. Our preliminary data indicate different expression of SOCS in untreated and IFN-β-treated MS patients. Our results suggest that SOCS-1 contributes to the IFN-β-mediated beneficial effects in MS and that SOCS proteins are distinctly expressed in human T cell subsets. We are currently testing the functional impact of SOCS-1 on human T cell responses.
MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy (147.12)
Authors: Liu Philip , Wheelwright Matthew , Teles Rosane , Komisopoulou Evangelina , Edfeldt Kristina , Ferguson Benjamin , Nehta Manali , Vazirnia Aria , Rea Thomas , Sarno Euzenir , Graeber Thomas , Modlin Robert
Published: January 2023

Leprosy is a human disease in which the clinical manifestations correlate with the host immune response to the pathogen, Mycobacterium leprae. In tuberculoid (T-lep) patients the infection is localized and Th1 cytokines predominate in lesions; whereas, in lepromatous patients (L-lep) the infection is disseminated and Th2 cytokines predominate. Evidence also suggests a role for the vitamin D-dependent antimicrobial pathway in contributing to host defense in leprosy. By profiling miRNAs in leprosy lesions, we identified 13 miRNAs differentially expressed in L-lep vs. T lep lesions. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target genes important in T-lep lesions, including twelve Th1 and antimicrobial pathway genes. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in M. leprae-infected monocytes and induced by the mycobacterial virulence factor phenolic glycolipid-I (PGL-I). Hsa-mir-21, by downregulating Toll-like receptor induced CYP27B1 and IL1B and upregulating IL-10, inhibited the vitamin D-dependent antimicrobial peptide gene expression. In contrast, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced antimicrobial peptide gene expression. The ability of the pathogen to upregulate hsa-mir-21 which targets multiple genes associated with the immunologically localized disease form provides an effective mechanism to escape the vitamin D antimicrobial pathway.