bluuebunny/crossref_metadata_2025 huggingface dataset:
Metformin attenuates the Type I Interferon response in human peripheral blood leukocytes.
Authors: Titov Anton A , Brusko Todd M. , Sobel Eric , Morel Laurence
Published: January 2023

Metformin (Dimethylbiguanide) is used for controlling blood sugar levels in patients with Type 2 Diabetes, primarily by suppression of hepatic gluconeogenesis. Recently, it has drawn attention in a clinical trial (ChiCTR-TRC-12002419) for its ability to reduce disease flares and lower effective prednisone dosage in Systemic Lupus Erythematosus (SLE) patients. We have previously demonstrated that a therapy combining metformin and a glucose inhibitor can reverse lupus in mouse models by normalizing CD4+ T cell metabolism. Studies in mouse and isolated ex vivo human CD4+ T cells have characterized metformin’s capacity to downregulate oxidative metabolism and skew differentiation away from Th1 and Th17 towards a Treg lineage. To investigate the mechanisms by which metformin modulates CD4+ T cells we performed a microarray study of CD4+ T cells from n=5 SLE patients and n = 5 healthy controls (HCs). CD4+ T cells were isolated from subjects and activated (anti-CD3, anti-CD28) in media or 1mM metformin for 24 hr. Multiple ANOVA analysis yielded 1,211 transcripts with a FDR <0.05 between groups. Pathway analysis determined that type I interferon stimulated genes (ISG) were significantly downregulated in the CD4+ T cells of both cohorts, but to a greater extent in the HC cohort. Downregulation of ISGs by metformin was validated in primary PBMCs stimulated with IFNα ex vivo by qRT-PCR. Luciferase reporter assays suggest that metformin reduces IL-6-induced activation of STAT3. These experiments provide evidence that the therapeutic potential of metformin in SLE may be multifaceted, including the modulation of type I IFN stimulated genes, as well as STAT3 signaling. Studies are ongoing to characterize the key mediators involved in these pathways.
The therapeutic potential of natural killer cells in treating systemic lupus erythematosus.
Authors: Reighard Seth D , Brunner Hermine , Waggoner Stephen N
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic disease defined by rampant autoantibody production and multi-organ pathology. Standard treatments for SLE are broadly immunosuppressive and often ineffective, as ~38% of patients develop lupus nephritis (LN) that can result in kidney failure and death. Current therapeutics may lack efficacy because they do not target the specific immune processes underlying SLE. For example, lupus autoantibodies can arise from aberrant germinal centers (GCs) driven by dysregulated follicular helper T cells (TFH). We have shown that natural killer (NK) cells suppress activated TFH during immunization, resulting in blunted GCs and antibody production. We hypothesize that an effective new strategy to treat SLE would involve harnessing of this NK cell-mediated humoral regulation. To assess whether therapeutic elevation of NK cell numbers can ameliorate disease, we used IL-2/αIL-2 complexes to expand endogenous populations of NK cells in the bm12 cGVHD mouse model of SLE-like disease. We also evaluated ex vivo activation, expansion, and infusion of NK cells as an immunotherapeutic means to ameliorate disease in mouse models of SLE. Finally, we are engineering human NK cells expressing a programmed death-ligand 1 (PD-L1)-based chimeric antigen receptor (CAR) that will facilitate cytolytic elimination of TFH and B cells that highly express programmed cell death protein 1 (PD-1). We plan to evaluate the capacity of these CAR NK cells to selectively kill PD-1+ targets and to reduce autoantibody-mediated LN in humanized mouse models of SLE. These CAR NK cells represent an innovative means to curtail autoantibody production, particularly during disease flares, and thereby promote disease remission in SLE.
Ebola virus: Development of Filovirus glycoprotein Fc fusion proteins as a vaccine candidate
Authors: Konduru Krishnamurthy
Published: January 2023

Krishnamurthy Konduru and Gerardo Kaplan

Laboratory of Emerging Pathogens, Division ofEmerging and Transfusion Transmitted Diseases, Center for Biologics Evaluationand Research, Food and Drug Administration, Silver Spring, MD 20993.

Recent Ebola virus(EBOV) epidemic in West Africa underscores the urgent need of vaccines ortherapeutics. Here we describe the development of EBOV and Marburg virus (MARV) subunit vaccines based on the extracellular domain of the viral glycoprotein (GP) fused to the Fc fragment of human IgG (EBOV gp-Fc or MARV gp-Fc) and expressed in mammalian cells. These GP fusion proteins undergo the complex post-translational processing observed in the membrane-bound GP. In vaccinated mice, EBOV gp-Fc induced high levels of total and neutralizing anti-GP antibodies and elicited T-cell immunity characterized by the production of IFNγ (Konduru et al. 2011). Vaccinated guinea pigs developed a strong humoral response as measured by total and neutralizing antibodies to GP, and were completely protected against EBOV lethal challenge (Konduru et al. 2016). We are currently extending our studies to the filo virus lethal challenge model in monkeys. Our preliminary data indicates that EBOV gp-Fc and MARV gp-Fc induces total and neutralizing antibodies as well as recall CD8+ T-cell immunity as assessed by the production of IFNγ and TNFα in PBMC. Further experiments, including protection against lethal challenge in monkeys, will be required to determine whether Filovirus GP-Fc fusion proteins could be developed as a candidate vaccine.
Modulation of epitope-specific antibody response by HIV Env immune-complex vaccines
Authors: Kumar Rajnish , Tuen Michael , Jiang Xunqing , Itri Vincenza , Pazner Susan Zolla , Kong Xiang-Peng , Hioe Catarina
Published: January 2023

HIV-1 envelope (Env) antigen is an important target for both neutralizing and non-neutralizing antibodies (Ab) against the virus. In the RV144 vaccine trial, correlate for reduced risk of HIV acquisition is the presence of binding but non-neutralizing Ab to V1V2 and V3 loops of HIV Env gp120. However, induction of high titer, durable, and cross-reactive Ab responses against these Env regions by vaccination is still an elusive goal.

Our past studies have shown that V3 immunogenicity can be significantly enhanced by vaccination with gp120/mAb immune complexes (IC) vs gp120 alone. Current study further examined parameters that confer enhanced immunogenicity to IC by testing in mice IC vaccines made with different HIV Env gp120 (clade B vs clade E) and mAb of different specificities (V3, V2, or CD4 binding site (CD4bs)). In addition, IC made of clade C gp140 and CD4bs mAb were also tested. The data showed that Greater titers of cross-reactive V3-binding Ab were induced by IC made of clade B gp120 and mAb to V2 or CD4bs as compare with gp120 alone, although the gp120/CD4bs IC was more potent in eliciting neutralizing V3 Ab. IC made with clade E gp120 or clade C gp140 did not show the same effects.Greater Ab responses to V1V2 were induced by IC made of clade E gp120 vs gp120 alone, regardless of the mAb used for IC, but the Ab were not neutralizing. IC made with clade B gp120 and clade C gp140 did not enhance V1V2 Ab titers.

These results demonstrate the capacity of Env/mAb IC vaccines to modulate induction of Ab responses to V1V2 and V3, and this activity is dependent on both Env proteins and mAb used to form IC. Further exploration using a cocktail of IC is proposed to improve Ab repertoires generated upon vaccination.
IL-33 enhances the induction, durability, and breadth of the antibody response to a DNA/protein-based HIV Env vaccine
Authors: Kobie James , Sarkar Sanghita , Piepenbrink Michael , Basu Madhubanti , Keefer Michael , Thakar Juilee , Hessell Ann Jones , Haigwood Nancy Logan
Published: January 2023

Induction of a sustained and broad antibody (Ab) response is a major goal in developing a protective HIV vaccine. DNA is an attractive priming strategy when combined with protein or viral vector boosting; however, DNA priming alone fails to induce a substantial Ab response, limiting its potential as a stand-alone immunogen. Using the VC10014 DNA/protein vaccine consisting of gp160 plasmids and gp140 proteins derived from an HIV clade B infected subject who developed broadly neutralizing serum Abs, and which has been previously shown to induce Tier 2 heterologous neutralizing Abs in rhesus macaques, we screened a panel of factors in C57BL/6 mice for their ability to enhance the Env-specific Ab response. IL-33, an alarmin, emerged as the lead candidate. The addition of recombinant IL-33 during the gp160 DNA priming phase dramatically changed the kinetics of the Ab response, inducing serum gp120-specific IgG (>1:12,500 titer) after a single DNA immunization, which was not detectable in mice that did not receive IL-33 (p<0.0001). Then following boosting with DNA/protein coimmunization in Alum, gp120-specific IgG levels were extraordinarily durable, remaining stable even at 6 weeks after final immunization compared to mice not receiving IL-33 (p<0.0001), in which they rapidly declined. IL-33 also increased the cross-clade breadth and avidity of the serum gp120-specific IgG response. Analysis of the Env-specific B cell immunoglobulin repertoire and the IL-33 mechanism of action is ongoing. These results demonstrate the profound effect that priming conditions can have on the quality of the Env-specific antibody response.
Spike nanoparticle and recombinant adenovirus 5 vaccines induce specific antibodies against the Middle East respiratory syndrome coronavirus (MERS-CoV)
Authors: Park Hyo-Jung , Lee Eun-Young , Jung SeoYeon , Ko Hae Li , Lee Sang-Myeong , Nam Jae-Hwan
Published: January 2023

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a single-strained RNA virus and is included in Betacoronavirus. While, in the Arabian Peninsula, it has shown an endemic pattern, a sporadic pattern was observed elsewhere. In 2015, there was big outbreak of the MERS-CoV in Korea. However, since we still have no clinically available vaccine to prevent the MERS-CoV infection, such a vaccine is urgently required. In the present study, we developed two types of MERS-CoV vaccines: a spike nanoparticle vaccine produced by an insect culture system and a recombinant adenovirus 5 vaccine expressing spike protein. Both vaccines induced total IgG, IgG1, and IgG2a against spike protein of the MERS-CoV. Thus, both vaccines can trigger Th1 and Th2 immune responses. Interestingly, as compared to spike nanoparticle formulation with Alum without RNA adjuvant, spike nanoparticle formulation with Alum and RNA adjuvant showed higher titers of IgG1 and IgG2a at 10 days after boosting immunization. Moreover, T cell proliferation in splenocytes of immunized mice was well induced after the spike nanoparticle treatment. Thus, RNA adjuvant may be effective to increase vaccine efficacy against the MERS-CoV. Taken together, the results of the present study suggest that the developed two type of MERS-CoV vaccines–the spike nanoparticle vaccine and the recombinant virus vaccine–may be helpful to prevent the spread of the MERS-CoV.
Immunomodulation by chicken NK-lysin-derived peptide, cNK-2 on chicken macrophages and monocytes.
Authors: Kim Woohyun , Lillehoj Hyun S. , Min Wongi
Published: January 2023

Chicken NK-lysin (cNK-lysin) is a homologue of human granulysin. Human granulysin is found in the cytolytic granules located in human natural killer and cytotoxic T lymphocytes. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core α-helical region of cNK-lysin, have antimicrobial activity against apicomplexan parasites such as Eimeria spp., via membrane disruption. Besides of antimicrobial activity of AMPs, the immunomodulatory activity of AMPs mediated by their interactions with host cells is increasingly recognized. Thus, in this study, we investigated whether cNK-2 modulates the immune response in the chicken macrophages and monocytes by evaluating the induction of chemokines, anti-inflammatory properties, and activation of signalling pathways. cNK-2 induced the expression of CCL4, CCL5 and interleukin-1β (IL-1β) in HD11 cells, chicken macrophage cell line, and CCL4 and CCL5 in primary monocytes. We also determined that cNK-2 suppresses the lipopolysaccharide-induced inflammatory response by abrogating IL-1β expression. The immunomodulatory activity of cNK-2 involves the mitogen-activated protein kinases-mediated signalling pathway, including p38, extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinases, as well as the internalization of cNK-2 into the cells. These results indicate that cNK-2 is a potential novel immunomodulating agent rather than an antimicrobial agent.
A non-prototypic leukocyte immune-type receptor (LITR) is expressed in response to virus infection in the channel catfish, Ictalurus punctatus
Authors: Blackmon Laura Elizabeth , Wilson Melanie , Bengten Eva
Published: January 2023

The channel catfish, Ictalurus punctatus, is a well-defined comparative model used to study innate and adaptive immunity. Leukocyte immune-type receptors (LITRs) are unique to teleosts and represent a large polymorphic and polygenic family of immunoregulatory receptors phylogenetically related to mammalian LRC-encoded proteins. LITRs are currently classified as either type I or type II receptors with inhibitory or activating signaling capabilities, respectively. Notably, some LITRs, as defined by their reactivity with anti-LITR monoclonal antibody CC41, are distinct markers of a cytotoxic cell population which undergoes expansion during the antiviral response against Channel Catfish Virus (CCV). Recent genomic analyses have identified a new LITR subset, and in contrast to the previously described inhibitory type I and activating type II LITRs, members of this non-prototypic LITR subset lack typical D1-D2 immunoglobulin domains. In addition, members of this subset are not CC41-reactive. Interestingly, increased message expression of a member of this subset, termed LITR-NP, is observed in catfish clonal G14D T cells after infection with CCV. When G14D cells are treated with UV irradiation, heat shock or serum starvation, however, LITR-NP message expression is not induced. This expression pattern suggests that LITR-NP may serve as a target ligand for NK receptors in catfish.
Probing host immune response to porcine reproductive and respiratory syndrome virus (PRRSV) infection using 3′RNAseq and NanoString arrays
Authors: Walker Kristen , Hong Linjun , Reecy James M. , Fritz-Waters Elyn , Dong Qian , Rowland Robert R. R. , Ireland Derek D.C. , Verthelyi Daniela , Tuggle Christopher K. , Dekkers Jack C.M. , Lunney Joan K.
Published: January 2023

Porcine reproductive and respiratory syndrome (PRRS) is economically the most important disease of pigs worldwide and, when combined with porcine circovirus (PCV2) coinfection, increases severity of disease. Our goal is to assess the role of host genetics in determining efficacy of PRRS vaccine responses and on resistance/susceptibility to coinfection challenge. We performed 3′RNA-seq and NanoString analyses to explore the blood transcriptome and identify mechanisms and biomarkers involved. We used a nursery pig model in which 50% of 200 pigs received a PRRS vaccination (Vx); after 28 days all were challenged with PRRSV and PCV2. Each pig was genotyped (80K SNP chip) and deeply phenotyped, including repeated blood sampling for viremia level (VL), and immune proteins, and weight gain (WG). Blood RNA was analyzed to identify differentially expressed (DE) genes associated with anti-viral response phenotypes. Previous work identified a PRRS resistance marker on swine chromosome 4 (SSC4) so, for these studies, samples from 7 sib sets (AA or AB for SSC4 marker; with/without Vx; 28 pigs total) were used for RNA preparation from Tempus tube-preserved blood samples collected following vaccination (4, 7, 11, 14, 21 days) and challenge (0, 4, 7, 11, 14, 21, 28 days). Samples were subjected to 3′RNA-seq and swine immune-focused, 220 gene NanoString codeset analyses. Data will be presented on DE gene expression, and particularly on immune related genes, that may contribute to controlling PRRS vaccination and PRRSV and PCV2 challenge responses. Further studies are aimed at identifying genes and biomarkers that regulate anti-viral response phenotypes. Funding: USDA ARS, USDA NIFA grant # 2013-68004-20362.
A noninvasive cutaneous delayed type hypersensitivity test is effective for evaluating the effects of handling on immunological stress levels in rats
Authors: Redmond Sarah , Tingle April , Cooper Diamond , Ugarte Claudia Paniagua , Hayes Dayna
Published: January 2023

Rats are a common model for human physiology and psychology, yet routine handling associated with animal maintenance and treatment administration has been shown to impact behavioral and physiological measures in rats, which raises concerns about their use as a model. Adolescent and adult female rats (Sprague-Dawley, n=6 per group) were subjected to handling treatments (restraint, tickling, or playful handling) to evaluate the potential to minimize the physiological impacts of handling stress. Treatments were administered every other day for 21 days, and body weights, blood smears, and ultrasonic vocalizations were collected throughout this time and for 14 subsequent days without handling treatments. A noninvasive delayed type hypersensitivity test was performed by sensitizing rats to 2,4-Dinitrochlorobenzene on abdominal skin and later assessing the response to ear lobe exposure by measuring ear thickness. The greatest increase in lobe thickness was consistently observed at 48 hours post exposure, with few animals showing inflammation that persisted more than 72 hours. Assessments in the middle of the handling period (day 10) and at the end of the handling period (day 21) showed significantly less swelling for adolescent rats receiving the restraint treatment as compared to rats in the tickling or playful handling groups. This effect was observed in both adolescent and adult rats, however the adolescent rats showed more significant changes in circulating blood cell populations and body weight than did adults. No differences were detected outside of the handling treatment period. These results further establish that routine handling can impact immunological measures in rats and demonstrate efficacy of this noninvasive assay.
Primary bovine CD4+ T cells rapidly reprogram metabolism to meet energetic demands during activation
Authors: Eder Jordan , Sacco Randy E
Published: January 2023

T lymphocytes undergo a metabolic reprogramming from predominantly oxidative phosphorylation in the resting state to aerobic glycolysis in an activated state. Efforts aimed at understanding T cell metabolism have concentrated primarily on humans and mice, however T cell metabolism in large animals, such as cattle and pigs, has yet to be examined in detail. Food production animals have been selected for maximal growth at minimal cost. Understanding the link between immunity and metabolism would be beneficial to producers in improving health status and longevity of their herd. As large animals can serve as alternative models in translational research, understanding species-specific immunometabolism is critical. Here, we characterized the metabolic processes in bovine CD4+ cells by interrogating mitochondrial respiration and glycolysis using the Seahorse Extracellular Flux Analyzer. We show that bovine CD4+ cells tend to have lower basal oxygen consumption rates (OCR) as well as lower extracellular acidification rates (ECAR) compared to mouse CD4+ T cells. Despite this, bovine CD4+ cells have an increased metabolic potential, % spare respiratory capacity, and % glycolytic reserve in comparison to mouse CD4+ T cells. Bovine CD4+ cells do not have as pronounced a mitochondrial or glycolytic response in comparison to the mouse CD4+ cells, but would be capable of rapidly modifying their cellular metabolism in the event of environmental stressors.
Relievable effect of dietary Allium hookeri on LPS induced intestinal inflammation response in young broiler chickens
Authors: Lee Youngsub , Lee Sunghyen , Gadde Ujvala Deepthi , Oh Sungtaek , Lillehoj Hyun S.
Published: January 2023

A study using 150 one-day-old broilers was conducted to assess the effects of Allium hookeri (AH) root and fermented root on inflammatory responses and intestinal barrier integrity of LPS challenged broiler chickens. Birds were randomly assigned to six groups (n = 25 birds/treatment) and fed standard diets supplemented with AH root or fermented root at two different levels (1% or 5%). Control birds were provided with non-supplemented basal diets. At 7 days of age, 5 groups (n = 125) in each dietary treatment were injected with LPS (1 mg/kg body weight), and the remaining 25 birds were injected with sterile PBS for negative control. LPS challenge significantly reduced average body weight gain at 24hr post-injection compared to PBS control, and increased by 1% with fermented root supplementation compared to LPS-challenged control. Serum α-1-AGP levels and transcript levels of IL-1b, IL-8, TNFSF15 and LITAF in jejunum were significantly higher in LPS-injected chickens, α-1-AGP levels in serum were reduced by AH root or fermented root (1, 5%) supplementation. Cytokine levels (IL-1b, IL-8 and LITAF) were also down-regulated by AH root and fermented root (1 and 5%) supplementation. However, TNFSF15 reduction was only significant by fermented root (1 and 5%) supplementation. The reduced expression of tight junction proteins (JAM2 and occludin) and MUC2 by LPS challenge was induced by root or fermented root (1% and 5%) supplementation, but there was no significant effect in ZO1. These findings demonstrated that dietary AH root or fermented root influences anti-inflammatory activity and tight junction protein expression in LPS-induced chickens.
Role of lipid inositol phosphatase SHIP-1 in Germinal Center B cell selection and function
Authors: Trivedi Nikita H , Luo Wei , Hawse William F , Shlomchik Mark J
Published: January 2023

Germinal Centers (GC) are sites within secondary lymphoid organs in which B cells undergo proliferation and differentiation. The PI3K signaling pathway is a central modulator of GC B cells (GCBC) selection and function. The PI3K pathway can be regulated by several enzymes including Src homology 2-containing inositol 5’-phosphatase (SHIP-1). SHIP-1 is known to be hyper-phosphorylated in GCBCs. Upon activation, SHIP-1 hydrolyzes phosphatidyl inositol triphosphate (PIP3) to generate phosphatidyl inositol 3,4 biphosphate (PIP2). PIP2 can bind to signaling proteins that are crucial to BCR signaling such as TAPP1/2 and Akt/PKB. To ascertain the role of SHIP-1 in survival and selection of GCBC we used a tamoxifen inducible Cre system to delete SHIP-1 from B cells during an ongoing GC response to NP-CGG. SHIP-1 deletion led to bi-modal expression of SHIP-1 in GCBC expressing intermediate (SHIP-1 int) and low levels of SHIP-1 (SHIP-1 lo) compared to WT controls (SHIP-1 hi). SHIP-1 int GCBC showed reduced phosphorylation of signaling proteins S6 and increased phosphorylation of Btk upon BCR crosslinking with anti-u antibody. SHIP-1 lo cells were un-responsive to BCR crosslinking and had the highest frequency of dead cells and Caspase 3 positive cells among all 3 groups. This data suggests that SHIP-1 expression and activity is crucial to GCBC signaling and survival. The role of PIP2 in regulating BCR signaling in GCBC remains to be elucidated.
Small size of membrane-tethered ligands regulates phosphatase segregation in γδ TCR triggering
Authors: Li Fenglei , Choudhuri Kaushik
Published: January 2023

Ligand recognition by the T cell antigen receptor (TCR) is central to effective immunity. Unlike αβ T cells, γδ T cells do not recognize pMHC antigens or use coreceptors CD4/8. Instead they recognize invariant membrane-tethered ligands, which are upregulated at sites of inflammation and infection. The substantial structural similarities between αβ and γδ TCRs prompted us to investigate whether they share triggering mechanisms. However, recent studies show that binding-induced γδ TCR conformational changes are not induced by physiological ligands, leaving open the possibility that other mechanisms are involved. We investigated the stress-inducible murine surface protein T22, that is recognized by the G8 γδ TCR - since detailed biophysical and structural information is available for this system. Here we tested whether ‘size-based’ kinase-phosphatase segregation (also known as the kinetic-segregation model) mediates G8 TCR signal initiation. Elongation of the T22 ectodomain leads to failure of TCR phosphorylation and IL-2 release. Electron microscopy showed that elongated T22/G8 leads to increased intermembrane spacing at the immunological synapse. Conversely, truncation of the large CD45 ectodomain attenuated triggering, pointing to a critical dependence on ‘close contact’ for phosphatase exclusion. To establish the physiological relevance of our findings, we confirmed that T22 elongation abrogates triggering in primary G8 γδ T cells, and using a second γδ TCR (KN6). We are currently completing investigations on the role of kinetic-segregation in hapten-specific and human γδ TCR triggering. Taken together, our findings establish that γδ TCR triggering occurs primarily through the kinetic-segregation mechanism.
Role of the butyrophilin juxtamembrane region in ligand binding and T cell activation
Authors: Wiemer Andrew J. , Nguyen Khiem , Li Jin , Puthenveetil Robbins , Lin Xiaochen , Hsiao Chia-Hung Christine , Vinogradova Olga
Published: January 2023

The B7 protein butyrophilin 3A1 (BTN3A1) is an immune receptor for pathogen-derived small isoprenoid diphosphates such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP). Binding to these ligands promotes a multifaceted immune response including activation of Vγ2Vδ2 T cells. Here, we use small-angle x-ray scattering (SAXS) to visualize the internal domain of BTN3A1 as it undergoes a conformational change following ligand binding. To better understand the cause of the conformational change, we used nuclear magnetic resonance (NMR) spectroscopy to determine that the juxtamembrane region is important to BTN3A1 activation by HMBPP. The spectral location of the affected amide peaks, partial NMR assignments, and juxtamembrane mutants (ST297AA or T304A) investigated confirm that the backbone amide of at least one threonine residue (T304), adjacent to conserved serine, comes close to HMBPP, while double mutation of non-conserved residues (ST297) perturbs the local fold of the protein. Threonine residue mutation reduces the ability of BTN3A1 in K562 cells to trigger activation of primary human Vγ2Vδ2 T cells in response to HMBPP. Our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that is mediated by the juxtamembrane region and required for activation. These results provide insight into the unique activation mechanism of butyrophilin-type immune receptors.
Notch ligands DLL4 and Jagged1 exert differential effects in allergic airway inflammation
Authors: Huang Miao-Tzu , Chen Yi-Lien , Lien Chia-I , Hsu Li-Chung , Chiang Bor-Luen
Published: January 2023

Notch is a pleiotropic signaling family that has been implicated in pathogenesis of allergic airway diseases; however, the distinct function of individual Notch ligands remains elusive. By using an OVA-induced murine asthma model, we investigated whether Notch ligands, Jagged1 and DLL4, exert differential effects during allergic airway inflammation. We found that whilst Jagged1 inhibition mitigated Th2-dominated airway inflammation, blockage of DLL4 aggravated the Th2-mediated asthma phenotypes. Additionally, blockage of Jagged1 signaling enhanced IL-17 production and neutrophilic airway infiltration. In vitro, exogenous Jagged1 induced Th2-skewed responses, whereas augmented DLL4 signaling displayed a dual role by promoting expansion of both Tregs and Th17. In vivo, DLL4 blockage impaired Treg differentiation which plausibly resulted in exaggerated asthma phenotypes. On the contrary, administration of DLL4-expressing antigen-presenting cells promoted endogenous Treg expansion and ameliorated the allergic responses. Therefore, whilst Jagged1 induces Th2-skewed inflammation, DLL4 elicits an essential self-regulatory mechanism via Treg-mediated pathway that overcomes Jagged1-induced Th2 responses and facilitates resolution of the airway inflammation. These findings uncover a disparate function of Jagged1 and DLL4 in allergic airway diseases, suggesting the feasibility of Notch ligand-specific targeting in therapy of allergic airway diseases.
IL-7 plays as a critical factor for the maintenance of allergen-specific memory CD4 T cells in the lung and airways
Authors: Yeon Seung-min , Jung Yong Woo
Published: January 2023

Memory T cells can respond rapidly against repeated antigen exposure, and maintain their population with extended period of time. While these characteristics of memory T cells have mainly been studied during viral infections, their roles in allergic diseases remain to be determined. Since allergic patients suffer relapses and exacerbations of the diseases by intermittent exposure to allergens, we hypothesized that allergen-specific memory CD4 T cells are present in the lung and airways and the crucial survival factors for these cells are provided in their microenvironments. In order to test this hypothesis, we transferred ovalbumin (OVA)-specific T cells into naïve Balb/c mice intravenously and induced allergic inflammation by treating these mice with a mixture of house dust mite and OVA intranasally. We found that allergen-specific memory CD4 T cells were present and survived more than 70 days in the lung and airways. In order to understand how these cells maintain their homeostasis, we blocked IL-7 signaling by treating these mice with an anti-IL-7Rα antibody. The numbers of allergen-specific memory CD4 T cells decreased in the mediastinal lymph node, lung and airways after IL-7Rα blockade. Furthermore, we found that allergen-specific memory CD4 T cells in the lung and airways induced airway inflammation regardless of T cell recruitment from circulation. In conclusion, the allergen-specific memory CD4 T cells are present in the allergic lung and airways, and the IL-7 signaling plays a critical role in the maintenance of these cells.
Estrogen contributes to sex differences in M2-polarization during asthma
Authors: Keselman Aleksander M , Fang Jessie , White Preston B. , Heller Nicola M
Published: January 2023

Asthma is a chronic inflammation of the airways that affects over 300 million people worldwide. Asthma exhibits sex differences, affecting mostly boys in childhood and women in adulthood. Alveolar macrophages have emerged as major mediators of allergic lung inflammation. Macrophages express receptors that recognize and respond to estrogen. We hypothesized that estrogen enhances M2 polarization of alveolar macrophages and thereby contributes to asthma. We found increased expression of the canonical mouse M2 marker YM1 by alveolar macrophages from allergic female mice compared to male mice. Consistently, bone marrow derived macrophages (BMMs) from female mice exhibited higher levels of YM1 and ARG1 than did BMMs from male mice following stimulation with IL-4. This sex difference is reflected on the epigenetic level as M2 promoters in BMMs from female mice exhibited enhanced acetylated H3 compared to BMMs from male mice following stimulation with IL-4. Pretreatment of BMMs from female mice with estrogen receptor ligands enhanced IL-4-induced M2-gene expression. Using ovariectomized (OVx) and LysMCREERaflox/floxmice, we show that an absence of either estrogen or the estrogen receptor-a (ERa) impairs M2 polarization following challenge with OVA. Thus, macrophages exhibit sex differences in their ability to polarize to an M2-phenotype in vitro and in vivo. Estrogen and ERa engagement augment IL-4-induced M2-gene expression. Together these data suggest that sex and hormonal factors contribute to sex differences in macrophage responses during asthma. Understanding the role of estrogen signaling in M2-polarization is paramount for identifying novel therapeutic targets to better treat women with asthma.
Pathogenic epigenetic programming of expanded B cell subsets in systemic lupus erythematosus
Authors: Scharer Christopher , Blalock Emily , Barwick Benjamin G , Gandham Bhanu , Neary Bridget , Sanz Ignacio , Boss Jeremy M
Published: January 2023

Systemic lupus erythematosus (SLE) is characterized by multiple B cell abnormalities that contribute to disease pathogenesis through the production of autoantibodies. The recent characterizations of immune cells from SLE patients suggest that distinct B cell subsets are expanded and that defects in epigenetic programming may be a contributing factor to disease etiology. Using high-dimensional flow cytometry, five B cell subsets were isolated from cohorts of SLE and HC subjects. To create a molecular map of each cell type, the transcriptome was defined by RNA-seq and the epigenome by dual DNA methylation (RRBS) and chromatin accessibility (ATAC-seq) profiling. These data reveal cell-type relationships that suggest pathogenic B cell subsets are programmed to differentiate down a different trajectory than normal B cells. Across all SLE B cell subsets a distinct disease signature was observed in each of the three data sets. Resting naïve B cells displayed a similar SLE signature to expanded B cell subsets indicating that a disease programming exists early in the differentiation pathway. Distinct epigenetic footprints were identified for transcriptional networks active in SLE B cells, revealing the unique environmental signals these cells receive. These results provide insights into the molecular programming of human B cell subsets, identify loci that may contribute to the expansion of distinct pathogenic B cell subsets, and suggest that early alterations in programming predispose B cells towards a disease fate.
Role and Regulation of CD11chi T-bet+ B cells in SLE
Authors: Wang Shu , Wang Jingya , Kumar Varsha , Naiman Brian , Karnell Jodi L. , Gross Phillip S. , Rahman Saifur , Manna Zerai , Hasni Sarfaraz , Siegel Richard , Sanjuan Miguel A , Cancro Michael , Kolbeck Roland , Ettinger Rachel
Published: January 2023

We examined a large cohort of systemic lupus erythematosus (SLE) patients and found an unusual B cell subset highly expanded. These B cells displayed a unique phenotype and expressed antigens not present on other B cell populations, including high densities of CD11c, FcRL5, and the T-box transcription factor, T-bet, but unexpectedly, were CD40lo and CD24−. Although these CD11chi cells were largely negative for the memory B cell antigen CD27, their telomere length was consistent with memory, rather than naïve B cells. Notably, the increase of CD11chi B cells in SLE significantly correlated with disease severity scores as well as with other markers of disease activity including serum IL-21. While memory B cells characteristically express low densities of IL-21R, these memory CD11chi B cells were IL-21R bright. In culture, IL-21 was a potent driver of CD11c expression when B cells were co-stimulated through the B cell receptor and CD40. Lastly, CD11chi B cells in SLE significantly correlated with blood plasma cells as well as a distinct set of autoantibodies, and importantly, were efficiently driven to differentiate into plasma cells in response to activated T cells. These data indicate that among the many roles of IL-21 in regard to B cell activation and differentiation, this pleiotropic cytokine also regulates CD11chi B cells, and presumably contributes to autoimmunity through the differentiation of autoreactive CD11chi B cells in SLE.
B cell αv integrins regulate development of autoimmunity in murine lupus models
Authors: Lacy-Hulbert Adam , Raso Fiona , Acharya Mridu
Published: January 2023

B cell activation by self-antigen and signaling through Toll-like receptors (TLR) such as TLR7 and TLR9 has been implicated in the development of autoimmunity. We have recently identified a mechanism by which αvβ3 integrin limit excessive B cell responses to antigens containing TLR ligands, including self-antigens. We have previously reported that mice in which αv integrins are selectively deleted from B cells develop increased levels of autoantibodies to RNA and double stranded DNA.

Based on these findings, we hypothesized that deletion of αv integrins would accelerate TLR dependent autoimmunity. To study this further, we crossed mice lacking αv integrins on B cells (αv-CD19 mice) with mice expressing increased levels of TLR7 (TLR7.1tg mice) that develop Lupus-like autoimmunity. We find that the resulting αv-CD19 TLR7tg mice had increased spleen weight in comparison with age-matched TLR7tg littermates. αv-CD19 TLR7tg mice also had increased number of IgG2c+ plasma cells and increased autoantibodies to RNA, all indicative of enhanced B Cell activation and development of autoimmunity. In addition, deletion of αv integrin on B Cells in this model leads to enhanced activation of T Cells. Currently, we are investigating the role of αv integrins on B Cell subpopulations in driving T Cell activation and antigen presentation in this model of autoimmunity.

B Cells contribute to autoimmunity by activating T Cells in addition to producing autoantibodies and cytokines. These studies allow us to understand how altered B Cell TLR signaling can lead to enhanced immune activation in autoimmunity and highlight the importance of αv integrins in regulating this process.
FDCs regulate self-reactive memory B cells through CD21
Authors: Das Abhishek , Heesters Balthasar , Bialas Allison , O’Flynn Joseph , Carroll Michael C
Published: January 2023

B cells are the key mediators in many autoimmune diseases like systemic lupus erythematosus (SLE). Yet little is known about the cells and factors that are vital for maturation and sustenance of autoreactive B cells. One of the hallmarks of autoimmunity is the formation of germinal centers (GCs), which are the site where self-reactive B cells expand and differentiate. Follicular dendritic cells (FDC), which are central to GCs, serve as an important source of antigen and chemokines and are essential for maintenance of GCs and B cell memory in response against foreign antigens. Whether FDCs play a similar role with self-antigens is not known. Using the 564Igi lupus strain in which autoimmunity is Type I interferon alpha (IFNa)-dependent, we found out FDCs are crucial for self-reactive GCs and secretion of autoantibody. Unexpectedly, we found that FDC uptake of nucleolar antigens via the CD21 receptor triggered endosomal TLR7 signaling and induction of IFNa, which is essential for escape of tolerance by self-reactive B cells. We postulated that self-antigen complexes were internalized via CD21. To test this idea, mice were treated with a neutralizing antibody specific for CD21 (clone 7G6). The treatment effectively “purged” FDC of self-antigen complexes leading to a reduction in interferon secretion, restoration of B cell tolerance and reduction in GC. Furthermore, using the Aid-cre-YFPflox-reporter strain, which permanently marks GC+ B cells, crossed with 564Igi, we found that blockade of CD21 resulted in a significant loss of self-reactive GC and memory B cells. These combined findings identify FDC as a novel therapeutic target for lupus.
Engagement of TLR4 with MAM superantigen promotes HMGB1-mediated induction of IRF7 and IFNα in synoviocytes of a mouse model of human RA
Authors: Mu Hong-Hua , Xia Neil
Published: January 2023

Mycoplasma arthritidis mitogen (MAM), a potent superantigen (SAg) secreted by M. arthritidis, is known to contribute to arthritis in mouse models of human rheumatoid arthritis (RA). Type I interferons (IFNs) are known to play an important role in RA. IFN gene transcription is activated by IFN-regulatory factors (IRFs) in response to innate immune recognition. We previously demonstrated that mouse strains with differing expression of the innate immune receptor TLR4 or/and TLR2 differ in their response to MAM. In this study, we investigated if MAM, or MAM with an autoantigen collagen II, can stimulate synovial fibroblasts (SF) to mount inflammatory responses in a mouse arthritis model. We found that in vitro stimulation of naïve SF with MAM in an arthritis susceptible mouse strain greatly increased level of IRF7 and IFNα. HMGB1, a heat-shock stress protein was also detected to produce in the cell culture of MAM-stimulated SF. In vivo studies showed mice injected with CII and MAM developed severe arthritis and exhibited a high HMGB1 level. An increased HMGB1 level was linked to the upregulation of the intracellular IRF7 and IFNα, and IRF7 was effectively inhibited by an anti-HMGB1 treatment, suggesting HMGB1 dependence. This led to the reduction of inflammatory mediators IL-6 and MMPs in inflamed joints of arthritis mice. Thus, our results suggest that MAM is capable of selecting the SF for local joint inflammation by promoting HMGB1 and IRF7 following interaction with TLR4. Our findings clearly demonstrates microbial agents, such as MAM SAg, can modify host tissue components, such as CII and stimulate synoviocytes, thus promoting local joint inflammation in autoimmune arthritis.
Elevated frequency of producing IL-17A Neutrophils in peripheral blood from patients with RA
Authors: Gonzalez-Orozco Maria , Juarez-Estrada Ana , Vazquez-Castellanos Gabriel , Barbosa-Cobos Rosa Elda , Limon-Camacho Leonardo , Ortiz-Navarrete Vianney
Published: January 2023

Introduction

Rheumatoid Arthritis (RA) is a systemic and chronic inflammatory disease, which affects 1–1.5% of population worldwide. Synovial membrane, cartilage and bone are the main sites affected by inflammation. The inflammatory process in RA leads to increased production of inflammatory cytokines IL-8, TNF-a, IL-6, IL-1b, IL-15, IL-18 e IL-17A in synovial fluid and serum. In addition to Th17, others cells such as gd T cells, iNKTs, mast cells and neutrophils also produce IL-17A. In mouse model of RA, IL-17A producing neutrophils seems to be important for the onset of the disease. Thus, we hypothesize that the population of IL-17A producing neutrophils is increased in patients with RA.

Objective

To analyze the frequency of IL-17A producing neutrophils in peripheral blood of patients with RA.

Methods

Peripheral blood from 68 patients with rheumatoid arthritis and 15 healthy donors has been collected, erythrocytes were lysed and peripheral blood leukocytes were stained for surface marker CD177 and for intracellular detection of IL-17A. Cells were analyzed by flow cytometry. It was also evaluated the frequency TH17 cells by flow cytometry using standard protocol.

Results

IL-17A producing neutrophils are increased in peripheral blood of patients with RA (0–19.5% Mean 1.66%) compared with healthy donors where no neutrophils IL-17A+ were observed (0–0.1% mean 0.05%). Surprisingly, Th17 cells were not detected in peripheral blood of patients with RA.

Conclusion

Neutrophils might be the most important source of IL-17A during the inflammatory response observed in patients with RA since we do not detect TH17 cells in these patients.
Antibody to HA4 of gingipain of Porphyromonas gingivalis is a possible biomarker for rheumatoid arthritis
Authors: Tsay Gregory J , Siao Shih-Hong , Lee Yi-Hsuan , Sun Justin , Chang Yu-Chao , Chen Jonathan , Lan Joung-Liang , Chang Jinghua Tsai , Zouali Moncef
Published: January 2023

Porphyromonas gingivalis (Pg) may play a role in the pathogenesis of rheumatoid arthritis (RA). Arg-gingipain A (RgpA), one of the virulence factors of Pg contains catalytic domain, hemagglutinin domain and sub-hemagglutinin domain including HA-1, HA-2 &HA-3, and HA-4. We detected the antibody against hemaglutinin domain (anti-HA), catalytic domain, HA-1, HA-2 &HA-3, and HA-4 in patients with autoimmune diseases and periodontitis by ELISA and Western blot

Methods

RgpA hemagglutinin domain and its subdomains were cloned respectively. ELISAs and Western blot using these recombinant proteins were developed and antibody reactivities examined in patients with RA, SLE, healthy normal controls, and individuals with PD.

Results

By ELISA, no serum from patients with RA, PD or normal controls was found to react with RgpA catalytic domain. In analysis of anti-RgpA hemagglutinin domain antibodies, 42 of 155 RA patients (27.1%), 16 of 48 PD patients (33.33%) were found to react with RgpA hemagglutinin domain. Anti-HA1 antibody, 39 of 155 RA patiens (25.16%), 6 of 48 PD patients (12.5%). Anti-HA2&3 antibody, 43 of 155 RA patients (27.74%), 10 of 48 PD patients (20.83%). Anti-HA4 antibody, 51 of 155 RA patients (32.9%), 2 of 36 SLE patients (5.56%), 7 of 48 PD patients (14.58%).

By Western Blot, serum from patients with both RA and PD reacted to HA (150kDa), HA1 (72kDa), and HA2/3 (45kDa), respectively. But only serum from patients with RA reacted to HA4(55kDa), not serum from patients with PD, SLE or healthy controls.

Conclusion

Only Serum from patients with RA recognize HA4. HA4 at the C-terminal domain of RgpA hemagglutinin of Pg is a target of RA. Anti-HA4 antibody is a candidate predictive biomarker for RA.
Neuropsychiatric symptoms of patients with systemic lupus erythematosus caused by intracisternal deposited lupus IgG through meningitis
Authors: Deng Guo-Min , Xie Changhao , Guo Yacong , Fang Xang , Qiao Wei
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by high levels of autoantibodies and multi-organ tissue damage. Although neuropsychiatric symptoms are frequent in patients with SLE, its pathogenesis remains unclear. In this study, we investigated pathogenesis of neuropsychiatric symptoms in SLE patients. We analyzed the medical records of 1131 patients with SLE and conducted animal experiments. We found that fifty-nine patients showed NPSLE clinical manifestation including headaches, seizure disorder, cognitive dysfunction, cerebrovascular disease, etc. Lupus-prone mice spontaneously develop meningitis and apoptosis of neurons. IgG is a major contributor in meningitis. This meningitis depends on the dose of IgG, but is not associated with the kind of autoantibody and disease activity. Monocyte/macrophage and TNF play important role in the development of this meningitis. Lupus IgG induces the apoptosis of neurons directly. The deposited IgG in brain tissue exerts an important role in the pathogenesis of NPSLE. This finding will promote the development of an effective therapeutic strategy for NPSLE patients.