bluuebunny/crossref_metadata_2025 huggingface dataset:
Concordance of increased B1 cell subset and lupus phenotypes in mouse and human dependent on BLK expression genotypes (BA3P.114)
Authors: Wu Ying-Yu , Georg Ina , Díaz-Barreiro Alejandro , Varela Nieves , Lauwerys Bernard , Kumar Ramesh , Bagavant Harini , Castillo-Martín Mireia , Salem Fadi , Marañón-Lizana Concepción , Alarcón-Riquelme Marta
Published: January 2023

Polymorphisms in the BLK gene have been associated with autoimmune diseases, including systemic lupus erythematosus (SLE), with risk correlating with reduced expression of BLK. How reduced expression of BLK causes autoimmunity is unknown. Using Blk+/+, Blk+/-, and Blk-/- mice, we show that aged female Blk+/- and Blk-/- mice produced higher anti-dsDNA IgG antibodies and developed immune complex-mediated glomerulonephritis, compared to Blk+/+ mice. Starting at young age, Blk+/- and Blk-/- mice accumulated increased numbers of splenic B1a cells, which differentiated into class-switched CD138+IgG-secreting B1a cells. Increased infiltration of B1a-enriched cells into the kidneys was also observed in aged Blk+/- and Blk-/- mice. In human, we found that healthy individuals had BLK genotype-dependent levels of anti-dsDNA IgG antibodies as well as increased numbers of a B1-like cell population, CD19+CD3-CD20+CD43+CD27+, in peripheral blood. Furthermore, we describe the presence of B1-like cells in the tubulointerstitial space of human lupus kidney biopsies. Taken together, our study reveals a previously unappreciated role of reduced BLK expression on extraperitoneal accumulation of B1a cells in mice, and the presence of IgG autoantibodies and B1-like cells in human.
MH7A human synovial cell migration is dependent on the expression of B cell activating factor (BA3P.115)
Authors: Lee Jiyoung , Lee Jiyoung , Park So-Jin
Published: January 2023

Rheumatoid arthritis (RA), one of autoimmune diseases, is characterized by histological hallmarks such as hyperplasia of the synovial lining cells, mononuclear cell infiltration, angiogenesis, and osteoclastic bone destruction. Synoviocyte migration and invasion play a role in the chronicity and severity of RA. B cell activating factor (BAFF) which plays a role in maturation and maintenance of B cells is also associated with autoimmune RA. However, limited information is available on the effect of BAFF on synoviocyte migration. Here, we investigated whether BAFF could regulate synoviocyte migration in the presence of absence of TNF-a, the main pro-inflammatory cytokine in RA using MH7A synovial cells that is established by transfection with the SV40 T antigen (Miyazawa et al., 1998). Cell migration was assessed using Boyden chamber assay. When BAFF expression was inhibited with BAFF-siRNA, MH7A cell migration was decreased gradually in the presence of absence of TNF-. While BAFF expression was dependent on p38 kinase activation, no changes in BAFF expression was detected by the inhibition of JNK phosphorylation. In conclusion, our results suggest for the first time that migration of synovial cells in RA could be controlled by BAFF expression.
Differential responses to autoantibody deposition in CD45E613R BALB/c and C57Bl/6-129 F1 glomeruli is mediated by alterations in the autoantibody repertoire and inflammatory renal mononuclear cells (BA3P.110)
Authors: Hai Si-Han , Hermiston Michelle
Published: January 2023

The mechanisms regulating the transition from autoreactivity to autoimmunity are poorly understood. CD45E613R mice express a point mutation in the juxtamembrane wedge that results in dysregulated phosphatase activity. Despite similar hyperactive ITAM-mediated signaling in multiple cell lineages, the phenotypic consequences of this mutation vary with genetic context. While both BALB/c and C57Bl/6-129 F1 CD45E613R mice develop similar high titer anti-nuclear antibodies (ANA) and immune complex deposition in the glomeruli, only F1 mice develop glomerulonephritis (GN). Here, we leverage these distinct phenotypes in the context of a common point mutation to define mechanisms leading to GN. BALB/c ANA are primarily anti-dsDNA while F1 ANA are anti-dsDNA, -H3 and -Sm/RNP. Consistent with this profile, F1 B cells have increased TLR7/9 signaling. Treatment of RAW264.7 cells with aged F1 but not BALB/c CD45E613R sera induces an inflammatory phenotype. Additionally, IL-10, IL-12, MCP-1 and RANTES are elevated in aged F1 CD45E613R mice. Analysis of young, autoantibody-free mice shows increased CD11b+ CD11c+ F4/80+ renal mononuclear cells surrounding F1 but not BALB/c CD45E613R glomeruli. Upon stimulation, only F1 CD45E613R renal mononuclear cells produce TNFα and IFNγ. Together, these data support a model in which an expanded antibody repertoire combined with altered kidney immune cell subsets and altered cytokines mediate the transition from autoreactivity to autoimmunity.
Anti-insulin B cells in T-independent germinal centers (BA3P.111)
Authors: Williams Jonathan , Henry-Bonami Rachel , Hulbert Chrys , Thomas James
Published: January 2023

Previous studies demonstrate B cells that harbor IgM-restricted anti-insulin transgenes are anergic. However, most pathogenic insulin autoantibodies are IgG, suggesting that class switch recombination (CSR) is an important biological process in mediating loss of tolerance and autoimmunity. This breach in the B cell compartment is widely assumed to be T cell-dependent (TD). To examine peripheral tolerance for CSR-competent anti-insulin B cells, we generated C57BL/6 mice that harbor anti-insulin VDJH-125 site-directed to the native Ig H chain locus (VH125SD). Contrary to expectations, anti-insulin B cells fail to produce IgG anti-insulin Abs following TD immunization of VH125SD mice with heterologous insulin. In contrast, anti-insulin B cells produce both IgM and IgG2a anti-insulin Abs following immunization of VH125SD mice with a T cell-independent (TI) conjugate of insulin to Brucella abortus ring test antigen (BRT). Anti-insulin B cells undergo clonal expansion in vivo and emerge as IgM+ and IgM- GL7+ Fas+ germinal center (GC) B cells following immunization with insulin-BRT, but not BRT alone. These data demonstrate that combined BCR/TLR-4 stimulation reverses anergy in anti-insulin B cells and promotes antigen-specific GC responses and production of IgM and IgG insulin autoantibodies. Environmental factors that lead to infection and inflammation could play a critical yet under-appreciated role in driving loss of tolerance and autoimmune disease.
Generation of NOD mice carrying R619W mutation in PTPN22 gene by CRISPR/Cas9-mediated genome engineering (BA4P.134)
Authors: Lin Xiaotian , Pelletier Stephane , Gingras Sebastien , Marquardt Kristi , Bottini Nunzio , Green Douglas , Sherman Linda
Published: January 2023

An allelic variant of protein tyrosine phosphatase nonreceptor 22 (PTPN22), PTPN22R620W, is strongly associated with type 1 diabetes (T1D) and several other autoimmune diseases in human, and increases the risk of developing T1D by 2-4 fold. Non-obese diabetic (NOD) mouse is an ideal spontaneous T1D model that shares with human many genetic pathways contributing to T1D; however, it has not previously been possible to introduce targeted mutations into NOD mice due to a lack of germline competent NOD embryonic stem cells. To overcome this technological hindrance, we used CRISPR/Cas9 technology to precisely mutate PTPN22 gene in its endogenous locus in NOD zygotes, and successfully produced 2 independent lines of NOD mice expressing PTPN22R619W variant, a murine ortholog of the human autoimmune-associated allele. We have also obtained a line carrying PTPN22 null allele. We have demonstrated the mutated PTPN22 genes in all lines have successfully germline transmitted. We have found a significant increase of T1D incidence in the heterozygous NOD PTPN22R619W mice, suggesting its strong effect in heterozygosity, which is very relevant to human T1D since PTPN22R620W is most commonly carried in heterozygosity. This is the first time the autoimmune-associated PTPN22 allele has been introduced into a spontaneous model of T1D and, to the best of our knowledge, the first time CRISPR/Cas9 technology has been used to genetically modify NOD mice.
Lupus nephritis develops in a RIP3-independent manner for both males and females (BA4P.136)
Authors: Corradetti Chelsea , Jog Neelakshi , Caricchio Roberto
Published: January 2023

Lupus nephritis is one of the most serious manifestations of lupus and is characterized by severe inflammation and necrosis, and if untreated leads to renal failure. Although lupus occurs more often in women, both sexes develop glomerulonephritis, which in males develops earlier and is more severe than in females. Previous work by our lab found that loss of Poly [ADP-ribose] polymerase 1 (PARP-1) activity, an enzyme involved in DNA repair and necrotic cell death, resulted in milder nephritis and higher survival rates among males only. A second necrotic pathway involves Receptor-Interacting Serine-Threonine Kinase 3 (RIP3), hence in this study we aimed to investigate the impact of RIP3 on nephritis and determine if this pathway is relevant in both sexes. To this end we used three inducible murine models of lupus nephritis: nephrotoxic serum (NTS)-induced nephritis, pristane-induced lupus and chronic graft versus host (cGVH) disease. We found that absence of RIP3 imparts no positive or negative effects on the development of lupus nephritis in all three models and in both male and female mice. In addition, in vivo experiments done in absence of both RIP3 and PARP-1 did not result in any additive effects between the two pathways, conferring protection only when PARP-1 was absent. Based on these studies, we conclude that RIP3 does not appear to play a significant role in lupus nephritis, nor does it appear to interact with PARP-1 to accelerate, worsen or ameliorate the disease.
STAT4 controls GM-CSF production by both Th1 and Th17 cells during EAE (BA4P.140)
Authors: Harrington Laurie , Nozell Susan , Rajbhandari Rajani , Benveniste Etty , McWilliams Ian
Published: January 2023

Factors mediating Th1 and Th17 CD4 T cell driven autoimmunity remain unclear. In the mouse model of Multiple Sclerosis, experimental autoimmune encephalomyelitis (EAE), mice genetically deficient in STAT4 are resistant to disease. In contrast, deletion or inhibition of the Th1-associated cytokines IL-12 or IFNγ, which act upstream and downstream of STAT4, respectively, does not ameliorate disease. This highlights a non-canonical role for STAT4 during EAE. Recent reports have indicated that GM-CSF, a cytokine produced by both Th1 and Th17 effector CD4 T cell subsets, is critical for EAE pathogenicity. Importantly, the requirement for STAT4 in mediating GM-CSF production has not yet been investigated. Herein, we demonstrate that STAT4 controls GM-CSF production by both Th1 and Th17 CD4 T cells during EAE, and this is CD4 T cell intrinsic. Furthermore, STAT4 mediates CD4 T cell derived GM-CSF production independently of the cytokines IL-12 and IL-23. Importantly, we show that STAT4 directly interacts with the Csf2 locus in effector CD4 T cells during EAE. Overall, these studies illustrate the previously unrecognized role of STAT4 to regulate GM-CSF production by both Th1 and Th17 effector cell subsets during EAE pathogenesis.
Induction of arginase-1 by TNF-α in oligodendrocytes is controlled by SHP-1 (BA3P.119)
Authors: Minchenberg Scott , LaRocca Daria , Massa Paul
Published: January 2023

Multiple Sclerosis (MS) is a debilitating neurological disease characterized by inflammatory demyelination in the central nervous system (CNS), and tumor necrosis factor (TNF-α) has been implicated in its pathogenesis. TNF-α is toxic to the myelin forming oligodendrocytes and their connected myelin sheaths, and its expression is elevated in MS lesions. However, the precise mechanism for TNF-α-mediated toxicity to oligodendrocytes remains unknown. Moreover, the role of regulatory molecules, including SHP-1, in controlling both TNF-α signaling and susceptibility to TNF-α-mediated demyelinating diseases in the CNS is unclear. Here, we show that compared to normal littermate controls, oligodendrocytes of SHP-1-deficient mice (motheaten), which are highly susceptible to inflammatory demyelination, display a significant increase in inflammatory gene expression in response to TNF-α. Unexpectedly, the most highly induced of these inflammatory genes is arginase-1, an enzyme that converts arginine to L-ornithine with major effects on allergic-type immune responses. To elucidate the mechanism underlying this induction, we are determining how TNF-α induces transcriptional activation of the arginase-1 promoter in an SHP-1-dependent manner. Further, we are investigating if abnormal expression of arginase-1 in oligodendrocytes is responsible for the oligodendrocyte dysfunction and increased demyelination observed in SHP-1-deficient mice.
Chronic liver fibrosis triggers autoantibody production by B cells in a MyD88-dependent pathway (BA4P.126)
Authors: Thapa Manoj , Tedesco Dana , Elrod Elizabeth , Han-Hwan Jin , Grakoui Arash
Published: January 2023

Our recent finding suggests that B cells contribute to liver fibrosis through hepatic stellate cells (HSCs) activation. While antibodies produced by the activated intrahepatic B cells contribute to autoimmunity is not known, mixed cryoglobulinemia and other B cell disorders are the leading extrahepatic manifestations of advanced liver diseases in humans. Here, our aim was to determine whether increased antibody production during liver fibrosis could potentially lead to systemic autoimmunity by production of autoantibodies and immune complexes (IC). We studied the role of B cells in autoimmunity using a carbon tetrachloride (CCl4)-induced mouse model of fibrosis and a genetically engineered multidrug resistance gene 2 knock out (Mdr2-/-) mice, which constitutively develop progressive liver fibrosis. In both fibrosis models, the frequency of B cells in the liver was elevated and B cells exhibited increased state of activation as measured by CD44 and CD86 expressions, constitutive IgG production and elevated TNF-a production. By using antinuclear antibody (ANA) staining with HEp-2 stabilized substrate and C1q standard ELISA, we detected the presence of antinuclear antibodies (ANA) and IC respectively in the serum of C57BL/6 mice treated with CCl4 for six weeks and Mdr2-/- mice at 25 week. Interestingly, the targeted deletion of MyD88 signaling in B cells during liver fibrosis resulted in reduced ANA in the serum suggesting the critical role of MyD88 in systemic autoimmunity.
Regulation of chronic lung inflammation to house dust mite allergen by Wnt antagonist (CAM1P.144)
Authors: Bothwell Alfred , Chae WookJin , Teixeira Alexandra , Chan Pamela , Hao Liming , Rothlin Carla , Krause Diane
Published: January 2023

Recruitment of leukocytes in response to allergen is an important step in the initiation of airway inflammation. Here we demonstrate that the Wnt antagonist Dkk-1 regulates the formation of leukocyte-platelet aggregates (LPAs) following house dust mite (HDM) allergen challenge. We show that circulating Dkk-1 is elevated after recurrent HDM allergen challenges and the source is activated platelets. The increase of P-selectin glycoprotein ligand-1 (PSGL-1) expression in leukocytes was mediated by Dkk-1 while other cell adhesion molecules such as ICAM-1 or LFA-1 were only marginally affected. The formation of LPAs was markedly reduced in Dkk-1 hypomorphic doubleridge (Dkk-1d/d) mice that had a 90% reduction of circulating Dkk-1. Recurrent allergen challenges in Dkk-1d/d mice showed significantly reduced infiltration of leukocytes including neutrophils in the lung compared to wildtype mice. Importantly, we show that type 2 cytokines (e.g. IL-4, IL-5 and IL-13) from CD4 T cells in mediastinal lymph nodes (med LNs) of Dkk-1d/d mice were substantially reduced compared to wildtype mice following allergen exposure. Consistently, we observed Gata-3 and c-Maf induction in Th2 cells did not occur in med LN CD4 T cells from Dkk-1d/d mice. Therefore, our results demonstrate that the Wnt antagonist from activated platelets regulates the early stage of leukocyte infiltration in the challenged tissue, thereby regulating inflammation and type 2 immune responses.
A novel IL233 fusion cytokine in a therapeutic approach for acute kidney injury (CAM1P.155)
Authors: Stremska Marta
Published: January 2023

Since we found that interleukin (IL)-2 upregulated IL-33 receptor-ST2 on CD4+ T-cells, we hypothesized that IL-2 and IL-33 may cooperate to expand endogenous Tregs. Indeed Tregs expressed ST2 and IL-33 contributed to increase Treg-expansion. We designed a fusion cytokine (IL233) containing the activities of IL-2 and IL-33 for better targeting of Tregs. We expressed the cytokines in E.coli, purified them to homogeneity and tested them in mouse model of ischemia reperfusion injury (IRI). C57BL/6 male mice were pretreated (ip) with different doses of cytokines prior to 26 min of bilateral ischemia and 24 hours of reperfusion and kidneys were characterized for function (plasma creatinine) and acute tubular necrosis. IL-2 and IL-33 in combination or as IL233 fusion cytokine, but not alone protected mice from IRI, with IL233 being more efficient than the combination. Our data suggests that the IL233 treatment first increases splenic levels of Tregs followed by their mobilization. Since both IL-2 and IL-33 also promote T-helper (Th)-2 response, an increase in IL-4 and IL-5, and a decrease in inflammatory cytokines IFN-γ and TNF-α were observed, indicating a shift towards a protective Th2 response. Adoptive transfer studies revealed higher degree of protection in mice injected with Tregs obtained from mice pretreated with IL233. Thus, IL233 fusion cytokine attenuates kidney inflammation to protect from IRI and bears strong potential to be a therapeutic agent for acute kidney injury.
Inhibitory effect of serum amyloid A on receptor activation of nuclear factor κB ligand-induced osteoclast formation (CAM1P.160)
Authors: Bae Yoe-Sik , Oh Eunseo , Lee Ha Young , Kim Hak Jung , Park Yoo Jung
Published: January 2023

Serum amyloid A (SAA) is a major acute-phase protein produced from the liver. Since the level of SAA rapidly increases up to approximately 1,000-fold during inflammation, it has been known as a marker of several chronic inflammatory diseases such as atherosclerosis. In this study, we find that SAA strongly inhibits osteoclast formation induced by RANKL. SAA not only markedly blocked the expression of several osteoclast-associated genes including TRAF6 and OSCAR but also strongly induced the expression of negative regulators such as MafB and IRF-8. Moreover SAA decreased c-fms expression levels on the cell surface during osteoclastogenesis via shedding of the c-fms extracellular domain. SAA also restrained fusion of osteoclast precursors by blocking intracellular ATP release. This inhibitory response of SAA is not mediated by well-known SAA receptors such as formyl peptide receptor 2, Toll-like receptor 2, or Toll-like receptor 4. These findings provide insight into a novel inhibitory role of SAA in osteoclastogenesis, and suggest that SAA is an important endogenous modulator which regulates bone homeostasis.
Anti-inflammatory effects of essential oils extracted from Chamaecyparis obtusa in inflammatory models of mice and raw 264.7 cells (CAM1P.165)
Authors: Park Yujin , Yoo Seung-Ah , Kim Wan-uk , Cho Chul-Soo , Yoon Chong-Hyeon
Published: January 2023

Although the biological activities of essential oil extracted from Chamaecyparis obtusa (EOCO) are not yet fully understood, antimicrobial, antifungal and anti-inflammatory effects have been reported. In this study, we investigated the anti-inflammatory effects of EOCO in two inflammatory models of mice, carrageenan-induced paw edema and thioglycollate-induced peritonitis, and lipopolysaccharide (LPS)-stimulated Raw 264.7 macrophage cells. Levels of pro-inflammatory cytokines were analyzed by ELISA, expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were determined by western blotting, and nitrite concentration was measured using Griess reagent. The thicknesses of mice paw edema induced by carrageenan and interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels in paw homogenates were significantly decreased in the EOCO (5 and 10 mg/kg) group as compared with control group. In mice with thioglycollate-induced peritonitis, EOCO (5 and 10 mg/kg) treatment reduced the numbers of total cells and suppressed tumor necrosis factor (TNF)-α, IL-1β, and IL-6 levels in peritoneal fluid. EOCO also decreased nitric oxide (NO), TNF-α and IL-6 production and inhibited iNOS and COX-2 expressions in LPS-stimulated Raw 264.7 cells. These data showed that EOCO has anti-inflammatory effect both in vivo and in vitro, and this effect may be related with inhibition of inflammatory mediators. In conclusion, EOCO could be an effective therapeutic agent in inflammatory diseases.
IL-1 signaling regulates skin inflammation through directly activating and indirectly chemoattracting dermal γδ T17 cells (CAM1P.147)
Authors: Cai Yihua , Xue Feng , Liu Na , Fleming Christopher , Yan Jun
Published: January 2023

Interleukin (IL)-1β has been demonstrated to play an important role in skin inflammation. The mechanisms by which IL-1β signaling pathway regulates skin inflammation remain elusive. Previously we have demonstrated the critical role of dermal γδ T17 cells in psoriasis pathogenesis, therefore we hypothesized that IL-1β may regulate skin inflammation through the activation of dermal γδ T cells. To test this hypothesis, we established a neonatal thymocytes reconstitution system in which IL-1R signaling is selectively deficient in dermal γδ T cells or the other cells. The reconstituted mice with IL-1R signaling specific deficiency in dermal γδ T cells showed less IL-17 production in skin compared to control mice after IMQ application, but the epidermal thickness was not significantly changed. Similar phenotype was also found in the reconstituted mice with WT dermal γδ T cells, but IL-1R deficiency in all the other cells. These results indicate that IL-1R signaling in both dermal γδ T cells and the other cells is required to induce full-fledged skin inflammation. Indeed, we found that IL-1β stimulated keratinocytes (KCs) to secrete chemokines including CCL20. Therefore, IL-1β may regulate skin inflammation at two levels: directly activating dermal γδ T17 cells and stimulating KCs to secrete chemokines which may chemoattract peripheral γδ T cells to dermis, thus amplifying the inflammatory cascade. This work is supported by a grant from National Psoriasis Foundation.
Respiratory syncytial virus infection triggers HMGB1 release from lung epithelial cells to promote inflammatory cytokine production (CAM1P.152)
Authors: Hosakote Yashoda , Garofalo Roberto , Kurosky Alexander
Published: January 2023

Respiratory syncytial virus (RSV) is an important cause of lower respiratory tract infections in infants and young children. Oxidative stress is shown to play an important role in the pathogenesis of lung inflammation. We have shown that RSV induces oxidative stress by rapid generation of reactive oxygen species and downregulation of antioxidant enzyme expression resulting in lung inflammation. High mobility group box 1 protein (HMGB1) is a redox-sensitive multifunction protein that serves as both a chromatin protein and an extracellular signaling molecule. Although HMGB1 has been implicated in inflammatory diseases, there has been no report on its role in RSV related lung disease. The goal of this study is to dissect the mechanisms that induce the release of HMGB1 and associated cellular signaling in response to RSV infection. Human lung epithelial cells were infected with RSV and harvested over time to analyze HMGB1 expression by Western blot, immunohistochemistry and qRT-PCR. Our studies show for the first time that RSV infection induces the translocation of HMGB1 from cell nuclei to the cytoplasm and its subsequent release to the extracellular space demonstrating a novel mechanism of HMGB1 in the activation of cells to promote inflammatory cytokine release. Understanding of HMGB1 role in the setting of viral-induced oxidative stress may lead to the development of novel strategies to treat respiratory viral infections in pediatric and elderly population.
The atypical chemokine receptor ACKR4 facilitates dendritic cell migration during inflammation by scavenging CCL19 (CCR3P.205)
Authors: Bryce Steven , Asquith Darren , Bromley Shannon , Luster Andrew , Graham Gerard , Nibbs Robert
Published: January 2023

The migration of dendritic cells from tissues to draining lymph nodes is a critical step in the induction of peripheral tolerance and the initiation of adaptive immune responses. This is dependent on CCR7 expression by dendritic cells. In response to the chemokine CCL21, CCR7 directs dendritic cells into lymphatic vessels in the tissue, and permits their transit from the subcapsular sinus into the lymph node parenchyma. The CCR7 ligands CCL19 and CCL21 also bind ACKR4, an atypical chemokine receptor expressed by keratinocytes in the skin and lymphatic endothelial cells lining the subcapsular sinus. In mice, ACKR4 controls interfollicular CCL21 gradients, and enhances dendritic cell entry into the lymph node parenchyma from the subcapsular sinus. Here we report that Ackr4 deficiency disrupts CCR7-dependent dendritic cell arrival at skin-draining lymph nodes during cutaneous inflammation. We show that this, at least in part, is due to the defective departure of dendritic cells from inflamed skin, and is accompanied by dysregulation of bioavailable CCL19 and CCL21 in the skin. Strikingly, genetic deletion of CCL19 completely rescues the defective inflammation-driven trafficking of dendritic cells caused by Ackr4 deficiency. Thus, by regulating CCL19, ACKR4 helps maintain CCR7-dependent dendritic cell departure from inflamed tissues.
Metabolic inhibition by inositol-tetrakisphosphate delays thymocyte β-selection and renders it Notch-dependent (HEM2P.241)
Authors: Conche Claire , Westernberg Luise , Huang Yina , Rigaud Stephanie , Siegemund Sabine , Mukherjee Sayak , Nosaka Lyn'Al , Das Jayajit , Sauer Karsten
Published: January 2023

In β-selection, surface-expression of a pre-T cell receptor (pre-TCR) induces thymocyte metabolic activation, proliferation, survival and differentiation. This pivotal αβ T cell fate-determining event requires co-stimulatory Notch signals. Here, we show that this Notch dependence is enforced through antagonistic signaling by inositol-trisphosphate 3-kinase B (Itpkb) and phosphoinositide 3-kinase (PI3K). Itpkb produces soluble inositol-tetrakisphosphate which competes with the PI3K lipid-product PIP3 for binding to the effector-kinase Akt. Itpkb-/- thymocytes are pre-TCR hyperresponsive, hyperactivate Akt, downstream mTOR and metabolism, undergo an accelerated β-selection and can develop to CD4+CD8+ cells without Notch. This is reversed by inhibition of Akt, mTOR or glucose-metabolism. Thus, Itpkb restricts pre-TCR induced metabolic activation to enforce coincidence-detection of pre-TCR expression and Notch-engagement, and to prevent premature thymocyte maturation.
Development of regulatory T cells in the thymus: two paths to the same goal? (HEM2P.237)
Authors: Owen David , Mahmud Shawn , Farrar Michael
Published: January 2023

Mature thymic regulatory T cells (Tregs) are defined by dual expression of Foxp3 and CD25 on CD4SP thymocytes. However, the cellular pathways by which CD4 thymocytes differentiate into Tregs remain incompletely understood. Previous reports demonstrated that Treg development occurred via a two-step process. In step one, strong TCR signaling induces expression of CD25 leading to a CD25+Foxp3- Treg progenitor (TRP). In step two, exposure of this TRP to IL2 results in conversion to a CD25+Foxp3+ Treg. Recently, work by Singer and colleagues have defined a CD25-Foxp3lo TRP that can also be converted by IL2 into CD25+Foxp3+ Tregs. A key question is why two distinct cellular pathways involving distinct TRP populations are needed to reach the same endpoint. Using a combination of maturation marker analysis and RAG2-GFP reporter mice we observed that CD25+Foxp3- TRP arise more rapidly than the CD25-Foxp3lo TRP following positive selection. Analysis of Nur77-GFP reporter mice revealed that these two progenitor populations also display distinct affinity spectrums for self-peptide. The CD25+Foxp3- TRP have a high and relatively narrow range of TCR affinities for antigen, while the CD25-Foxp3lo TRP have a lower but broader range of TCR affinity for antigen. Our results suggest that these two different Treg developmental pathways generate Tregs with distinct TCR repertoires that may have distinct function in protecting against autoimmunity and reactivity to commensal organisms.
Human resistin, a neutrophil-derived metabolic and inflammatory mediator correlates with decreased lung function and NETosis in cystic fibrosis airway disease (HUM1P.261)
Authors: Forrest Osric , Chopyk Daniel , Ingersoll Sarah , Yoo Dae-goon , Brown Milton , Tangpricha Vin , Tirouvanziam Rabindra , Rada Balazs
Published: January 2023

Resistin, a protein initially cloned from mouse adipocytes, is a strong antagonist of insulin signaling. In humans, it is located in both the azurophil and specific granules of PMNs and as a result can be elevated in metabolic and PMN-driven inflammatory disorders. Cystic fibrosis (CF) combines PMN-dominated airway inflammation with profound metabolic anomalies, but the presence of resistin in CF patients has not been studied thus far. Methods: We measured resistin levels using an ELISA on platelet-free plasma and airway fluid from CF patients and measured the presence of neutrophil extracellular traps (NETs) a marker of neutrophil activation in CF airway fluid. Results: Resistin levels in platelet-free plasma were significantly higher in CF subjects than in HC subjects. Resistin levels in expectorated CF sputum were 2 to 3 orders (100-500 fold) of magnitude higher than in plasma. Interestingly, we found a strong negative correlation between sputum resistin and lung function (Spearman Rho=-0.81, P<10-4). Similarly resistin was also shown to positively correlate with NETs in CF sputum. Conclusions: Resistin levels are abnormal in CF plasma and strikingly elevated in CF airway fluid during chronic disease. These results show, as also suggested by prior studies, that extracellular resistin is associated with PMN-driven CF airway inflammation, including NET release. Mechanistic studies looking at the precise impact of resistin on metabolism and inflammation in CF are warranted.
ICOS deficiency results in defective ILC2 expansion, impaired Th2 responses, and increased mortality after bleomycin-induced lung injury (HUM1P.264)
Authors: Hrusch Cara , Casaos Joshua , Bonham Catherine , Blaine Kelly , Bryazka Dana , Jaffery Mohammad , Takahashi Stephenie , Noth Imre , Sperling Anne
Published: January 2023

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreversible lung scarring and loss of pulmonary function. RNA analysis has shown that patients with IPF have reduced ICOS expression in peripheral blood mononuclear cells. We now show that ICOS surface expression on CD4 T cells positively correlates with lung function in patients with IPF. However, whether decreased ICOS is a result of disease progression or playing a role in the pathogenesis is unclear. Therefore, using bleomycin to induce pulmonary fibrosis in mice, we found that ICOS was decreased in peripheral CD4 T cells similar to our findings in patients. In contrast, ICOS was increased on lung CD4 T cells by 3d post-bleomycin and remained elevated through 21d at the height of collagen deposition. Compared to wild-type B6 mice, ICOS-deficient mice had increased weight loss and mortality after bleomycin challenge, suggesting a protective role for ICOS. Strikingly, ILC2s expanded in the lung at 3d post-bleomycin in B6 mice, but not in ICOS-deficient mice. In B6 mice, ILC2 expansion was associated with CD4 T cell infiltration at 5d and Th2 cytokine production in the draining lymph nodes at 21d. However, ICOS-deficient mice that survived the initial bleomycin insult failed to recruit T cells to the lungs and had less IL-13 in the draining lymph nodes. Overall, these results indicate that ICOS expression promotes lung tissue repair by promoting both innate and adaptive type-2 immune responses.
Staphylococcal superantigens trigger rapid human IL-17A production by a novel population of memory T cells (HUM1P.268)
Authors: Szabo Peter , Goswami Ankur , McCormick John , Haeryfar S. M.
Published: January 2023

Toxic shock syndrome (TSS) is a life-threatening illness characterized by fever, hypotensive shock and multi-organ failure within hours of onset. TSS is caused by exposure to bacterial toxins known as superantigens (SAgs), which are produced by common Gram-positive bacteria such as Staphylococcus aureus. During infection, SAgs can activate of up to 20% of all T cells, resulting in massive and rapid release of pro-inflammatory cytokines, systemic intravascular coagulation, organ damage and death. However, the exact cellular mechanisms underlying the rapidity of this potentially fatal inflammatory reaction are far from clearly understood. In our preliminary experiments, human peripheral blood mononuclear cells (PBMCs) stimulated with SAgs in vitro showed immediate up-regulation (up to 2000-fold) of mRNA for the pro-inflammatory cytokine IL-17A. SAg-stimulated PBMCs also produced substantial amounts of IL-17A protein within hours of activation. The cells responsible for the rapid production of IL-17A were identified as a subset of memory T cells exhibiting a novel phenotype. Furthermore, neutralizing IL-17A at the onset significantly reduced both morbidity and mortality in a humanized mouse model of TSS. Our results reveal a critical role for memory T cells in TSS and define a novel contribution by IL-17A to the initiation and pathogenesis of the syndrome.
Repeated challenge with the hapten oxazolone provokes persistent vulvar mechanical sensitivity, labiar mast cell accumulation and nerve overgrowth in previously sensitized ND4 mice (HUM1P.270)
Authors: Chatterjea Devavani , Landry Jasmine , Dhanwada Jyothi , Mengistu Hanna
Published: January 2023

Vulvodynia is a chronic vulvar pain condition affecting up to 8-16% of 18-60 year-old women. Epidemiological studies have associated an increased risk of developing vulvodynia with a history of seasonal and insect allergies. We have previously demonstrated that acute labiar exposure to the hapten allergen oxazolone provokes increased vulvar mechanical sensitivity in ND4 Swiss mice. Here, we show that repeated oxazolone challenge produces persistent vulvar mechanical sensitivity in sensitized mice for at least12 days after 10 allergen exposures. This persistent sensitivity is accompanied by mast cell accumulation and overgrowth of cutaneous neurites both of which persist for at least 14 days after the final allergen exposure. These changes are accompanied by an early and transient increase in mRNAs encoding CADM1 - an adhesion molecule that facilitates nerve-mast cell synapses - in the skin and mRNAs encoding the inflammatory cytokine IL-6 in the spinal cord. Taken together, these neuroimmune biomarkers may serve as potential diagnostic/therapeutic targets for specific allergy-driven subsets of chronic vulvar pain.
FcγR3A genotype influences the inhibition of activated T cell proliferation by infliximab and adalimumab (HUM1P.313)
Authors: Casinghino Sandra , Cole Susan , Bono Christine , Al-Derzi Mazin
Published: January 2023

The anti-tumor necrosis factor (TNF) antibodies, infliximab and adalimumab are efficacious in Crohn’s disease (CD) patients but their relevant mechanism(s) of action (MOA) are not fully elucidated. Neutralization of TNF activity is a key efficacy mechanism, but may not be the only relevant MOA for efficacy in CD. Fc receptor-mediated dampening of the immune response has been proposed as one of the alternative hypotheses. Two FcγR3A-158 polymorphisms (158V and 158F) have been shown to influence binding affinity of IgG1 antibodies, and CD patients with V/V genotypes have increased biological responses to infliximab. Previous work by others showed that in a mixed lymphocyte reaction (MLR), infliximab and adalimumab induced formation of immunosuppressive macrophages in an Fc region-dependent manner and that these macrophages inhibited proliferation of activated T cells. We ran MLRs with FcγR3A genotype-matched donor pairs to determine if Fc receptor genotype influenced the anti-inflammatory response by infliximab and adalimumab. Our results showed that regulatory macrophages were induced regardless of FcγR3A genotype but that decreases in T cell proliferation were more pronounced with V/V donor pairs than with V/F or F/F genotypes. In conclusion, our work suggests that infliximab and adalimumab may be more effective at inducing an anti-inflammatory state in individuals with the 158V polymorphism of FcγR3A.
Effects of the Bruton’s tyrosine kinase inhibitor ibrutinib on humoral immunity in patients with chronic lymphocytic leukemia (HUM1P.258)
Authors: Sun Clare , Lee Yuh Shan , Lipsky Andrew , Farooqui Mohammed , Herman Sarah , Salem Dalia , Stetler-Stevenson Maryalice , Yuan Constance , Aue Georg , Wiestner Adrian
Published: January 2023

Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia. Ibrutinib, a covalent inhibitor of Bruton’s tyrosine kinase (BTK), is highly active in B cell malignancies, including CLL. Inactivating mutations in BTK cause X-linked agammaglobulinemia. We evaluated the impact of ibrutinib on immunoglobulin (Ig) levels in CLL patients (pts) treated at NIH. Consistent with previous reports, IgG remained stable during the first 6 months, but decreased from a median of 707 mg/dL at baseline to 611 mg/dL at 24 months (17% reduction, n=25, p<0.0001). In contrast, there was a sustained increase in IgA and a transient increase in IgM. Next, we assessed serum free light chains (FLCs) to distinguish the effects on clonal from non-clonal B cells. In κ-clonal CLL cases, the clonal (κ) FLC decreased from a median of 6.71 to 1.42 mg/dL after 6 months (n=39, p<0.0001). Interestingly, the non-clonal (λ) FLC increased, suggesting the recovery of normal B cell function. Consistently, we found an increase in normal B cells in blood and bone marrow, including early B cell precursors. In comparison, CLL pts (n=16) treated with anti-CD20-based chemoimmunotherapy also showed a decrease in IgG at 24 months (12% reduction, p=0.0052), and surprisingly, an improvement in IgA levels, suggesting that the latter effect is not drug-specific but due to tumor reduction. The consequences of long-term BTK inhibition on immune function require further study.
Dendritic cells are functionally relevant targets of IL-25 in allergic lung inflammation (HYP2P.320)
Authors: Siebenlist Ulrich , Claudio Estefania , Tassi Ilaria , Wang Hongshan
Published: January 2023

Asthma is a chronic inflammatory disease of the airways, typically triggered by allergens and often associated with type 2 responses. Skewing towards type 2 inflammation is due to release of one or more of the cytokines IL-33, TSLP and IL-25 by epithelial cells upon encounter with allergens or helminthes or when stressed. All three cytokines stimulate innate lymphoid cells type 2 (ILC2) to rapidly produce the classic inflammatory type 2 cytokines IL-13 and IL-5, and administration of any one of these cytokines into lungs initiates a strong inflammatory asthma-like response. IL-25 is the only member of the IL-17 cytokine family involved in type 2 responses. To understand its contributions to allergic lung inflammation, we first investigated for mechanisms by which IL-25 initiates responses on its own. In addition to ILC2s, we unexpectedly found that lung resident dendritic cells (DCs) rapidly responded. To assess the relevance of this finding, we made use of mice in which IL-25 cannot signal DCs. Activation of DCs by IL-25 was required for production of IL-9 by T cells, a cytokine linked to asthma as well. Importantly, IL-25-mediated activation of DCs was also required for IL-9 production by T cells in the context of house dust mite (HDM)-induced pulmonary inflammation, a physiologic inducer of asthma in patients. Our findings identify lung resident DCs as novel and functionally relevant targets of IL-25, even in the context of the broadly acting HDM allergens.
IκBζ regulates human lung epithelial cytokine responses to house dust mite antigens (HYP2P.330)
Authors: Sundaram Kruthika , Wewers Mark
Published: January 2023

Rationale: Asthma is a chronic lung disease characterized by inflammation of bronchial epithelium. House dust mite (HDM) is a common allergen that triggers asthma. IκBζ is a newly recognized member of the NFκB family that is induced in lung epithelial cells to regulate their production of cytokines. We hence analyzed how IκBζ might modulate HDM‘s induction of IL-6 and GMCSF in lung epithelial cells. Methods: We co-cultured primary human bronchial epithelial cells or BEAS2B cells with HDM alone or with HDM-treated human monocytes, macrophages or dendritic cells and measured GMCSF, IL-6 and IκBζ protein expression. Results: Bronchial epithelial cells expressed IκBζ and released IL-6 and GMCSF only when co-cultured with HDM treated monocytes. However, these epithelial cells did not respond to direct HDM stimulation when cultured alone or to co-culture with HDM-treated macrophages or dendritic cells. The monocyte co-culture response correlated with its IL-1β release and was blocked by IL-1 receptor antagonist and the TLR4 antagonist Rhodobacter sphaeroides LPS. GFP tagged IκBζ accumulated in the nucleus of epithelial cells and si-IκBζ suppressed the epithelial release of IL-6 and GMCSF. Conclusions: Monocytes release IL-1β in response to HDM thus inducing the lung epithelial cell IκBζ-mediated expression of asthma associated inflammatory cytokines. We propose that control of IκBζ expression deserves future study as a key regulator of HDM induced asthma.
PARP inhibition blocks chronic asthma symptoms in a house dust mite asthma model and differentially modulates human CD4+T cell function (HYP2P.332)
Authors: Ghonim Mohamed , Pyakurel Kusma , Ibba Salomi , Wang Jeffery , Rodriguez Paulo , Lammi Matthew , Zea Arnold , Mansy Moselhy , Ochoa Augusto , Boulares Hamid
Published: January 2023

Asthma incidence is on the rise. In the United States alone, more than 20 million individuals suffer from the disease. A sizable portion of these asthmatics do not respond to the existing drugs. Accordingly, the need for new therapies is an immediate a necessity. One of the most essential goals for treatment is the identification of drugs that block the release or effects of Th2 cytokines. Using HDM-based chronic model, we show that a single administration of olaparib, a PARP inhibitor, was effective in decreasing lung eosinophilia. Two additional administrations of the drug provided better protection and significantly reduced airway hyper-responsiveness. These results were confirmed using similarly exposed PARP-1−/− mice. Such protective effects may be mediated by a significant reduction in Th2 cytokines and a concomitant increase in T-regulatory cells. In addition, we show that PARP is highly activated in lung tissues and PBMCs from asthmatics demonstrating the relevance to the human disease. Using human CD4+T cells, we show that PARP inhibition significantly reduced GATA-3 expression, Th2 cytokine production without affecting production of IFN-γ, T cell proliferation, or the overall numbers of Th1 or Th2 cells. However and similarly to our animal studies, PARP inhibition markedly increased the number of human T-regulatory cells. Our results provide an additional support to the potential of PARP inhibition as a novel strategy for the treatment of asthma
Innate CD4+ T cells associated with development of virus- induced demyelinating disease (INC1P.362)
Authors: Olson Julie , Penm Steven
Published: January 2023

Theiler’s murine encephalomyelitis virus (TMEV) infection of susceptible mice results in a persistent infection in the central nervous system (CNS) that leads to the development of a chronic progressive demyelinating disease associated with a pro-inflammatory immune response. TMEV- induced demyelinating disease serves as a mouse model for multiple sclerosis. TMEV infection of resistant mice leads to clearance of the virus from the CNS without development of demyelinating disease. We have previously shown that the innate immune response to TMEV directly affects development of demyelinating disease in susceptible mice. We recently identified a population of CD4+ T cells that infiltrate into the CNS during the innate immune response to TMEV in susceptible mice. Most interestingly, the resistant mice infected with TMEV do not have innate immune infiltration of CD4+ T cells into the CNS. The CNS infiltrating innate immune CD4+ T cells in the TMEV- infected susceptible mice were NK1.1-, αβ+ and CD3+. The innate immune CD4+ T cells expressed high levels of IL-17A and IFNγ but low levels of IL-22. Furthermore, susceptible mice expressed high levels of IL-23, IL-1β, and IL-6 during the innate immune response to TMEV which promotes IL-17A expression in CD4+ T cells. Therefore, the innate immune response in susceptible mice promotes the infiltration of innate CD4+ T cells into the CNS which express pro-inflammatory cytokines associated with development of demyelinating disease.
Basal forebrain cholinergic signaling regulates peripheral cytokine responses (INC1P.349)
Authors: Pavlov Valentin , Silverman Harold , Dancho Meghan , Regnier-Golanov Angelique , Ochani Mahendar , Hanes William , Olofsson Peder , Chavan Sangeeta , Golanov Eugene , Prado Vania , Prado Marco , Tracey Kevin
Published: January 2023

Brain cholinergic signaling regulates peripheral cytokine release and inflammation (Nat Rev Endocrinol, 2012, 8:743). We have previously shown that the centrally-acting acetylcholinesterase inhibitor galantamine suppresses peripheral pro-inflammatory cytokine release through a brain muscarinic receptor-mediated and vagus nerve-dependent mechanism. Here, we provide insight into the brain mechanisms of this regulation. VAChTNkx21-Cre-flox/flox mice with selective, basal forebrain cholinergic neuron genetic ablation of the vesicular acetylcholine transporter (an important determinant of acetylcholine release), and control VAChTflox/flox mice, were treated with galantamine or saline and subjected to endotoxemia. Galantamine (3mg/kg) i.p. administration suppressed serum TNF levels in control mice only. In addition, serum TNF and IL-10 in VAChTNkx2 1-Cre-flox/flox mice were significantly higher (2.9-fold, P<0.05) and lower (57%, P<0.05) respectively, as compared to control mice. Furthermore, electrical stimulation of the medial septum, an important basal forebrain cholinergic region, significantly lowered serum TNF (79%, P<0.01) and other pro-inflammatory cytokine levels in endotoxemic mice, as compared to sham-stimulation. These results demonstrate a previously unrecognized role of basal forebrain cholinergic signaling in the regulation of peripheral inflammation.