bluuebunny/crossref_metadata_2025 huggingface dataset:
The upregulation of genomic imprinting DLK1-Dio3 miRNAs in lupus-prone mice is associated with DNA hypomethylation (BA4P.133)
Authors: Dai Rujuan , Lu Ran , Ahmed S Ansar
Published: January 2023

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) regulation are now increasingly recognized as vital contributors to lupus pathogenesis. Our recent studies revealed a large cluster of miRNAs, which is located at the genomic imprinted DLK1-Dio3 domain was dramatically increased in splenocytes from MRL-lpr mice when compared to control MRL mice. In this study, we found that MRL-lpr mice have reduced DNA methylation levels in whole splenocytes and also in purified splenic CD4+ T and CD19+ B cells when compared to MRL control, which was associated with the upregulation of DLK1-Dio3 miRNAs in lpr lupus cells. DNA demethylation treatment with 5-Aza-2’-deoxycytidine significantly increased DLK1-Dio3 miRNAs in splenic cells (especially Con A-activated cells) from MRL mice, but not MRL-lpr mice. This data further confirmed the contribution of DNA hypomethylation to the augmented expression of DLK1-Dio3 miRNAs in lupus splenic cells. Although the contribution of DLK1-Dio3 miRNAs to autoimmune disease remains unknown, our preliminary data revealed that inhibition of selected DLK1-Dio3 miRNA such as miR-379 reduced LPS-induced IFNγ in MRL-lpr splenocytes, suggesting a role of miR-379 in the regulation of inflammation. Together, our data is the first to show DNA methylation regulation of genomic imprinting DLK1-Dio3 miRNAs in autoimmune lupus, which may provide novel insight into epigenetic mechanisms in lupus pathogenesis.
PPARγ controls effector T cell functions to prevent autoimmunity in gender different way (BA4P.135)
Authors: Park Hong-Jai , Kim Do-Hyun , Kim WonJu , Choi Je-Min
Published: January 2023

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that regulates lipid and glucose homeostasis. Although several studies have demonstrated the intrinsic roles of PPARγ in adaptive immunity, its regulatory functions in T cells are not fully elucidated and controversial. We investigated the role of PPARγ in effector T cell functions in CD4-PPARγKO mice and analyzed the mice phenotype. Interestingly, mild autoimmune phenotypes with increase of follicular helper T cells, germinal centers, and auto-antibody production were found in female CD4-PPARγKO mice, however the male mice did not show any significant phenotypes. Whereas female PPARγ-deficient T cells were hyper-reactive to TcR stimulation to produce more cytokines and to proliferate vigorously, the male PPARγ-deficient T cells produced less or comparable cytokines. This gender different response might be due to the decreased expression of Bcl-2 and IL-7Rα in male PPARγ-deficient T cells, and diminished estrogen receptors (ERs) expression in female PPARγ-deficient T cells. Additionally, PPARγ agonist pioglitazone treatment showed selective inhibition on male Th17 cells while it showed pan-decrease of all effector T cell differentiation including female Th1, Th2, and Th17 cells. Collectively, these results suggest that PPARγ has a gender difference in the regulation of effector T cell functions and suggest different strategy is needed for the autoimmune therapeutics for specific gender.
p21 suppresses autoimmunity by regulating ERK1/2 phosphorylation, NF-κB activation and IFNγ production in hyperactivated T cells (BA4P.141)
Authors: Balomenos Dimitrios , Vazquez-Mateo Cristina , Weber Kathrin , Martínez-A Carlos
Published: January 2023

Peripheral tolerance is essential for suppressing autoimmunity and depends on the regulation of T cell activation, proliferation and apoptosis. p21, known originally as a cell cycle inhibitor, is implicated in effector/memory T cell responses and is considered a suppressor of lupus-like autoimmunity. However, the mechanism by which p21 regulates T cell responses and autoimmunity remains unknown. We investigated these questions by studying T cells of C57BL/6 p21-/- and C57BL/6-lpr p21-/- mice. p21 regulated activation of repeatedly stimulated effector/memory T cells in a cell cycle-independent manner, but had no effect on naïve T cells. We identified a previously non-described role for p21, in controlling T cell activation via downstream MAPK and NF-κB pathways in the cytoplasm. Lack of p21 caused hyperactivation of ERK1/2 and NF-κB in reactivated T cells. Importantly, p21-deficiency led to increased IFNγ production due to an effect of p21 on the IL-12- and IL-18-dependent IFNγ induction. Stimulation of TCR-transgenic T cells with specific antigens in vivo confirmed that p21 is involved in effector/memory T cell activation. Lack of p21 dramatically increased the mild lupus symptoms of C57BL/6-lpr mice to full-blown disease and death in C57BL/6-lpr p21-/- mice. The mechanism by which p21 regulates disease severity was based on its control of T cell activation and IFNγ production. Analysis of these p21 functions might be of therapeutic value for Lupus.
The murine Pbx1-d lupus susceptibility allele accelerates mesenchymal stem cell differentiation and impairs their immunosuppressive function (BA4P.143)
Authors: Morel Laurence , Lu Shun , Zeumer Leilani , Sorensen Heather , Riva Alberto , Croker Byron , Wallet Shannon
Published: January 2023

Pbx1-d is a dominant negative splice isoform of the gene Pbx1 that corresponds to the NZM2410 lupus susceptibility locus Sle1a1. Pbx1 is required to maintain stem cell self-renewal, including that of mesenchymal stem cells (MSCs). MSCs have immunosuppressive functions that require stem cell maintenance. We tested the hypothesis that the expression of Pbx1-d favors MSC differentiation and impairs their immunosuppressive functions. We demonstrate that Sle1a1 MSCs express high levels of Pbx1-d as compared to congenic C57BL/6 (B6) MSCs. Sle1a1 MSCs grew faster and differentiated significantly more rapidly into osteoblasts than B6 MSCs. This corresponded to a significant decrease in the expression of genes associated with stemness and an increase in the expression of genes associated with differentiation. In addition, Sle1a1 MSCs express a gene expression profile associated with an enhanced innate immunity and inflammation. Suppression of Ig production from TLR-activated B6 B cells and IL-2 secretion from activated B6 CD4+ T cells was significantly impaired is Sle1a1 MSCs as compared to B6 MSCs. B6.Sle1a1 MSCs showed intermediate activity in suppressing lupus immunophenotypes in three different mouse models. Together these data suggest that the expression of the lupus susceptibility allele Pbx1-d isoform impairs MSC functions, which may contribute to lupus pathogenesis both through a defective immunosuppression and the promotion of a pro-inflammatory environment.
B7-H1 regulates autoimmunity in αCTLA-4, bm12 transfer and pristane-induced models through distinct sexually dimorphic and non-dimorphic effects (BA4P.128)
Authors: Gupta Harshita , Clark Curtis , Lin Pei , Pandeswara Srilakshmi , Hurez Vincent , Curiel Tyler
Published: January 2023

Females have more autoimmunity through poorly understood mechanisms. Regulatory T cells (Tregs) defend against autoimmunity. We show that B7-H1 signals dampen estrogen (E2)-driven Treg mTOR activation through estrogen receptor-β, maintaining Treg function in E2. B7-H1-/- female mice (but not males or WT of either sex) developed αCTLA-4-induced autoimmunity, rescued by E2 removal or B7-H1+/+ dendritic cell (DC) transfer. Monocytes or B or T cells also gave protective B7-H1 signals to restore Treg function in E2. B7-H1-mediated E2 protection was during Treg differentiation and was not via CD80, PD-1 or Treg B7-H1. E2 also reduced male B7-H1-/- Treg function in vitro and in vivo. Sex-matched B7-H1-/-bm12 splenocyte transfer (minor MHC mismatch) caused lupus symptoms (albuminuria, alopecia) earlier in B7-H1-/- females than B7-H1-/- males or WT. B7-H1-/- males also sickened faster than WT males, showing sex-independent B7-H1 effects. Transfer of B7-H1+/+bm12 splenocytes into B7-H1-/- mice did not induce rapid symptoms, suggesting rescue by B7-H1 signals in bm12 cells. In pristane-induced lupus, B7-H1-/- females had earlier proteinuria than WT females. Plasmacytoid DC or granulocytes from female B7-H1-/- produced more IFN-α in vitro with pristane+TLR7 agonist than WT females. Thus, B7-H1 regulates autoimmunity through diverse sexually-dimorphic and non-dimorphic mechanisms. Defining B7-H1 effects in autoimmunity will help understand female predilection and immunopathogenesis.
Impact of weight loss on obese adipose tissue immune cell function (CAM1P.154)
Authors: Zamarron Brian , Mergian Taleen , Martinez-Santibanez Gabriel , Lumeng Carey
Published: January 2023

Obesity induces inflammation in adipose tissue involving the recruitment and activation of macrophages, CD8+ and CD4+ T cells. These cells play a critical role in the development of insulin resistance seen with obesity. Weight loss can improve insulin resistance and health, but little is known concerning the effects of weight loss on immune activity in adipose tissue. Diet switch from a 60% high-fat diet to 13% normal-fat diet was used to induce weight loss in diet-induced obese C57Bl/6 mice. Adipose tissue fibrosis and macrophage accumulation was evaluated by histology. Leukocyte populations were analyzed by flow, including sorting and cytokine expression comparisons of adipose tissue macrophages before and after weight loss. Weight loss improved glucose tolerance and reduced serum insulin. However extensive adipose fibrosis developed after diet switch and was associated with macrophage accumulation. Analysis of macrophages after weight loss showed they retained a pro-inflammatory gene expression profile and had high surface marker expression of T cell co-stimulatory markers, similar to macrophages found in obese adipose tissue. These adipose macrophages were comprised of both old and newly recruited cells that continued to be activated after weight loss. The continued inflammatory activation of leukocytes after weight loss may help explain the variation seen in metabolic improvements and long-term weight management of patients in response to weight loss interventions.
Grem1 is a key regulator of synovial hyperplasia in rheumatoid arthritis. (CAM1P.162)
Authors: Han Eun-Jin , Choi Sussanna , hwang daehee , you sungyong , Kim Wan-uk
Published: January 2023

Fibroblast like synoviocyte plays critical roles is the major cell in the inflammatory processes of Rheumatoid arthritis. We attempted to identify a key regulator that can drive aggressive phenotypes of RA FLS. For this, we performed an integrative transcriptome data analysis using three datasets from independent RA studies, a panel of normal tissues, and NCI60 cancer cell line panel. Through the analysis, we identified Gremlin (GREM1), which is robustly up-regulated in RA and predominantly expressed in FLS. GREM1 is identified as bone morphogenetic proteins antagonist and is known to bind BMPs. To investigate potential roles of GREM1 in RA, we first examined the level of GREM1 expression in synovial tissues, synovial fluid (SF) and FLS from RA. Immunohistochemistry and ELISA revealed up-regulation of GREM1 protein in RA compared to OA. Proinflammatory cytokines also induced the expression of GREM1 in RA FLS and their expressions were significantly correlated with GREM1 expression in RA SF. We next investigated cellular functions of GREM1 using RA FLS. GREM1 deficieny by siRNA elicited significant suppression of FLS proliferation, migration and invasion. In contrast, stimulation with rhGREM1 to RA FLS increased cell survival and invasion. Furthermore, we found that GREM1 activates AKT1 and MAPK signaling cascade, as well as antagonizes BMP2. This study demonstrated that GREM1 is a key regulator responsible for synovial hyperplasia and FLS migration.
Proinflammatory role of tissue-resident fibroblastic reticular cells (CAM1P.146)
Authors: Klementowicz Joanna , Tang Qizhi
Published: January 2023

Fibroblastic reticular cells (FRC) play an important role in maintaining immune homeostasis and orchestrating immune responses by producing chemokines and cytokines that promote immune cell migration, organization, function, and survival in secondary lymphoid organs. However, little is known about their function in the development and maintenance of inflammation in the tissue. Here we show that FRC-like cells can be found in inflamed islets of Langerhans in type 1 diabetes-prone NOD mice. No FRC are found in non-inflamed islets and crucially their number increases with severity of islet infiltration. Islet FRC share many characteristics of lymph node FRC, including CCL19 and IL-7 expression. However, in contrast to lymph node FRC, islet FRC are a major source of IFNγ-induced CXCL9 in inflamed islets, indicating their more proinflammatory phenotype. In addition, co-culture of islet FRC with activated T cells results in increased expression of proinflammatory molecules including granzymes, perforin, and IFNγ by T cells. Finally, LTβR-Ig treatment leads to dramatic reduction of islet FRC, which is associated with a significant loss of CD4 and CD8 T cells among islet infiltrates as early as 3 days after treatment. These data indicates FRC-like cells in inflamed tissue actively contribute to the local inflammatory responses and may act as a viable therapeutic target for the treatment of type 1 diabetes.
Unprocessed IL-36a co-operates with IL-1 in psoriasiform skin inflammation (CAM1P.148)
Authors: Jensen Liselotte , Milora Katelynn , Fu Hangfei
Published: January 2023

Psoriasis has been linked to overzealous IL-1 and IL-36 signaling; however, whether these cytokines have redundant, interrelated or independent functions have not been examined. It is also unknown if the IL-36 cytokines undergo processing in vivo. Here, we examined IL-36 expression, functional involvement and interaction with IL-1/IL-1R1 signaling in a mouse model of drug induced psoriasiform skin inflammation. Normal skin did not express detectable levels of IL-36a, IL-36b, or IL-36g protein. All 3 cytokine mRNAs were up-regulated during inflammation; however, only IL-36a secretion could be detected in explant skin cultures. Significantly less (25%, p<0.01) was released from IL-1R1 deficient skin. This demonstrates that IL-1 regulates IL-36a expression in vivo. Interestingly, the released IL-36a was not processed. Using knockout (KO) mice for each individual IL-36 cytokine, we found that IL-36a, but not IL-36b and IL-36g, significantly contributed to pathology. Specifically, we observed thinner epidermis and decreased dermal inflammation in IL-36a KO mice compared to wild type. Treated IL-36a/IL-1R1 double KO mice exhibited even thinner epidermal and dermal thickness. Neutrophil recruitment to the epidermis was impaired in the IL-36a KO mice, but could be rescued with application of the chemokine CXCL1. In summary, we conclude that IL-1 dispatches unprocessed IL-36a, which in turn boosts the pro-inflammatory activity of IL-1 signaling during psoriasis-like skin inflammation.
Integrin αvβ8-mediated TGFβ activation by effector regulatory T cells is essential for suppression of T cell-mediated inflammation (CAM1P.150)
Authors: Travis Mark , Kelly Aoife , Smedley Catherine , Bauché David , Campbell Simon , Marie Julien , Worthington John
Published: January 2023

Foxp3+ regulatory T-cells (Tregs) play a pivotal role in suppressing self-harmful T-cell responses to prevent inflammatory disease. An important cytokine in the development and function of Tregs is TGFβ, a latent cytokine that must be activated to function. Compelling evidence suggests that T-cells need to respond to TGFβ in order for Treg-mediated suppression to occur. Yet, how TGFβ is regulated to promote this suppression is poorly understood. We now show that both mouse and human Tregs have high expression of the TGFβ-activating integrin αvβ8, which enables these cells to activate high levels of latent TGFβ. However, deletion of integrin αvβ8 expression from Tregs resulted in no overt phenotype at rest, indicating that the integrin plays little role in controlling immunity during homeostasis. Strikingly, we find that integrin αvβ8 expression and function is highly upregulated on an activated/effector Treg subset marked by KLRG1, and that Tregs lacking expression of integrin αvβ8 were unable to function as suppressive cells during models of colitis and delayed type hypersensitivity in vivo. Thus, we have uncovered an essential mechanism by which effector Tregs suppress T-cells during inflammation, highlighting a key role for Treg-mediated activation of latent TGFβ in suppression of self-harmful T-cell responses. Our work therefore highlights a novel pathway that could be therapeutically targeted to treat inflammatory disease.
IL-1 family members induce pro-inflammatory cytokine release in costimulated T cells outside of TCR stimulation (CCR3P.209)
Authors: Tsurutani Naomi , St. Rose Marie-Clare , Mittal Payal , Adler Adam , Vella Anthony
Published: January 2023

Toll-like receptor stimulation induces production of pro-inflammatory cytokines such as IL-1 or IL-12 that act as a 3rd signal in programming antigen-specific effector T cell differentiation. Signal 3, however, can stimulate already differentiated effector T cells to release cytokines such as IFN-g or IL-4. The identity of signal 3 mediators is important to better understand how signal 3 can drive T cell function in the absence of an obvious TCR ligand, which could occur during infection or cancer. We discovered that IL-36 play this role when T cells were co-stimulated with TNF super family members and had differentiated into effectors. By RT-PCR it was found that IL-2 induced IL-36 receptor gene expression and that IL-36 stimulation, in the absence T cell receptor stimulation, released prodigious levels of IFN-g. To understand how signal 3 responses were related to cellular function we used an RNA-POL ChIP-seq approach and found that optimally costimulated T cells were more prone to possess a higher metabolic state. Measurement of glycolysis in these T cells confirmed that they were actively able to consume glucose, but as this activity diminished so did the capacity to respond to IL-36. Thus, the data suggest that T cells can use an “innate” response pathway such as the IL-36R pathway but only when they are undergoing a strong glycolytic response. Therefore, cytokine therapy for effector T cells is possible as long as they are in the correct metabolic state.
The TORC1-activated proteins, p70S6K and GRB10, regulate IL-4 signaling and M2 macrophage polarization by modulating phosphorylation of insulin receptor substrate-2 (CCR3P.214)
Authors: Heller Nicola , Fang Xi , McCormick Sarah , Gowda Nagaraj , Thompson Joshua , Lamworth Jessie , Warren Kristi
Published: January 2023

The abundance of M2 macrophages in the airways of both human asthmatics and mouse models correlates with disease severity. M2 macrophage differentiation is induced by IL-4 and IL-13. The cytosolic adaptor, IRS-2, is tyrosine phosphorylated (pY-IRS-2) following engagement of the type I IL-4 receptor and it mediates M2 gene expression. Therefore, the goal of this study was to determine negative regulators of IRS-2 activity. We found an inverse relationship between tyrosine and serine phosphorylation of IRS-2. IRS-2 was highly serine phosphorylated (pS-IRS-2) in unstimulated cells and pS-IRS-2 levels decreased as pY-IRS-2 increased following IL-4 stimulation. Inhibiting serine phosphatase activity caused a dramatic increase in IRS-2 serine phosphorylation and IL-4-induced M2 gene expression was ablated. By using phospho-serine kinase arrays and inhibitors of serine/threonine kinases, we showed that TORC1 and p70S6K inhibition but not TORC2, JNK or GSK3β inhibition, resulted in prolonged pY-IRS-2. p70S6K inhibition also augmented expression of the hallmark human M2 macrophage genes, CD200R, CCL22, and TGM2. Lastly, we showed interaction between IL-4Rα, γC, IRS-2 and the TORC1-activated protein GRB10. Using siRNAs to GRB10, we enhanced pY-IRS-2, prolonged IL-4 receptor expression and increased M2 gene expression in human monocytes after IL-4 stimulation. These data highlight p70S6K and GRB10, downstream of TORC1, as key negative regulators of IL-4 signaling in human monocytes.
An alternatively spliced IL-15 isoform modulates cell activation via inducing differential IL-15 receptor-dependent signaling pathways (CCR3P.203)
Authors: Ku ChiaChi , Chang Yi-Hsuan , Lee Tsung-Lin , Chien Yun , Lin Yu-Jei , Kung John
Published: January 2023

IL-15 is a pleiotropic cytokine mediating proliferation and survival of memory CD8+ T cells, natural killer (NK) and NKT cells as well as enhancing cytokine production. Deregulated and excessive expression of IL-15 has been implicated in promoting pathophysiologic state for human inflammatory diseases and malignant transformation. In a routine phenotype-driven screen, we identified a point mutation in exon 7 of the IL-15 gene in Pedigree 191 of N-ethyl-N-nitrosourea (ENU) mutagenized mice which expressed a depressed level of CD44 in CD8+ T cells (called DM for deficiency in memory) and resulted in an increased expression of alternatively spliced IL-15 isoform (IL-15ΔE7). Mechanical stimulation of DM skin reduced keratinocyte activation. Ectopic expression of IL-15ΔE7 in WT skin profoundly blocked neutrophil infiltration to inflamed skin induced by abrasion, sodium dodecyl sulfate or immiquimod treatment, suggesting a regulatory role of IL-15∆E7 in mediating proinflammatory response in skin. While treating T cells with recombinant soluble IL-15∆E7 protein activated a transient but delayed activation of MAPK/p38, IL-15∆E7 failed to activate phosphorylation of STAT5 and its downstream target bcl-2 expression. The capacity of IL-15ΔE7 in inducing differential signaling has pointed IL-15ΔE7 as a potential therapeutic agent for treating inflammatory disorders.
Immune-related trends in bio-medical literature (CCR3P.208)
Authors: Geifman Nophar , Bhattacharya Sanchita , Butte Atul
Published: January 2023

Bio-medical knowledge, including immunology-related knowledge, evolves and changes over time. Examining such changes, along with other immune-related patterns and trends may reflect changes in the field and may assist in detecting new and un-addressed areas of research. We have recently developed a cytokine-centered knowledgebase focusing on the role of cytokines in diagnostics and therapeutics and their association with immune cell types and a variety of different diseases. As a part of its initial development, the knowledgebase was extensively populated with information extracted from relevant biomedical literature. Here, we present interesting cytokine-, cell- and disease-related trends found in the literature (and captured in our knowledgebase). For example, the association of Rheumatoid Arthritis (RA) with tumor necrosis factor alpha (TNFa) has increased over time; on the other hand, at the same time, there was a dramatic decrease in the number of publications linking RA with Interleukin 1 indicating a shift in the focus of RA-cytokine related research. Such changes over time in immune-related topics as well as the association of cytokines and diseases across different tissue types, age groups and ethnic groups demonstrates the usefulness of the knowledgebase in capturing immune-related patterns and focusing at possibly useful avenues of research.
TSLP plays roles that are distinct from IL7 in normal human B cell development (HEM1P.227)
Authors: Payne Kimberly , Milford Terry-Ann , Francis Olivia , Baez Ineavely , Coats Jacqueline , Dovat Sinisa , Morris Chistopher , Su Ruiijun
Published: January 2023

The role of TSLP in human B cell development is largely unknown and its function in context of IL7 is unstudied. We found that TSLP can replace IL7 in providing a signal essential for in vitro production and proliferation of human CD19+ Pax-5+ B cell progenitors. To study the role of TSLP in human B cell development, in the context of IL7 stimulation, we developed a novel xenograft model. Mouse IL7 is active on human cells, however, this is not the case for TSLP. We took advantage of this to develop a xenograft model system comprised of mice that provide human TSLP (+T mice) and control mice that do not (-T mice). Using this model we found that TSLP increases proliferation and upregulates Mcl-1 in CD34+CD19-IL7Ra progenitors resulting in a 3-4 fold expansion of the CD34+ pro-B cell compartment in +T as compared to -T mice. This expansion is maintained during subsequent stages of B cell development, partially due to increased protection from apoptosis that occurs independently of Bcl-2 family pro-survival proteins. While TSLP stimulates in vitro proliferation of pro-B cells, in vivo proliferation of B lineage precursors was similar in +T and -T mice suggesting redundancy with IL7. B cell subsets in normal pediatric bone marrow samples show progenitor ratios and Mcl-1 expression that mirrors that in +T xenograft mice. These data provide evidence that TSLP function in human B cell development includes both unique activities as well as activities redundant to those of IL7.
Bmi1 maintains the self-replenishing ability of B-1a cells in postnatal mice. (HEM1P.230)
Authors: Yoshimoto Momoko , Kobayashi Michihiro , Gao Rui , Yoder Mervin , Liu Yan
Published: January 2023

B-1a cells are innate immune cells that reside in the peritoneal cavity. We have recently reported that some B-1 cells originate from hemogenic endothelial cells in the early mouse embryo, and are independent of HSC-derived hematopoiesis. B-1a cells are also considered to possess self-replenishing capacity. The mechanisms that permit B-1a cells to undergo self-replenishment in vivo independent of HSCs remain unclear. Bmi1 is a polycomb group protein that is known to be critical for self-renewal of adult BM HSCs. In Bmi1-/- postnatal BM, the number of HSCs was markedly reduced and long-term reconstituting ability was lost. We hypothesized that Bmi1 is also critical for B-1a cell self-replenishment ability. Bmi1 mRNA expression was higher in B-1a cells than other lymphoid subsets in adult wild type mice. In Bmi1-/- mice, absolute cell numbers in the spleen, BM, and peritoneal cavity were decreased but the percentage of each mature lymphoid population was not altered except for B-1a cells. The percentage of peritoneal B-1a cells was 4-7 fold less than wild type. Bmi1-/- B-1a cells displayed impaired self-replenishing ability upon transplantation into the peritoneal cavity of recipient mice. Ink4a-Arf is one of the known target genes of Bmi1, and its expression is increased in Bmi1-/- HSCs and B-1a cells. The percentage and numbers of B-1a cells were restored in Ink4a-/-Arf-/-Bmi1-/- mice. These data suggest that Bmi1 maintains the self-replenishing ability of B-1a cells.
STAT5 antagonism of B cell superenhancer networks initiates progenitor B cell leukemia and predicts patient survival (HEM1P.222)
Authors: Farrar Michael , Katerndahl Casey , Heltemes Harris Lynn , Willette Mark , Henzler christine , Yang Rendong , Silverstein Kevin , Frietze Seth , Schjerven Hilde , Ramsey Laura , Hubbard Gregory , Muschen Markus , Kornblau Steven
Published: January 2023

B cell Acute Lymphoblastic Leukemia (B-ALL) arises from the transformation of progenitor B cells. The transcription factor STAT5 is required for transformation but how STAT5 mediates this effect is unclear. Previous studies suggested that STAT5 only acts to promote survival of progenitor B cells. However, other roles for STAT5 in B cell development and B-ALL have not been explored. Here we show that STAT5 activation drives leukemia in cooperation with defects in a linear signaling pathway emanating from the pre-BCR, including Blnk, Btk, Prkcb, Nfkb1, and Ikzf1. Using microarray analysis and ChIP-Seq we demonstrate that STAT5 disrupts the function of superenhancer binding transcription factor networks that normally promote B cell development. STAT5 versus NFkB or IKAROS binding to these enhancers largely had opposing effects on target gene expression. The antagonism between STAT5 and IKAROS or NFκB has direct clinical relevance as the balance between these transcription factors affects patient outcome; patients with high ratios of active STAT5 to NFκB or IKAROS had more aggressive disease characterized by shorter remission and decreased survival. Thus, our studies illustrate how modest perturbations in two opposing transcriptional programs can have dramatic consequences for B cell development and transformation and how the degree of antagonism between these transcriptional programs ultimately predicts patient outcome to therapy.
Inhibition of B lymphopoiesis mediated by adipocytes, myeloid-derived suppressor cells, and IL-1 (HEM1P.223)
Authors: Kennedy Domenick , Knight Katherine
Published: January 2023

B lymphopoiesis declines with age in many mammals, correlating with an increase in adipose tissue in the bone marrow (BM). We showed that adipocyte-derived factors inhibit B lymphopoiesis in vitro, and in this study, we investigated the mechanism by which this occurs. In cultures of mouse BM cells with OP9 stromal cells, we found that adipocyte conditioned medium (ACM) induces the generation of CD11b+Gr1+ myeloid cells, which when added to OP9 cultures also inhibited B lymphopoiesis. These ACM-induced CD11b+Gr1+ cells express arginase (Arg1) and iNos (Nos2), and suppress T cell proliferation, suggesting that they are myeloid-derived suppressor cells (MDSCs). Addition of arginase and iNos inhibitors to BM cultures containing ACM-induced MDSCs did not restore B lymphopoiesis, suggesting that MDSC-mediated inhibition is independent of arginase and iNos. Transwell and conditioned medium experiments showed that MDSC-mediated inhibition of B lymphopoiesis occurs through soluble factors. Using a cytokine array, we detected IL-1α and IL-1β in MDSC-conditioned medium. Addition of IL-1α/β to BM cultures resulted in the loss of B lymphopoiesis, while addition of anti-IL-1α and anti-IL-1β to BM cultures containing MDSCs restored B lymphopoiesis. These data identify IL-1 as the major inhibitory factor responsible for MDSC-mediated inhibition of B lymphopoiesis. This study identifies IL-1 as a potential therapeutic target for restoring B lymphopoiesis in aged individuals.
Sodium tanshinone IIA sulfonate inhibits cigarette smoke-induced airway inflammation via regulating cystic fibrosis transmembrane conductance regulator-mediated pathway (HUM1P.263)
Authors: Lu Wenju , Li Defu , Gong Xuefang , Wang Jian , Zhong Nanshan
Published: January 2023

Cystic fribrosis transmembrane conductance regulator (CFTR) is an important anti-inflammatory protein and confers to development of chronic obstructive pulmonary disease (COPD). Sodium tanshinone IIA sulfonate (STS) has been reported to be anti-inflammatory under various stimuli exposure, however, the underlying mechanisms were not fully understood. In this study, we found exposure to cigarette smoke (CS) led to COPD in mice, manifested by reduction in lung function, increases of inflammatory cell counts in bronchial alveolar lavage fluid , and alveoli enlargement; these changes were associated with reduced expression and chloride current of CFTR, greater activation of ERK1/2 and Nrf2, and greater levels of IL-6 and IL-8 in the lung. Treatment of STS aerosol (10mg·kg-1·day-1) attenuated these responses in COPD mice. In 16HBE human bronchial epithelial cells, exposure to CS extract resulted in reduced CFTR expression, activation of ERK1/2 and Nrf2, and increased IL-6 and IL-8 release; these were potentiated by rise of intracellular concentration of Cl- (70 mM), pretreatment with CFTR inhibitor inh-172 (10 mM) or Nrf2 activation, yet prevented by knockdown of Nrf2, inhibition of ERK1/2 activation, or STS (25uM) treatment. These results indicate that STS could inhibit CS-induced COPD development by preventing CS-induced CFTR reduction, thus inhibiting Cl--ERK1/2-Nrf2 mediated pro-inflammatory cytokine production in airway epithelial cells.
Meta-analysis of atopic dermatitis studies (HUM1P.269)
Authors: Bhattacharya Sanchita , Butte Atul
Published: January 2023

Several independent studies been conducted to understand the genetic and environmental factors influencing the cause of atopic dermatitis, which is an inflammatory skin disorder. To leverage the power of combining the studies we downloaded seven publicly available datasets on atopic dermatitis from ImmPort data portal (https://immport.niaid.nih.gov/). These studies include wide range of molecular and cellular measurements like ELISA, flow and complete blood count (CBC) across multiple cohort and various sample types. We have taken the meta-analysis approach to understand the factors influencing the disease phenotype by integrating the datasets measured under different conditions to find the associations between independent and combined effects. The molecular and cellular measurements were normalized such that data is comparable across studies. We have found a strong association of certain inflammatory cytokines in atopic dermatitis patients under specific conditions, and perturbations in certain cell types of these patients. We also evaluate the influence of gender, age and vaccine response in atopic dermatitis patients with respect to healthy controls.
Comparative studies connecting IFN dysregulation with pathway dysregulation in 2 autoinflammatory diseases, SAVI and CANDLE (HUM1P.274)
Authors: Marrero Bernadette , Liu Yin , Huang Yan , Almeida De Jesus Adriana , Goldbach-Mansky Raphaela
Published: January 2023

Type I interferon dysregulation is associated with some autoinflammatory diseases. Gain-of-function mutations in TMEM173 encoding Stimulator of Interferon Genes (STING) lead to constitutive production of Type I IFN and the clinical syndrome, STING associated vasculopathy with onset in infancy (SAVI). In another IFN mediated disease, Chronic Atypical Neutrophilic Dermatosis with Lipodystrophy and Elevated Temperature (CANDLE), disease causing mutations cause global proteasome dysfunction and increase in IFN message. The cellular source of the IFNs in blood and the dominant isoforms remain unclear. We examined the expression of 5 type I IFN genes among FACS sorted PBMCs: T-, B-, NK cells and monocytes in SAVI (n=2) and total PBMCs from CANDLE patients (n=2) by qRTPCR. We found in SAVI that monocytes expressed the highest level of type I IFN message. Among the IFN genes tested and normalized to healthy monocytes, patients’ IFNβ is over 250-fold higher than IFNα; following the notion that STING pathway activation leads to IFNβ transcription. Depleting monocytes from SAVI PBMCs reduced 90% of the IFN message, confirming that monocytes not dendritic cells are the main source of IFNs. In contrast, CANDLE patients’ IFN alpha and beta genes are comparably upregulated. Although IFN dysregulation is identified in blood, determining the cellular origin of the IFN production and the pathways that drive IFN dysregulation will allow us to develop better targets for therapy.
The role of IL-22 signaling in the pathogenesis of necrotizing enterocolitis (HUM1P.314)
Authors: Good Misty , Ma Congrong , Kumar Pawan , Pociask Derek , Ozolek John , Kolls Jay
Published: January 2023

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and the specific molecular mechanisms responsible for NEC development remain unclear. NEC is characterized by intestinal epithelial barrier disruption and impaired mucosal healing. Recent evidence suggests a critical role for IL-22 in the regulation of intestinal epithelial barrier integrity and gut inflammation in inflammatory bowel disease. Thus, we hypothesized that IL-22 signaling plays an important role in NEC pathogenesis. To test this, we subjected wild type and IL-22Ra1 intestinal specific knock out mice (villin-cre X IL22ra1fl/fl) to an experimental model of NEC using gavage formula feeds and intermittent hypoxia. NEC severity was assessed by intestinal mucosal cytokines and a NEC severity score assigned by a blinded pathologist. We discovered the IL-22Ra1 intestinal specific knock out mice demonstrated earlier NEC-related mortality than WT mice. Further, histology on the IL-22Ra1 intestinal specific knock out mice demonstrated disruption in the intestinal mucosal architecture and upregulation of pro-inflammatory cytokines. Taken together, these data suggest that IL-22 signaling may play an important role in attenuating the immune response in NEC pathogenesis. These findings raise the possibility for novel therapeutic approaches in the management of this devastating disease by exploring the efficacy of IL-22 treatment for NEC and the mechanisms involved.
Understanding the hygiene hypothesis mechanism (HYP2P.331)
Authors: Johnson Jenny , Jones Mark , Cobb Brian
Published: January 2023

As the western world becomes increasingly sterilized, the rate of allergic diseases, such as asthma, have increased dramatically. Alternatively, children born vaginally, raised on a farm, or with older siblings have a reduced risk of developing a multitude of allergic conditions. This phenomenon, known as the hygiene hypothesis, has become well established; however, the cellular mechanism by which this exposure protects the individual remains unclear. We have found that the commensal bacterium B. fragilis, in particular the capsular polysaccharide PSA, can protect against airway inflammation characteristic of asthma. The population of CD4+ T cells responding to PSA treatment was found to be clonal in nature, with a CD4+Foxp3-CD45RblowCD44+CD62L- (TEM) phenotype. These cells show potent inhibition of asthma in an IL-10-dependent fashion upon transfer to naïve recipients. Additionally, analysis of top clones revealed increased prevalence of zwitterionic motifs in the CDR3 loop sequences of PSA experienced T cells, which corresponds to a need for a similar motif in PSA itself for MHCII presentation. Collectively these data show that a polysaccharide antigen induces clonal expansion of effector T cells which act to suppress asthmatic inflammation, thereby adding further evidence to the beneficial effects of commensal flora on immune homeostasis as predicted by the hygiene hypothesis.
Crucial role for CD11c+ interstitial macrophages in the development of TSLP-mediated allergic airway inflammation (HYP2P.321)
Authors: Lai Jen-Feng , Thompson Lucas , Ziegler Steven
Published: January 2023

Allergic asthma is a common chronic Th2-driven airway inflammatory disease. TSLP is an epithelial-derived cytokine recognized as a potent factor in the development of asthma. TSLP is known to influence the ability of dendritic cells to enhance Th2 T cell responses, however many of the mechanisms underlying TSLP-induced airway inflammation remain undefined. In addition to dendritic cells, the lung tissues include other MHC class II-expressing phagocytic populations that may influence T cell responses. Here we demonstrate that pulmonary CD11c+ interstitial macrophages (IM) (CD11cintF4/80+Nur77+CD11b+CX3CR1hiCCR2loMHCII+/-) express high levels of TSLPR and recruited to the lung tissue in response airway priming in the context of TSLP and antigen. TSLP-mediated eosinophilic allergic airway inflammation is attenuated by ablation of CD11c+ IM or by selective deficiency of TSLPR signaling in these cells. More importantly, CD11c+ IM are sufficient for the development of this response in the absence of DC. These findings indicate a crucial role for CD11c+ IM in the development of TSLP-mediated allergic airway inflammation. By elucidating the cellular mechanisms of airway inflammation, these data may contribute to more effective therapeutic approaches for treatment of asthmatic disease.
Macrolide therapy suppresses key features of experimental steroid-sensitive and steroid-insensitive asthma (HYP2P.342)
Authors: Essilfie Ama-Tawiah , Horvat Jay , Kim Richard , Mayall Jemma , Pinkerton James , Beckett Emma , Starkey Malcolm , Simpson Jodie , Foster Paul , Gibson Peter , Hansbro Philip
Published: January 2023

Background Steroid-insensitive endotypes of asthma are an important clinical problem requiring effective treatments. They are associated with non-eosinophilic inflammatory responses and bacterial infections. Macrolide therapy is effective in steroid-insensitive endotypes, such as non-eosinophilic asthma, however the mechanisms of how they work is unknown. Objectives To determine the efficacy of macrolide and non-macrolide antibiotic treatments in infection-induced, severe, steroid-insensitive allergic airways disease (SSIAAD). Methods Mouse models of Chlamydia and Haemophilus lung infection-induced SSIAAD were used to investigate the effects of clarithromycin and amoxicillin treatment on immune responses and AHR in steroid-sensitive AAD and SSIAAD compared to dexamethasone treatment. Results Amoxicillin and clarithromycin had similar anti-microbial effects on infection. Amoxicillin did not suppress either form of AAD, but restored steroid sensitivity in SSIAAD by reducing infection. In contrast, clarithromycin alone widely suppressed inflammation and AHR in both steroid-sensitive and SSIAAD. This occurred through reductions in both Th2 responses that drive steroid-sensitive, eosinophilic AAD, and TNF-α and IL-17 responses that induce neutrophilic SSIAAD. Conclusions Macrolides have broad anti-inflammatory effects that are independent of their anti-microbial effects. The specific responses suppressed are dependent upon the responses that dominate during disease
Severe asthma, airway re-modelling and induced bronchial associated lymphoid tissue produced using novel dust mite specific CD4 T cell receptor transgenic mice (HYP2P.327)
Authors: Kemeny David , Chua Yen , Liong Ka , Koh Hui , Tang Yafang , Zhou Qian , Wong Hok Sum , Huang Chiung-Hui , Wong Wai Shiu Fred , Chua Kaw
Published: January 2023

Patients with severe asthma are very difficult to manage are present a major medical challenge. Development of clinical asthma requires many years of patient exposure to allergens. Mouse asthma models require weeks and are normally transitory and do not lead to allergen-induced dyspnea. We have followed an alternative approach to conventional immunization through the transfer highly polarized CD4 T cell receptor transgenic T cells specific for the dust mite allergen Blo t 5 followed by thrice weekly intranasal recombinant challenge with recombinant allergen to induce persistent high levels of eosinophilia (4 million/mouse lavage) and serum IgE (40,000 ng/ml) and specific IgG1 (30,000 ng/ml). Intranasal challenge of these mice with 25ug of allergen is well tolerated, but a fourfold increase to 100ug induces respiratory symptoms within 15 minutes. These mice show evidence of mucus plugging, increased collagen deposition, mucus hypersecretion and the formation of bronchial associated lymphoid structures (iBALT). Neutralization of IL-4 or IL-13 with monoclonal antibody reduced the response and a combination of these two was most effective. Many drugs reduce respiratory inflammation in mice yet fail to benefit asthmatic patients. We hope that the model described here may help identify new/better drugs for the treatment of patients with severe asthma.
A West Nile virus NS4B P38G mutant induces MAVS-dependent type 1 interferon and the host anti-viral responses (INC1P.353)
Authors: Luo Huanle , Xie Guorui , Diamond Michael , Gale Michael , Barrett Alan , Wang Tian
Published: January 2023

The nonstructural (NS) proteins of West Nile virus (WNV) are important determinants of viral pathogenesis. WNV NS4B-P38G mutant, which has a P38G substitution in the NS4B protein is highly attenuated in mice, but induces stronger type 1 interferon (IFN) and T cell responses than the wild-type WNV. Here, we investigated mice lacking the RIG-I-like receptor adaptor gene Mavs, and found increased viremia and lethality following WNV NS4B-P38G infection. MAVS-/- mice had a reduced IFNβ production and impaired primary T cell response after infection. WNV NS4B-P38G also induced lower levels of type 1 IFNs and cell surface expression of MHC class II and co-stimulatory molecules on MAVS-/- dendritic cell (DC)s. IRF-3, -5, and -7 are the key transcription factors responsible for mediating the type I IFN and ISG response in mDCs during wild-type WNV infection. PCR array analysis showed a significantly decreased expression of IFNβ and most IFN stimulated gene (ISG)s, except IRF3 and IRF5 in WNV NS4B-P38G-infected MAVS-/- DCs compared to the wild-type group. Moreover, DCs of IRF3-/- x IRF7-/- double knockout or Irf3-/-×Irf5-/- ×Irf7-/- triple knockout mice had either unaffected or a higher IFNβ expression after WNV NS4B-P38G infection. Collectively, our results suggest that MAVS signaling is essential for the induction of type 1 IFN and T cell responses upon WNV NS4B-P38G mutant infection and this could occur through an IRF-3 and IRF-5-independent pathway.
Influenza induces GM-CSF deficiency: lung-specific overexpression after infection confers protection (INC1P.355)
Authors: Halstead Eric , Guo Weichao , Yang Linlin , Hu Sanmei , Umstead Todd , Hartshorn Kevan , White Mitch , Chroneos Zissis
Published: January 2023

GM-CSF is an essential cytokine for normal lung immune homeostasis. In the current studies we examined the role of GM-CSF and its lung-specific conditional overexpression on the host immune response to influenza. Transgenic tet-GM+/+ mice were intranasally infected with A/PR8/34 H1N1 IAV, BALF was collected and single cell lung suspensions were generated using collagenase/DNase digestion. IAV infection induced a prolonged period of GM-CSF deficiency in the lung environment. "Early" (3 days prior to infection) induction of GM-CSF led to protection characterized by significantly enhanced weight recovery as compared to non-induced mice, as well as increased numbers of CD11c+ cells, including CD11b+ and CD11b- dendritic cells as well as enhanced recovery of CD11c+ Siglec-F+ AMs. Interestingly, the weight recovery curves for "late" (3 days post-infection) as compared to "early" GM-CSF overexpression were similar suggesting that GM-CSF may have a role in the resolution of inflammation. Indeed, local GM-CSF restoration caused decreased the frequencies of inflammatory Ly-6C+ monocytes/macrophages and Ly-6G+ neutrophils. Lastly, "late" GM-CSF overexpression accelerated secretion of anti-IAV IgA antibody in BALF. These results indicate viral-induced GM-CSF deficiency impairs recovery from influenza infection, yet can be corrected with exogenous administration of GM-CSF.
Intracellular activation of complement 3 mediates intestinal tissue damage during mesenteric ischemia (INC1P.356)
Authors: Kannan Lakshmi , Tsokos Maria , Dalle Lucca Jurandir , Tsokos George
Published: January 2023

Ischemia of the intestine followed by reperfusion leads to significant local and remote organ injury (IRI) attributed to inflammatory elements acting during the reperfusion phase. Whether ischemia contributes to IRI has not been addressed. Careful evaluation of intestinal tissue following ischemia we noticed significant local injury, which was associated by increased production of complement 3 (C3) by intestinal epithelial cells. C3-deficient mice failed to exhibit similar injury and while cobra venom factor pre-treatment, which depleted all complement in the circulation, eliminated all IRI, but it failed to mitigate injury during the ischemia phase. C3 was activated within the epithelial cells during ischemia as indicated by immunocytochemistry and Caco2 (colon epithelial cancer cells) produced C3a after LPS stimulation. Production of C3a was decreased in the presence of cathepsin inhibitors. In conclusion, intestinal epithelial cells when subjected to ischemia can produce and activate C3 and initiate intestinal damage, which is amplified during the reperfusion phase.