bluuebunny/crossref_metadata_2025_split huggingface dataset:
Minimizing PCR bias in high-throughput adaptive immune repertoire sequencing assays (P3389)
Authors: Carlson Chris , Emerson Ryan , Sherwood Anna , Desmarais Cindy , Chung Moon , Parsons Joe , LaMadrid-Herrmannfeldt Marissa , Livingston Robert , Rieder Mark , Robins Harlan 
Published: January 2023

Abstract
The human adaptive immune system is a primary defense against the vast world of potential pathogens. To counter this, B and T cells rearrange a diverse repertoire of surface receptors. This system functions in part by clonal expansion, so measuring changes in the abundance of both unique and total clones is necessary to understand the immune response. We developed immune profiling assays to amplify and sequence rearranged receptors. To efficiently capture receptor diversity we use a multiplex PCR approach. However, small variations in annealing kinetics can have large impacts on amplification efficiency causing PCR amplification bias (PAB). To address this issue, we developed a method to quantify and correct PAB introduced by multiplex PCR. We demonstrate this method using our T cell receptor γ (TCRG) assay. Our method uses a quantitated, synthetic immune repertoire standard consisting of every possible Variable(V) and Joining(J) gene combination. Following multiplex TCRG PCR amplification, we assessed the difference between the synthetic VJ composition before and after amplification and quantified PAB. We adjusted primer concentrations and redesigned primers to generate an unbiased multiplex PCR assay. Using this method we reduced PAB for the TCRG assay from a 340-fold to a 4.5-fold dynamic range. Now the abundance of receptor sequences is representative of the frequency of each receptor chain in the input sample, allowing for accurate quantification of the TCRG repertoire.
Live cell RNA detection aids in the understanding of cellular responses (P3362)
Authors: Weldon Don , Ko Alex , Williams Yuko , Koong Victor 
Published: January 2023

Abstract
Understanding the immune response can be aided by analyzing the genes that immune cells express in response to stimuli. Since all cells from the same orgainism have the same genetic components it is the selection of which genes are expressed that gives each cell its unique characteristics. Once a cell senses cues from its environment it begins to respond by transcribing its DNA into RNA which is later translated into protein. In order to monitor responses at the earliest possible point the RNA levels of a given gene can be assessed giving the researcher insight to how the cell has chosen to react before the protein is translated. Detecting gene expression has traditionally been limited to technologies that examine RNA in lysed or fixed cell populations. The ability to detect gene expression in live cells would allow for more physiologically relevant information. We have developed a novel RNA detection technology capable of detecting specific mRNA and miRNA in live, intact cells. It allows for carrier-free cellular uptake of the reagent, followed by detection of target RNA, with the ability to perform downstream analysis in the same sample.
Cell type-specific role of autophagy against Citrobacter rodentium infectious colitis (P3268)
Authors: Inoue Jun , Hoshi Namiko , Nishiumi Shin , Fujishima Yoshimi , Masuda Atsuhiro , Shiomi Hideyuki , Yoshida Masaru , Azuma Takeshi 
Published: January 2023

Abstract
Recently, genome-wide association studies linked some autophagy-related genes to increased inflammatory bowel disease susceptibility, which suggests that autophagy has an important role in the control of gut immunity. To examine the function of autophagy in different cell compartments in the intestine, we generated the Atg7, an essential gene for autophagy induction, conditional knockout mice in the intestinal epithelium (Atg7FL/FL;Villin-Cre mice). We injected mice with LPS and tested the inflammatory response in the intestine. Atg7FL/FL;Villin-Cre mice exhibited stronger activation of NF-κB and higher levels of TNF-α and IL-1β than WT mice. We also tested the autophagy function in fibroblasts NIH3T3 cells overexpressing mutant Atg4B, which have defect in autophagosome closure, and found that those cells showed enhanced NF-κB activation to LPS stimulation as well. These results suggest that autophagy deficiency in non-hematopoietic compartment can lead to excess inflammatory response against intestinal microorganisms. To confirm this point, we infected mice with C. rodentium and found that Atg7FL/FL;Villin-Cre mice showed aggravated histological intestinal injuries and higher TNF-α, IL-1β and IFN-γ levels in the colon than WT mice. Our findings suggest that autophagy in non-hematopoietic cells play an important role in the regulation of inflammation and host defense against C. rodentium. We are currently testing the function of Atg7 in the hematopoietic cell compartment.
Mice deficient in IL-25 are less susceptible to dextran sulfate sodium-induced colitis (P3270)
Authors: Li Yanfei , Wang Anjiang , Grinchuk Viktoriya , Smith Allen , Ramalingam Thirumalai , Wynn Thomas , Urban Joseph , Shea-Donohue Terez , Zhao Aiping 
Published: January 2023

Abstract
IL-25 is a member of IL-17 cytokine family and is highly expressed in gastrointestinal epithelial cells. The biological activities of IL-25 have been associated with promotion of Th2 but inhibition of Th1/Th17 immunity. Whether IL-25 plays a role in the development of colonic inflammation is unresolved. In this study, we investigated the contribution of IL-25 to DSS-induced colitis in mice. C57BL/6 WT and IL-25-/- mice received 3% DSS in the drinking water ad libitum and were euthanized eight days later. When compared to WT mice, IL-25-/- mice lost significantly less body weight and had delayed appearance of diarrhea and bloody stool, as well as less shortened colon length. Histological evaluation of H&E-stained tissue sections of colon showed that IL-25-/- mice had only mild inflammation, while severe inflammation developed in WT mice based on the extent of focal crypt lesions, loss of goblet cells, and infiltration of inflammatory cells. IL-33 purportedly contributes to intestinal inflammation and qPCR analysis of colonic cytokine expression showed an up-regulation of IL-33 in DSS-treated WT but not IL-25-/- mice. In addition, there were significantly fewer transcripts of the pro-inflammatory cytokines (TNFα, IL-6, IL-17A) in the colon of DSS-treated IL-25-/- than WT mice. Overall, these data suggested that a deficiency in IL-25 partially protected mice from DSS-induced colitis, revealing a previously unidentified pro-inflammatory role for IL-25 in colonic inflammation.
Bone marrow precursors require β7 integrins to give rise to intestinal mononuclear phagocytes with tolerogenic potential (P3290)
Authors: Mora J. Rodrigo 
Published: January 2023

Abstract
Eduardo J. Villablanca 1, Jaime De Calisto 1, Patricia Torregrosa Paredes 1, 2, Barbara Cassani 1, Susanne Gabrielsson 2 & J. Rodrigo Mora 1 1 Gastrointestinal Unit, Massachusetts General Hospital & Harvard Medical School, Boston, MA; 2 Translational Immunology Unit, Karolinska Institutet, Stockholm, Sweden Intestinal mononuclear phagocytes, including dendritic cells (DC) and macrophages (MF), develop from at least two different bone marrow (BM) precursors. CD103- MF derive from lineage negative (Lin-) Ly6Chigh monocytes, whereas CD103+ DC, which metabolize vitamin A into all-trans retinoic acid (RA), derive from Lin-Ly6Clow BM precursors. However, how mononuclear phagocytes precursors are recruited to the intestinal mucosa remains unknown. Here, we show that BM Lin-Ly6Clow cells require β7 integrins to reconstitute intestinal mononuclear phagocytes and to give rise to RA-producing mesenteric lymph node DC. Interestingly, the BM contains a distinct population of α4β7+ Lin-Ly6Clow cells, which was markedly reduced in vitamin A-depleted mice. Importantly, mice lacking β7 integrins in the CD11c+ compartment showed decreased generation of antigen-specific regulatory T cells and were impaired in developing oral tolerance. Thus, BM progenitors require α4β7 to give rise to intestinal mononuclear phagocytes with tolerogenic potential.
A short-term model of COPD identifies a role for mast cell tryptase (P3242)
Authors: Hansbro Philip , Beckett Emma , Stevens Richard , Jarnicki Andrew , Kim Richard , Hanish Irwan , Hansbro Nicole , Deane Andrew , Keely Simon , Horvat Jay , Yang Ming , Oliver Brian , van Rooijen Nico , Inman Mark , Adachi Roberto , Soberman Roy , Hamadi Sahar , Wark Peter , Foster Paul 
Published: January 2023

Abstract
Cigarette smoke-induced chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory disorder of the lung. The development of effective therapies for COPD has been hampered by the lack of an animal model that mimics the human disease in a short time-frame.We have created a mouse model of cigarette smoke-induced COPD that develops the hallmark features of the human condition in a short amount of time. Tightly controlled amounts of cigarette smoke were delivered to the airways of mice, and the development of the pathological features of COPD were assessed. The roles of macrophages and mast cell (MC) tryptase in pathogenesis were evaluated using depletion and in vitro studies and MC protease-6 deficient mice. After 8 weeks of smoke exposure, wild-type mice developed chronic inflammation, mucus hypersecretion, airway remodeling, emphysema, and reduced lung function. These characteristic features of COPD were glucocorticoid-resistant and did not spontaneously resolve. Systemic effects on skeletal muscle and the heart, and increased susceptibility to respiratory infections also were observed. We demonstrate here that macrophages and tryptase-expressing MCs were required for the development of COPD. Recombinant MC tryptase induced pro-inflammatory responses from cultured macrophages. This model can be used to better understand multiple aspects of COPD pathogenesis.
TWEAK/Fn14 pathway promotes chronic colitis and fibrosis mediated by IL-13-TSLP axis (P3244)
Authors: Dohi Taeko , Kawamura Yuki , Kawashima Rei , Son Aoi , Oshio Tomoyuki , Wu Ping , Burkly Linda 
Published: January 2023

Abstract
Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a cytokine of the TNF ligand superfamily that is constitutively expressed by many immune cell types including monocytes/macrophages, DC, neutrophils, natural killer cells, and also T cells. The only known signaling receptor for TWEAK, FGF-inducible molecule 14 (Fn14), is a highly inducible molecule in epithelial cells (EC), endothelial cells, and other mesenchymal cell types wherein TWEAK signals through Fn14 to promote the canonical and noncanonical NF-κB, MAPK and potentially other pathways. We previously reported that TWEAK promoted colitis in mice by enhancing production of inflammatory mediators by EC. TWEAK also induced EC damage in the presence of IL-13, which is known as a pathogenic profibrotic T helper 2-type cytokine. In our current study of chronic colitis induced by repeated rectal injection of a hapten, we found that inflammation, fibrosis, and T helper 2-type immunity were significantly reduced in Fn14 knockout (KO) mice when compared with wild type (WT) control mice. Expression of thymic stromal lymphopoietin (TSLP) was lower in Fn14 KO colon EC than in WT EC. TWEAK/Fn14 was required for the induction by IL-13 of TSLP in colon explants. TWEAK and IL-13 synergistically promote embryonic fibroblast proliferation. Thus we propose an IL-13-TWEAK/ Fn14-TSLP axis as a key mechanism underlying chronic colitis with fibrosis.
IL-10 modulates DSS-induced colitis through a macrophage -ROS-NO axis (P3254)
Authors: Li Bofeng , Alli Rajshekhar , Vogel Peter , Geiger Terrence 
Published: January 2023

Abstract
IL-10 is a critical regulator of colitis development. Yet, its cellular targets and mechanisms of action are not fully resolved. To identify lineages and mechanisms responsible for IL-10's protective effects in colitis, we generated mice with cell type-restricted deficiencies in IL-10 responsiveness by using DSS colitis model with mice bearing deletions of the IL-10Rα in T cells, B cells, DCs, or macrophages. Mice with a macrophage-selective deletion of IL-10Rα (IL-10RαMdel) developed markedly enhanced colitis that did not significantly differ from disease in IL- 10-/- or IL-10Rα-/- mice; no impact of IL-10Rα-deficiency in other lineages was observed. IL-10RαMdel colitis was associated with increased mucosal barrier disruption in the setting of intact epithelial regeneration. Quantitation and phenotypic assessment of lamina propria macrophages did not identify differences from controls. IL-10Rα-deficient macrophages also did not display a competitive advantage over wild type cells in mixed chimeras. Pro-inflammatory cytokine production, and particularly TNF-α, was increased, though TNF-α neutralization failed to reveal a defining role for the cytokine in the aggravated disease. IL-10RαMdel lamina propria macrophages produced substantially greater levels of NO and ROS than control macrophages. Inhibition of these more dramatically reduced colitis severity in IL-10RαMdel mice, and largely eliminated the differential effect of DSS in IL-10RαMdel and control mice.
1,25-dihydroxyvitamin-D3 reversibly prevents EAE by modulating Th cells trafficking without affecting priming or effector functions (P5123)
Authors: Grishkan Inna , Calabresi Peter , Gocke Anne 
Published: January 2023

Abstract
Pharmacologic targeting of chemokine receptors alters Th cell trafficking and thus poses an attractive opportunity for amelioration of autoimmune diseases such as multiple sclerosis (MS). MS risk is associated with vitamin D deficiency, and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has been shown to prevent experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. However, the mechanism of its in vivo action is poorly understood. In the present study, we utilized FACS analysis and in vivo tracking of bioluminescent Th cells to investigate the effect of 1,25(OH)2D3 on Th cells in MOG EAE. We demonstrate that MOG-specific Th cells are successfully generated in the presence of 1,25(OH)2D3, are able to enter peripheral circulation, but are not present in the CNS. 1,25(OH)2D3 treatment does not affect the ability of Th cells to produce IFNγ and IL-17, but reversibly reduces their expression of CXCR3, a chemokine receptor essential for migration to the CNS. Remarkably, mice rapidly develop EAE upon treatment cessation, demonstrating that pathogenic Th cells are present but unable to cause disease while exposed to 1,25(OH)2D3. Herein, we provide novel evidence that 1,25(OH)2D3 prevents EAE by modulating encephalitogenic Th cells migration without affecting their priming or effector functions. The disease rebound further emphasizes that 1,25(OH)2D3 is not an immunosuppressive but rather a reversible immunomodulatory agent that specifically targets pathogenic Th cells in EAE.
Rexinoids as modulators of central nervous system inflammation (P5125)
Authors: Nowak Elizabeth , Chandraratna Roshantha , Dmitrovsky Ethan , Noelle Randolph 
Published: January 2023

Abstract
Transcription factors that interact with retinoid x receptors as heterodimers, such as retinoic acid receptors, liver x receptors, and peroxisome proliferator-activated receptors, demonstrate profound immunoregulatory activities. Therefore small molecule agonists and antagonists to these receptors are attractive drug candidates to control immunity. Studies in our laboratory show that in vivo treatment of mice developing experimental autoimmune encephalomyelitis, a model of central nervous system (CNS) inflammation, with the rexinoid IRX4204 delays the onset and reduces the severity of disease. In accordance with reduced disease severity, we find significantly decreased CNS infiltration of myeloid dendritic cells and CD4+ T cells. Studies to determine the mechanism of action of this drug are in progress.
Vitamin D reduces development and progression of virus-induced demyelinating disease (P5131)
Authors: Olson Julie , Hall Samuel 
Published: January 2023

Abstract
Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that is associated with an inflammatory immune response. Microglia are the CNS resident immune cells which have been shown to be activated during MS. Virus infections have been suggested as a causative agent for MS, and vitamin D insufficiency has been correlated with disease development. Thus, we wanted to determine whether higher vitamin D levels at the time of virus infection could alter development of demyelinating disease. Theiler’s murine encephalomyelitis virus (TMEV) induced demyelinating disease is an animal model of MS. TMEV infection of susceptible mice leads to development of a demyelinating disease associated with an inflammatory immune response in the CNS. Our studies showed that increased levels of vitamin D3 at the time of virus infection reduced development and progression of demyelinating disease. Vitamin D reduced the number of CD4+ T cells expressing IFNγ and increased the number of CD25+CD4+ T cells. Most interestingly, vitamin D reduced the expression of pro-inflammatory cytokines and increased the expression of IL-10 and type I interferons by microglia. The reduction in the inflammatory immune response by microglia was dependent on IL-10 induction of SOCS3. Overall, these results show that vitamin D reduced the inflammatory immune response in the CNS and reduced the development and progression of demyelinating disease following virus infection.
The peroxisome proliferator-activated receptor-γ agonist rosiglitazone in the prevention and treatment of murine systemic lupus erythematosus (P5135)
Authors: Aprahamian Tamar , Bonegio Ramon , Weitzner Zachary , Rifkin Ian 
Published: January 2023

Abstract
Peroxisome proliferator-activated receptor-γ agonists are known to have many immunomodulatory effects. We have previously shown that the peroxisome proliferator-activated receptor-γ agonist rosiglitazone is beneficial when used early in prevention of lupus and lupus-related atherosclerosis. In this study, we attempt to define the ability of peroxisome proliferator-activated receptor-γ agonists to ameliorate established autoimmune disease using two mouse models: the MRL.lpr mouse which is an established lupus-like model, and the gld.apoE-/- mouse, which is a model of accelerated atherosclerosis and lupus. We demonstrate that, in contrast to the marked amelioration of disease seen when rosiglitazone was started before disease onset, treatment with rosiglitazone after disease onset in MRL.lpr or gld.apoE-/- mice had minimal beneficial effect on the development of autoimmune phenotype; however, rosiglitazone treatment remained highly effective at reducing lupus-associated atherosclerosis in gld.apoE-/- mice on both normal diet or high cholesterol Western diet. These results suggest that beneficial effects of peroxisome proliferator-activated receptor-γ agonists on the development of autoimmunity might be limited to the early stages of disease but that atherosclerosis, a major cause of death in SLE patients, may be ameliorated even in established or severe disease.
Priming of regulatory DNA elements activates inflammatory cytokine genes synergistically with TLR signaling to increase transcription (P1230)
Authors: Qiao Yu , Giannopoulou Eugenia , Elemento Olivier , Ivashkiv Lionel 
Published: January 2023

Abstract
Synergistic activation of inflammatory cytokine genes by IFN-γ and Toll-like receptor signaling is important for innate immunity and inflammatory disease pathogenesis. Enhancement of Toll-like receptor signaling events, a previously proposed mechanism, is insufficient to explain massive activation of cytokine production in human macrophages. We found that IFN-γ induced sustained occupancy of STAT1 and IRF1 at proximal and distal regulatory regions of the TNF, IL6 and IL12B gene loci in human macrophages, which was associated with increased histone acetylation, an indicator of an active histone state. This opening of chromatin greatly facilitated further recruitment of other transcription factors and the assembly of the RNA polymerase II complex upon Toll-like receptor activation, which resulted in increased and prolonged transcription of these inflammatory cytokines. Genome-wide analysis revealed pervasive priming of regulatory elements by IFN-γ and linked coordinate priming of promoters and enhancers with synergistic induction of transcription. Furthermore, IFN-γ-mediated priming conferred sensitivity of cytokine gene transcription to inhibitors of Jak kinases or chromatin regulators of the bromo and extra terminal protein family. Our results provide a synergy mechanism whereby IFN-γ creates a primed chromatin environment to augment Toll-like receptor-induced gene transcription, and suggest therapeutic approaches that selectively target priming mechanisms.
The pro-inflammatory and anti-inflammatory TLR2 responses to Staphylococcus aureus can be uncoupled (P1242)
Authors: Peres Adam , Stegen Camille , Cousineau Benoit , Desrosiers Martin , Madrenas Joaquín 
Published: January 2023

Abstract
Microbial detection by pattern recognition receptors (PRRs) is typically associated with a pro-inflammatory cytokine response that primes adaptive immunity. However, some PRRs may also elicit an anti-inflammatory response. Most notably, LPS engagement to Toll-like receptor 4 (TLR4) leads to a PI3K-dependent internalization of TLR4, and the production of Type I interferons and, subsequently, of the anti-inflammatory cytokine IL-10. We have previously reported that peptidoglycan-embedded molecules in the gram-positive bacterium Staphylococcus aureus cell wall, acting on TLR2 on monocytes and macrophages, trigger a PI3K/Akt-dependent IL-10 response that can suppress adaptive T cell immunity. We now report that, unlike the anti-inflammatory TLR4 response, the TLR2-mediated anti-inflammatory IL-10 response is independent of actin-mediated receptor internalization and Type I interferon production. More importantly, the anti-inflammatory response triggered by S. aureus isolates through TLR2 can be uncoupled from the pro-inflammatory response, as they have different kinetics and TLR2 internalization requirements. Our data reveal a novel mechanism of TLR-mediated anti-inflammatory response that may be triggered by selective ligands on the staphylococcal cell wall. Identification of these anti-inflammatory TLR2 ligands may be a first step toward designing novel therapies to treat staphylococcal infections.