bluuebunny/crossref_metadata_2025 huggingface dataset:
New multiplex assay panels for simultaneous quantification of major proinflammatory chemokines in human and mouse samples (TECH1P.866)
Authors: Sun Binggang , Hu Jiaming , Yao Jingjing , Ji Shaoquan
Published: January 2023

Chemotactic cytokines or chemokines play pivotal roles in various processes such as immune surveillance, organ development, angiogenesis, and immune responses. Expression profiling of chemokines, especially those involved in inflammation and immune disorders, is important in achieving a deeper understanding of disease states. Using fluorescence-encoded beads, we have developed two multiplex immunoassay panels to simultaneously detect 13 human or mouse chemokines, respectively. Both human and mouse panels include MCP-1 (CCL2), RANTES (CCL5), IP-10 (CXCL10), Eotaxin (CCL11), TARC (CCL17), MIP-1α (CCL3), MIP-1β (CCL4), MIG (CXCL9), and MIP-3α (CCL20). In addition, the human panel contains ENA-78 (CXCL5), GROα (CXCL1), I-TAC (CXCL11) and IL-8 (CXCL8), and the mouse panel contains LIX (CXCL5), KC (CXCL1), BLC (CXCL13) and MDC (CCL22). Each antibody pair was selected for sensitivity, specificity, accuracy and reproducibility. Suitable for use on various flow cytometers, the assays have higher sensitivity and wider dynamic range than typical ELISA methods. Further advantages include small sample volume requirement, flexible assay configurations and time- and cost-effectiveness. In addition, the assays have been validated with expected changes in relevant biological samples. The chemokine panels can be used for serum, plasma, cell culture supernatant, and other sample types, offering useful tools for biomedical research and drug discovery.
Investigation of humoral immune response towards persisting Epstein-Barr virus infections in multiple sclerosis and chronic fatigue syndrome using peptide microarrays (TECH1P.868)
Authors: Castro Aaron , Pawlowski Nikolaus , Reimer Ulf , Jansong Janina , Löbel Madlen , Ruprecht Klemens , Scheibenbogen Carmen
Published: January 2023

Most humans carry a considerable number of persisting viral infections, frequently in a latent state. Chronic infections may intermittently reactivate especially under immune suppression and play an important role as trigger or cofactor for autoimmune diseases and cancer. Correlation of such infections with diseases is difficult. An analysis of antibody repertoire in chronic infection may provide information about patterns of virus reactivation. Following primary infection the herpes virus 4 (EBV) results in persistent mostly asymptomatic latent infection. About 95% of the adult population are EBV seropositive. EBV infections are known as cofactors for various diseases such as lymphomas, multiple sclerosis and chronic fatigue syndrome. The detailed mapping of humoral immune response in human serum samples allows a high resolution analysis of the antibody repertoire against EBV antigens. We developed a peptide microarray platform with peptide libraries of up to 6900 members. Here we present data using a library of peptide scans through major EBV antigens from incubations with serum samples of healthy human donors, patients diagnosed with chronic fatigue syndrome or multiple sclerosis. Even though there are shared epitopes between the specimens and cohorts, different epitope patterns are observed for a number of antigens and antigen areas. The patterns of antibody responses could be of diagnostic value for multiple sclerosis and chronic fatigue syndrome.
Multiparametric phenotyping and cell health analysis of immune cell sub-populations on a novel microcapillary cytometer (TECH1P.870)
Authors: Gillis Katherine , Khan Asima , Clor Julie , Tyagarajan Kamala
Published: January 2023

Cellular health enumeration and assessment of heterogeneous cellular sample such as whole blood and PBMCs often require many colors to accurately characterize subpopulations and are critical in defining immune cell handling as well as in functional studies ranging from cytokine secretion to proliferation. Traditional multi-color flow cytometers have posed challenges in multi-parametric experiments, managing compensation, and analyzing results. Existing solutions are expensive, cumbersome to run and require extensive training or dedicated personnel. In this study, we will present data from simplified methods allowing identification of multiple subsets including T,B, NK, naïve and memory, monocytes, and granulocytes while simultaneously assessing cell health for studies with activation agents, cytotoxic treatments or culture conditions on a novel instrument platform, the guava easyCyte™ 12. The guava easyCyte™ 12 is a novel small footprint cytometer, with 3 lasers including blue, red and violet. This allows for data on up to 12 parameters in a microplate or tube-based format. The system is based on principles of microcapillary cytometery, provides absolute count and percentage data without external beads, uses low sample volume and uses flexible and intuitive software. The combination of multicolor antibody panel and cell health markers with the powerful instrument platform enables obtaining critical sub-population level information for PBMC and whole blood samples.
A novel method of exploring the interaction mechanism between small molecul Sudan I and antibody binding (TECH1P.871)
Authors: Xuan Jingxiu , Zhang Chongjie , Huang Jian , Wei Dapeng
Published: January 2023

Sudan I is artificial synthesized compound(MW is 248.28 Da). As an illegally food additive, it is one of the potential carcinogens to human health and safety. The molecular modeling techniques was applied to explore the interaction mechanism of the small molecule and relevant McAb and to analyze the McAb’s specificity. The hybridoma of anti-Sudan I was successfully produced. The 3D-structures of anti-Sudan I and Sudan I, Sudan II, Sudan III and Sudan IV were modeling by Gaussian. The binding free energies between anti-Sudan1 and antigen including Sudan I, Sudan II, Sudan III and Sudan IV were separately -7.04 kcal/mol, -7.48 kcal/mol, -5.02 kcal/mol and -4.25 kcal/mol by Autodock 4.1 . The hydrogen bonds of antigen-antibody binding were generated apart from Sudan IV. The pocket size of anti-Sudan I Ab was 5.23 Å×6.64 Å×10.36 Å, and its volume was 359.56 Å3, and Sudan I, Sudan II, Sudan III and Sudan IV volumes were 228.28 Å3, 261.12 Å3, 323.76 Å3 and 356.56 Å3, respectively. Comparing Sudan I, II, III and IV‘s paths, Sudan I, II were similar but Sudan III, IV couldn’t enter unless they induce the change of the antibody’s conformation. From all above results, the anti-Sudan 1 McAb is specific to Sudan I. It is a novel attempt to explore the interaction mechanism between the small molecule and its relevant monoclonal antibody.
Haploid genetic dissection of immune molecule trafficking (TECH1P.836)
Authors: Shen Jingshi , Davis Eric , Dinarello Charles
Published: January 2023

To perform their expected functions, immune molecules must be targeted to specific cellular compartments such as the plasma membrane. Improper protein targeting can lead to major forms of immune disease such as immunodeficiency and persistent inflammation. Our group developed a novel haploid genetics approach in order to unravel protein targeting on a genome-wide scale. We take advantage of human myeloid cell lines (e.g. KBM7 and HAP1) that exhibit a haploid karyotype. Retroviral insertional mutagenesis of these cells offers an efficient method to generate a library of mutant cells with complete disruption of specific genes. After selections of desired phenotypes (e.g., mislocalization of a protein), the retroviral insertion sites of the mutant clones can be mapped by deep sequencing. We have used this haploid genetics approach to characterize the plasma membrane protein CD59, a key player in the complement system. We identified ~20 genes involved in the biosynthesis, maturation, and trafficking of CD59. These genes are involved in GPI lipid synthesis, oligosaccharide modification, as well as protein trafficking at both the early and late stages of the secretory pathway. This haploid genetic screen provides the first genome-wide map of a complement protein. Many factors identified in the screen represent novel components of the complement system, and may serve as therapeutic targets for treating immune disorders.
ImmuSort, a database on dynamic gene expression and electronic sorting of immune cells (TECH1P.840)
Authors: Wang Pingzhang , Yang Yehong , Han Wenling , Ma Dalong
Published: January 2023

With abundance of accumulated public biological data, it is worthy of consideration about how to fully use these data for wet-experiments. In the current study, we built an immunological database, ImmuSort. ImmuSort provides a convenient way to view global differential gene expression across various experimental conditions or disease states in immune cells, and enables an electronic sorting of these cells in association with a certain expressional intensity. Meanwhile, gene plasticity, comparison of expression level, biomarker discovery and valuation, identification of cell specific transcripts, etc., can also be accessible in the database. Importantly, ImmuSort database can contribute to the discovery of potential novel functional populations of immune cells, e.g., the association of CCL20 bright myeloid populations with inflammation, and provide clues for the function of human and mouse genes in the immune system. ImmuSort is the first product of our immunological database development project. It can be freely accessed on the web at http://202.85.212.211/Account/ImmuSort.html.
High-throughput high-accuracy immune repertoire sequencing (TECH1P.841)
Authors: Jiang Ning , Wu Di , Qu Mingjuan
Published: January 2023

Using VDJ recombination, the human immune system contains hundreds of billions of B and T cells that each expresses a unique antigen receptor through a gene recombination mechanism. This “immune repertoire” forms the base of a safety net that protects each individuals against infections and cancers, with particular lymphocytes proliferating in response to antigenic stimulation. Recently developed immune repertoire sequencing technology has enabled us to quantitatively study the immune repertoire in response to vaccination. However, high rate of errors generated from sequencing the PCR amplification process imposed a challenge on accurate determining repertoire diversity and somatic hypermutations. Recently, we developed a RNA barcoding technology that significantly reduced the error rate from 10-3 to 10-5 nt. This new method greatly improved the over-all diversity and mutation estimates and enabled us to answer many questions in human natural infections.
Impact of IL-2 and IL-10 on human CD4/CD8 T cell composition and Vβ repertoire in a mouse xenogenic GVHD model (TRAN1P.873)
Authors: Abraham Sojan , Choi Jang Gi , Manjunath N. , Shankar Premlata
Published: January 2023

High level of IL-10 is present in sera of patients with Graft-versus-host disease (GVHD) following allogenic bone marrow transplantation. Our studies with NSG mouse model of GVHD induced by transfer of human PBMCs also revealed high levels of IL-10 in the sera, particularly in presence of IL-2, which accelerates the disease. Blocking antibody to IL-10 was unable to protect the animals from GVHD progression. To directly test the effect of IL-10, GVHD was induced after hydrodynamic injection of plasmids encoding human IL-10 singly or in combination with IL-2. When administered alone, IL-10 treatment resulted in more severe GVHD manifestation. Further, in IL-2 accelerated GVHD, IL-10 was unable to ameliorate the course of the disease. There was a marked difference in the composition of the repopulating human T cells in blood and spleen of mice treated with IL-2 or IL-10 alone compared to a combination of the two cytokines. Singly, both cytokines induced expansion of CD4+ as well as CD8+ T cells with a relatively diverse repertoire but in combination, they induced large oligoclonal expansions of CD4+ T cells with very limited Vβ repertoire and near complete absence of CD8+ T cells. Taken together, our results demonstrate for the first time that IL-10 directly contributes to GVHD pathogenesis. Further, when used in combination with IL-2, IL-10 has a major impact on the repertoire of the responding T cells.
Small peptides elicit anti-tumor effects in CT26 model through blocking PD-L1/PD-1 (TUM2P.900)
Authors: Gao Yanfeng , Liu Beiyuan , Chang Haonan , Li Wenwen , Zhou Yang , Li Guodong , Qi Yuanming , Liu Lei
Published: January 2023

It is reported that PD-L1 plays an important role in the immune tolerance of tumor, which through its interaction with PD-1 to induce the T cell anergy and/or exhaustion. Blockade of PD-1/PD-L1 interaction by monoclonal antibody has achieved great success during clinical trial against cancer. Thus, development of novel compounds targeting PD-1/PD-L1 is considered to be very useful. In the present study, a series of small peptides which could bind to PD-L1 were firstly screened out by using phage display peptide library. Several peptides showed potent binding affinity towards PD-L1 and could compete with PD-1 protein to bind PD-L1 expressed on 293T cell surface. In the mouse CT26 colon carcinoma model, these peptides elicited potent anti-tumor activity when injected intratumorly. They could also prolong the survival rate with less effect on bodyweight loss compared to chemotheraputic agent 5-FU. Whether these peptides elicit anti-tumor effects through restoring the T cell function are now being investigated. These peptides could be used as novel anti-tumor agents combined with chemotherapy and radiotherapy.
Modulation of macrophage responses by aberrant lipoproteins in chronic inflammatory diseases. (HUM3P.241)
Authors: Smith Carolyne , Vivekanandan-Giri Anuradha , Playford Martin , Berthier Celine , Kretzler Matthias , Mehta Nehal , Pennathur Subramaniam , Kaplan Mariana
Published: January 2023

Lupus (SLE) patients are fifty times more likely to experience a cardiovascular event compared to age- and sex-matched controls. High levels of oxidized HDL (oxHDL) in SLE patients may contribute to this association. Under normal conditions, unoxidized HDL induces an anti-inflammatory response within macrophages (MØ). We addressed whether lupus oxHDL modifies this response and putative mechanisms associated with enhanced inflammation. Methods: Affymetrix gene expression analysis was performed on control MØs exposed to control or lupus HDL before an LPS challenge. Confirmation at the mRNA and protein level was performed. Signaling pathways involved in gene induction abnormalities were explored using molecular approaches. Results: MØs exposed to SLE HDL up-regulate IL-6, IL-12B and TNF-α at the mRNA and protein levels, compared to MØs exposed to healthy HDL. SLE HDL showed impaired induction of ATF3 mRNA and nuclear translocation. ATF3 activity was restored when the oxHDL binding protein LOX-1 receptor was inhibited with blocking antibodies. SLE HDL also showed a decrease activation of kinases upstream of ATF3 activation. Conclusion: Lupus HDL induces inflammatory responses in macrophages which may promote phenotypic proatherogenic changes and increase cardiovascular risk in this disease. Modifying the HDL proteome in SLE may decrease this risk.
Dysregulation of micro-RNAs involved in TLR pathway in peripheral blood mononuclear cells and synovial fluid mononuclear cells of patients with enthesitis related arthritis form of juvenile idiopathic arthritis (HUM3P.254)
Authors: SINGH SUSHMA , Aggarwal Amita
Published: January 2023

Toll like receptor (TLR) pathway plays a major role in inflammatory arthritis. MicroRNA (miR)-146a, miR-155, miR-21 regulate downstream molecules of TLR signaling like IRAK-1 and TRAF-6. TLR pathway genes are generally upregulated in ERA-JIA. We studied expression of miR-146a, miR-155, miR-21, miR-210 and miR-26a in PBMC and SFMC of patients with ERA. RNA was isolated from PBMC (n=21) & SFMC (n=11) of ERA patients and PBMC of controls (n=11). The miRNA and target gene expressions were analysed by TaqMan qRT-PCR assays. Among 21 patients, 20 were boys. Mean disease onset age was 10.3Y & disease duration was 6Y. All had arthritis, 14 had enthesitis, 4 had uveitis, 7 had IBP and 2 had family history of HLAB27 related disease. No expression difference was seen in miR-146a, miR-155, miR-210 and miR-26a in PBMC of patients and controls but miR-21 was increased in patients. To check inflammatory site specific expression, comparison of SFMC and PBMC was done showing downregulation of miR146a and upregulation of miR-155, miR-21 and miR-210 in SFMC. Expression of miR-26a did not show any change. MiR-146a target genes i.e. IRAK-1 and TRAF-6 were upregulated in SFMC compared to PBMC. There is dysregulation of miRNAs regulating TLR pathway in mononuclear cells of patients with ERA-JIA especially at the chronic inflammation site. Down regulation of miR-146a, a key regulator may lead to over-expression of TLR downstream molecules like IRAK-1 and TRAF-6 thus perpetuating inflammation in ERA.
Expansions of salivary gland CD4+ T cells from Sjögren’s syndrome patients: single-cell repertoire analysis and correlation with clinical measures of disease (HUM3P.255)
Authors: Joachims Michelle , Leehan Kerry , Lawrence Christina , Pan Zijian , Radfar Lida , Rasmussen Astrid , Scofield R. , Sivils Kathy , Thompson Linda , Farris A.
Published: January 2023

Sjögren’s syndrome is a systemic rheumatic disorder characterized by dry eyes, dry mouth and T cell infiltration of exocrine gland tissue. To determine the extent of salivary gland (SG) CD4+ memory T cell clonal expansion and whether these expansions are as frequent in peripheral blood (PB), a multiplex PCR method was used to amplify both the α and β T cell receptor (TCR) sequences from memory CD4+ T cells sorted from SG lip biopsy tissue and PB of 10 primary Sjögren’s syndrome patients. Over 3,000 TCR sequences were obtained from 50-115 (median 91) individual SG and 75-121 (median 104) individual PB T cells per patient. The percentage of cells that were part of clonal expansions was significantly higher in SG (median 11%, range 0-28%) compared to PB (median 0.9%, range 0-7%, p=0.003). Sequence analysis revealed: 1) highly homologous CDR3s among different expanded SG T cell clones within single patients, suggesting antigen-driven expansion and 2) unique cases of convergent recombination among unrelated patients, where different V segments/additions/deletions were utilized to make identical CDR3 amino acid sequences. The percentages of clonally expanded SG T cells correlated significantly with degree of SG fibrosis and reduced saliva production but not with systemic features of disease. SG clonal expansions detected in this study likely identify T cells involved in recognition of common antigen(s) and glandular dysfunction.
A combined genome-wide DNA methylation and transcription analysis identified genes related with pathogenesis of systemic sclerosis (HUM3P.257)
Authors: Li Quanzhen , Zhu Honglin , Luo Hui , Zuo Xiaoxia , Chen Tao , Raman Indu
Published: January 2023

Systemic sclerosis (SSc) is an autoimmune disease with unknown etiology. Studies have revealed epigenetic modification can contribute to susceptibility and pathogenic process in systemic autoimmune diseases by regulating gene activation or silencing. In this study, a genome-wide DNA methylation and transcription analysis was performed on PBMC from 20 SSc patients and 23 matched healthy controls (HC) using the Illumina Human Methy450 and HumanHT-12 Beadchips. Out of 485,000 CpG sites tested across the genome, 4848 hypomethylated and 216 hypermethylated CpG sites were identified from SSc as compared with HC, representing 2488 hypomethylated and 134 hypermethylated genes. Transcriptional microarray analysis identified 1023 significantly upregulated genes in SSc, of which 405 genes were hypomethylated, indicating a negative regulation of DNA methylation on gene expression. The hypomethylated genes in SSC include established autoimmune-related and novel genes, such as AIF1, ARID3A, IFI44L, PARP9, RSAD2, EPSTI1, EIF2AK2 and CYFIP2. Pathway analysis revealed enrichment of genes involved in wnt signaling pathway, leukocyte transendothelial migration, cell adhesion molecules and chemokine signaling pathway. The Wnt signaling pathway was represented by hypomethylation of Wnt5b, Wnt7a, Smad2, and Mapk9. The hypomethylation of genes involved in chemokine signaling pathway were correlated with the increased level of chemokines such as eotaxin, MCP-1 and RANTES in sera of SSc patients.
Obesity-induced inflammatory profile changes in mice and humans (HUM3P.263)
Authors: Mirsoian Annie , Erdembileg Annurad , Sckisel Gail , Berglund Lars , Murphy William
Published: January 2023

Obesity is increasingly becoming a pandemic affecting populations of all ages. Hallmark to obesity is a self-sustaining chronic proinflammatory response characterized by increased immune infiltrates into adipose tissue. However, most studies focus on the impact of metabolic factors upon disease risk. There is currently a paucity of data characterizing the activation and exhaustive phenotypes of immune infiltrates as impacted by obesity. Additionally, there is currently little to no data examining the immunological phenotype of young obese populations, which may provide therapeutic targeting for early disease prevention. In this study, we examined the inflammatory phenotype of lean versus obese mice as well as humans across increasing ages. We observed that increased acute-phase protein levels in young obese cohorts are similar in level to aged cohorts. Our results demonstrate for the first time that immunological changes induced by aging are similarly induced in young obese cohorts. Cytokine analyses demonstrate changes in magnitude of systemic IL-6 levels and PD-1 and Lag-3 expression on CD4 and CD8 T cell populations among young lean, obese, and aged. Importantly, we demonstrate that characterizing immunological systemic changes may provide a better scoring system over metabolic or vascular markers to account for early disease development, and may also provide therapeutic targets for early prevention of age-associated diseases.
CD40 expression on CD4+ T cells (Th40 cells) as a biomarker for multiple sclerosis (HUM3P.265)
Authors: Wagner David , Vaitaitis Gisela , Waid Dan
Published: January 2023

Multiple Sclerosis is a neurodegenerative disease with autoimmune components. Diagnosis is invasive and time consuming. The etiology is unclear as are clues for disease progression. The autoimmune components have largely been defined in the EAE mouse model. The problem has been defining a reliable biomarker for pathogenic cells. We described a unique T cell subset, Th40 cells that is highly auto-aggressive. We compared Th40 levels in clinically isolated syndrome (CIS), relapsing/remitting (RRMS), secondary progressive (SPMS) and primary progressive (PPMS) patients to non-autoimmune controls. MS subjects have significantly increased percentages of Th40 cells compared to controls. In CIS and RRMS Th40 cell levels cover a broad range while in SPMS and PPMS the range is centralized. Longitudinal studies show that Th40 cell levels remain elevated. Th40 cells produce IFNg, TNFa and IL-17. HLA haplotypes were diverse with only 50% of patients expressing the DR2 (DR15) haplotype or DR3 and DR4. Th40 cells are significantly elevated in MS regardless of HLA haplotype and responded to CNS antigens creating antigen signatures. The ratio of Th40 to or to Tr1 cells is 7 to 1. Th40 cells have a memory phenotype. The chemokine receptors CCR5 and CXCR3 were detected. Samples from patients on disease-modifying-therapy (DMT) demonstrated lymphopenia, but the Th40 percentage was not altered. Th40 cells provide a unique diagnostic tool for MS with significant improvement over current options.
Nedd4 family interacting protein 1 limits effector CD4 T cell abundance, cytokine production and inflammation. (HUM3P.248)
Authors: Layman Awo Akosua , Oliver Paula
Published: January 2023

When effector T cells fail to appropriately terminate signaling responses, autoimmune diseases such as multiple sclerosis and inflammatory bowel disease may occur. Signaling in a cell may be terminated by the ubiquitylation and degradation of key signaling transducers. The ubiquitylation cascade is a well studied sequence of orchestrated reactions by which ubiquitin is added on to a substrate protein to, among other things, target that protein for degradation. Ndfip1, or Nedd4 family interacting protein 1, activates the function of E3 ligases, which play important roles in the ubiquitylation cascade. Ndfip1 works with Itch and Nedd4-2 E3 ligases to degrade the IL-4 transcription factor, JunB. Thus in mice that lack Ndfip1, there is unrestricted IL-4 secretion, which leads to an inflammatory phenotype. However, this fails to explain why human SNPs in Ndfip1 have been linked to two autoimmune conditions where TH1 and/or TH17 effector function is unrestrained, namely, multiple sclerosis and inflammatory bowel disease. Mice lacking Ndfip1 show increased overall frequencies of activated T cells and of IFNγ+ and IL-17A+ CD4 T cells, which suggests a general defect in limiting effector T cell abundance. We present data indicating that Ndfip1 limits inflammation in two models of inflammatory bowel disease. Understanding how Ndfip1 limits effector T cell function could clarify how human diseases such as inflammatory bowel disease and multiple sclerosis develop.
Humanized mouse model of mast cell-mediated passive cutaneous anaphylaxis and passive systemic anaphylaxis (HYP4P.302)
Authors: Kenney Laurie , Bryce Paul , Falahati Rustom , Bebbington Chris , Tomasevic Nenad , Shultz Leonard , Greiner Dale , Brehm Michael
Published: January 2023

Mast cells are critical components of allergic responses in humans, and animal models that allow the in vivo investigation of human allergic responses and the evaluation of new human-specific therapeutics are urgently needed. Here we have developed a new humanized mouse model for human mast cell engraftment and human IgE-dependent allergic responses. This model is based on the NOD-scid IL2rgnull IL3/GM-CSF/SCF (NSG-SGM3) strain of mouse engrafted with human fetal thymus, liver and hematopoietic stem cells (termed the BLT model). Large numbers of human mast cells develop in NSG-SGM3 BLT mice and populate peripheral lymphoid tissues, mucosal tissues and the peritoneal cavity. The human mast cells in NSG-SGM3 BLT mice express CD117, tryptase, and IgE-receptor, undergo a calcium flux and degranulate in an IgE-dependent antigen-specific manner, and can readily be cultured and expanded in vitro. The NSG-SGM3-BLT mice develop a human mast cell dependent, antigen-specific IgE-mediated passive cutaneous anaphylaxis (PCA) response upon intradermal challenge. Moreover, we describe the first report of a human mast cell antigen-dependent passive systemic anaphylaxis (PSA) response in humanized mice following intravenous challenge with antigen. The NSG-SGM3 BLT mouse provides a readily available source of human mast cells and is a promising pre-clinical model to investigate the pathogenesis of human allergic responses and to testing new therapeutics.
Involvement of RIP2 in mast cell activation downstream of the high-affinity IgE receptor FcεRI (HYP4P.304)
Authors: Tigno-Aranjuez Justine
Published: January 2023

RIP2 is a dual-specificity kinase which is activated following the engagement of the peptidoglycan sensors NOD1 and NOD2. Despite abundant expression of RIP2 in various tissues and cell types, as well as having numerous reported binding partners, its role has primarily been demonstrated as important solely downstream of NOD1/2 signaling. We report that RIP2 potentially has a role downstream of signaling through the high affinity IgE receptor, FcεRI. We demonstrate that members of the Src family kinases as well as Syk, which are involved in FcεRI signaling, bind to and promote tyrosine phosphorylation of RIP2. Importantly, we show that loss of RIP2 affects mast cell activation downstream of FcεRI but not activation mediated through other pathways such as through purinergic receptor engagement. Lastly, use of Gefitnib, a potent RIP2 inhibitor, shows downregulation of FcεRI-mediated responses. Altogether, these data provide evidence for the involvement of RIP2 in a novel, non-NOD-mediated signaling pathway and suggest that Gefitinib or specific RIP2 inhibitors may be useful for the treatment of disorders in which mast cell FcεRI signaling play an important role such as in allergy or asthma.
Inhibition of CD23-mediated IgE transcytosis suppresses the initiation and development of airway allergic inflammation (HYP4P.308)
Authors: Palaniyandi Senthilkumar , Liu Xiaoyang , Periasamy Sivakumar , Ma Aiying , Tang Jin , Jenkins Mark , Tuo Wenbin , Song Wenxia , Keegan Achsah , Conrad Daniel , Zhu Xiaoping
Published: January 2023

The human low affinity IgE receptor, CD23 (FceRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains completely unknown whether CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo. To prove this, we first showed that epithelial CD23 in wild-type (WT) mice transcytosed either IgE or ovalbumin (OVA)-IgE complexes across the airway epithelial barrier. Neither type of transcytosis was observed in CD23-knockout (KO) mice. The OVA sensitization and aerosol challenge of the irradiated WT/WT or CD23KO/WT chimeric mice that CD23 expressed on radioresistant airway structural cells, mainly in epithelial cells, resulted in airway eosinophilia, lung damages including collagen deposition and significantly increased goblet cells, and increased airways hyperreactivity. In contrast, the absence of CD23 expression on airway structural or epithelial cells, but not on hematopoietic cells, of the bone-marrow chimeric mice (WT/CD23KO) significantly reduced OVA-driven allergic airway inflammation. Finally inhalation of a CD23-blocking B3B4 antibody to target exposed CD23 in AECs of the sensitized WT mice before airway challenge suppressed the salient features of asthma, including bronchial hyperreactivity. Our study further suggests that the functional inhibition of CD23 on AECs is a potential therapeutic means to intervene the asthma development.
Mast cells produce factor XIIIA and mast cell chymase reduces factor XIIIa activity during sepsis through proteolytic degradation (HYP5P.318)
Authors: Shubin Nicholas , Glukhova Veronika , Clauson Morgan , Truong Phuong , Vaisar Tomas , Abrink Magnus , Pejler Gunnar , White Nathan , James Richard , Piliponsky Adrian
Published: January 2023

Previously, we reported that mast cell (MC) chymase, and its mouse homologue, mast cell protease 4 (MCPT4), protect from septic morbidity and mortality by limiting inflammation via TNF degradation. Here, we identified another potential MCPT4 substrate; factor XIIIa (FXIIIa; the essential fibrin-crosslinking/clot stabilizing FXIII catalytic subunit). We found that extracellular FXIIIA (the pro-form of FXIIIa) protein levels were significantly increased in IgE-stimulated MCPT4-/- peritoneal MC (PMC) supernatants compared with WT PMCs (FXIIIA mRNA levels were unchanged). These data suggest that MCs both secrete FXIIIA and are capable of degrading it via MCPT4. Based on these results, and on published data demonstrating that FXIIIa activity correlated with sepsis severity, we hypothesize that chymase/MCPT4 mediated cleavage of FXIIIa participates in the previously-noted protective effect of chymase during sepsis. First, we verified that chymase is capable of degrading FXIIIA and reducing FXIIIa activity in-vitro and in-vivo. Additionally, we identified 6 chymase-driven primary cleavage sites in human FXIIIA. Finally, we observed that following experimentally-induced sepsis, plasma and peritoneal FXIIIA levels as well as plasma FXIIIa activity were significantly diminished in WT compared with MCPT4-/- mice. In conclusion, these data demonstrate that MCs produce FXIIIA and that MCPT4 reduces FXIIIa activity during experimental sepsis.
Splenic monocytes traffic to the thyroid and produce a novel TSHβ splice variant during acute Listeria infection in mice (INC4P.334)
Authors: Klein John , Montufar-Solis Dina
Published: January 2023

It has been known for over three decades that thyroid stimulating hormone (TSH) is produced by cells of the immune system; however, the functional role of that TSH has remained obscure. We recently identified a novel splice variant of the thyroid stimulating hormone β-subunit (TSHβv) that is primarily produced by cells of the immune system, in particular by myeloid cells in the bone marrow (BM) and spleen. During acute infection with virulent L. monocytogenes, TSHβv synthesis increased in CD14+, F4/80+ BM cells and spleen cells. In vitro culture of the mouse AM macrophage cell line with LPS or zymosan, or following stimulation with anti-CD14 or anti-TLR2 antibodies, resulted in increased synthesis of TSHβv. Three days post-infection with Listeria, there was an infiltration of CD14+, F4/80+ cells into the thyroid that was followed by an increase in intrathyroidal TSHβv gene expression on days 4 and 5 compared to non-infected mice. Following in vivo transfer of CFSE-labeled spleen cells from infected mice into normal non-infected mice, CFSE+ cells were present throughout the thyroid, demonstrating an active process of trafficking of TSHβv-producing spleen cell into the thyroid during an acute infection. These findings point to a novel mechanism of host metabolic regulation by the immune system during systemic bacterial infection, and they define a functional role for TSHβv in that process.
A novel population of IFN-γ-producing B cells promote innate defense against Listeria monocytogenes infection (INC4P.337)
Authors: Bao Yan , Cao Xuetao
Published: January 2023

Although B cells play important roles in the humoral immune response and the regulation of adaptive immunity, B cell subpopulations with unique phenotypes, particularly those with non-classical immune functions, should be investigated further. By challenging mice with Listeria monocytogenes, Escherichia coli, vesicular stomatitis virus, and TLR ligands, we identified an inducible CD11ahiFcγRIIIhi B cell subpopulation that is significantly expanded and produces high levels of IFN-γ during the early stage of pathogen infection. This subpopulation of B cells can promote macrophage activation via IFN-γ, thereby facilitating the innate immune response against intracellular bacterial infection. As this new subpopulation is of B-cell origin and exhibits the phenotypic characteristics of B cells, we designated these cells as IFN-γ-producing innate B cells. Dendritic cells were essential for the inducible generation of innate B cells from follicular B cells via CD40L-CD40 ligation, and increased Btk activation was found to be responsible for the increased activation of noncanonical NF-κB in innate B cells after CD40 ligation, with the consequent induction of additional IFN-γ production. The identification of this new population of innate B cells may contribute to a better understanding of B cell functions in anti-infection immune responses and immune regulation.
The role of ATG5-dependent autophagy during Rickettsia australis infection. (INC4P.339)
Authors: Bechelli Jeremy , Zhao Xuemei , Smalley Claire , Hwang Seungmin , Popov Vsevolod , Leoncio Leoncio , Walker David , Fang Rong
Published: January 2023

Rickettsiae are obligate intracellular bacteria that replicate in the cytoplasm of eukaryotic cells. We found that degradative autophagy is induced upon the infection of murine bone marrow macrophages (BMMs) with R. australis and the bacteria are specifically targeted by autophagosomes for degradation. Inhibition of autophagy using 3-methylamine (3-MA) significantly increased bacterial burden in BMMs. Consistently, rickettsial loads in livers of infected Atg5flox/flox Lyz-Cre mice were significantly greater than those of Atg5flox/flox littermates. As a consequence, host survival was significantly reduced in autophagy-deficient Atg5flox/flox Lyz-Cre animals, suggesting that macrophage autophagy mediates host cellular immunity against rickettsiae in vivo. In contrast, limited level of autophagy was induced in human endothelial cells, which are the major targets of rickettsiae. Taken together, our results suggest that R. australis induces autophagy in murine macrophages and plays a role in bacterial clearance both in vitro and in vivo. However, levels of autophagy in human endothelial cells early during infections appear to be lower compared to those in murine macrophages.
Improved antibacterial resistance of alcohol fed mice 7 days after alcohol abstinence (INC4P.323)
Authors: Ohama Hideko , Asai Akira , Ito Ichiaki , Yokohama Keisuke , Nakamura Ken , Tsuchimoto Yusuke , Fukunishi Shinya , Tsuda Yasuhiro , Higuchi Kazuhide , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

MRSA enteritis is one of the most serious complications which develop into sepsis in alcoholics. In this study, we examined the effect of alcohol abstinence on the host antibacterial resistance of the alcohol fed mice (AF-mice) orally infected with MRSA. AF-mice were BALB/C mice fed 5% ethanol-containing Lieber-DeCarli liquid diet ad libitum for 1 to 3 weeks. These mice were subjected to alcohol abstinence for 7days. Three to 6 days after alcohol abstinence, all of the mice were decontaminated with an antibiotic mixture and treated with a proton pump inhibitor. On day 8, these mice were orally infected with 107 CFU/mouse of MRSA. The effects of alcohol abstinence on the host antibacterial resistance of AF-mice were evaluated by bacterial growth in mesenteric lymph nodes (MLNs) and mortality rates. In the results, sever growth of MRSA was demonstrated in MLNs of AF-mice without alcohol abstinence, while the pathogen was not isolated from MLNs of alcohol abstinent AF-mice 4 days after the infection. All of AF-mice died within 10 days of MRSA infection, while 100% of alcohol abstinent AF-mice survived. CCL1+IL-10+F4/80+ cells (M2bMϕ) were isolated from MLNs of AF-mice, while alcohol abstinent AF-mice were shown to be a non carrier of M2bMϕ in their MLNs. These results indicate that impaired host antibacterial resistance of AF-mice against MRSA enteritis was effectively improved by alcohol abstinence for 7 days
Alarmin function of galectin-9 in murine respiratory tularemia (INC4P.341)
Authors: Sharma Jyotika , Steichen Anthony , Simonson Tanner , Salmon Sharon , Metzger Dennis , Mishra Bibhuti
Published: January 2023

Sepsis is a complex immune disorder that is characterized by systemic hyperinflammation. Alarmins, which are multifunctional endogenous factors, have been implicated in exacerbation of inflammation in many immune disorders including sepsis. Here we show that that Galectin-9, a host endogenous β-galactoside binding lectin, functions as an alarmin capable of mediating inflammatory response during sepsis resulting from pulmonary infection with Francisella novicida, a Gram negative bacterial pathogen. Our results show that this galectin is upregulated and released during tissue damage in the lungs of F. novicida infected septic mice. In-vitro, purified recombinant Galectin-9 exacerbated F. novicida-induced production of inflammatory mediators by macrophages and neutrophils. Concomitantly, Galectin-9 deficient (Gal-9-/-) mice exhibited improved lung pathology, reduced cell death and reduced leukocyte infiltration, particularly neutrophils, in their lungs. This positively correlated with overall improved survival of F. novicida infected Gal-9-/- mice as compared to their wild-type counterparts. Collectively, these findings suggest that exatracellular galectin-9 functions as a novel alarmin by augmenting the inflammatory response in sepsis development during pulmonary F. novicida infection.
Differential caspase regulation of actin dynamics during Legionella pneumophila infection (INC4P.324)
Authors: Caution Kyle , Akhter Anwari , Hassan Hoda , Tazi Mia , Ahmed Amro , Hoque Sheshadri , Ghonime Mohammed , Gavrilin Mikhail , Amer Amal
Published: January 2023

Legionella pneumophila (Legionella) is the causative agent of Legionnaires’ disease. Clearance of Legionella requires efficient phagolysosomal fusion. Regulation of the host actin cytoskeleton is crucial for proper vesicle trafficking. It has been determined that activation of caspases-1/11 are required for trafficking and fusion during infection. The molecular mechanisms of caspase-mediated clearance are unknown. It is this study’s objective to decipher the effects of caspase-1/11 on actin dynamics to promote phagolysosomal fusion and restriction. During Legionella infection, caspase-1 and -11-/- macrophages exhibited diminished F/G-actin ratios compared to WT counterparts. In addition, the absence of caspase-1 or -11 prevented the colocalization of the bacterium with F-actin, inhibiting fusion. In elucidating the molecular mechanism, it was found that caspase-1 and -11 differentially distinct molecules upstream of the actin regulator cofilin. We found that the activation of cofilin was affected by both caspases distinctly to promote phagolysosomal fusion, during Legionella infection. These data establish that inflammasome caspases differentially regulate actin polymerization during Legionella infection by modulating F-actin assembly via the activation of cofilin. Understanding the novel molecular mechanisms of caspase-mediated regulation of the host cytoskeletal network will provide novel targets to develop therapeutic interventions for numerous infectious diseases.
Shaping the lung IL-22 response during invasive aspergillosis (INC4P.325)
Authors: Reeder Kristen , Godwin Matthew , Zindl Carlene , Dunaway Chad , Weaver Casey , Steele Chad
Published: January 2023

Exposure to the opportunistic mold Aspergillus fumigatus may result in a wide range of infectious diseases. Among these infections, invasive aspergillosis is the most common and the most lethal. Our lab has previously shown a protective role for Dectin-dependent IL-22 production. In our current studies we sought to identify potential cell source(s) of IL-22 as well as factors critical for its production during invasive aspergillosis. Employing human CD4 IL-22 reporter mice, we show that iNKT cells and γδ T cells are sources of IL-22 48 h post-A. fumigatus exposure. In subsequent studies, we observed an early impairment in IL-22 production by lung cells from CD1d deficient (iNKT deficient) mice that returned to wild type levels by 48 h. In contrast, mice deficient in γδ T cells had relatively intact IL-22 production early after A. fumigatus exposure that became significantly attenuated by 48 h post-challenge. In order to identify factors critical for IL-22 production we interrogated the role of several pertinent transcription factors including: related orphan receptor-γ (RORγ), the aryl hydrocarbon receptor (Ahr), MyD88, and TRIF. Collectively, our data shows that iNKT cells and γδ T cells support early vs. late IL-22 production, which is fully dependent on RORγ and MyD88, partially dependent on Ahr, and independent of TRIF.
STING senses Brucella abortus DNA and triggers pro-inflammatory cytokine production and resistance against bacterial infection (INC4P.330)
Authors: Oliveira Sergio , Marim Fernanda , Barber Glen
Published: January 2023

Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING. Previous studies reported by our research group showed that B. abortus-induced IFN-b is dependent on STING. Herein, we demonstrated that STING is required for Brucella abortus control in vivo. STING KO mice showed enhanced susceptibility at three- and six-weeks post-infection compared to wild-type animals. Furthermore, we observed reduced levels of IFN-g production in spleen cells of STING KO compared to wild-type that corroborates with enhanced susceptibility. Moreover, additional results showed that STING plays an important role in the response to infection in vitro. Macrophages derived from STING KO mice showed reduction in IL-12 and IL-1b cytokine production, when infected with the bacteria or stimulated with bacterial DNA. Diminished production of IL-1b in KO macrophages implicates STING as possible regulator of inflammasome activation. Additionally, murine embryonic fibroblasts (MEFs) from STING KO mice produced much less IL-6 when stimulated with Brucella DNA compared to wild-type MEFs. In summary, we demonstrated the importance of STING as a cytosolic sensor during Brucella abortus infection and its contribution to bacterial control in vivo.
Effects of lipid signaling on innate immune networks during influenza infection. (INM3P.360)
Authors: Oguin Thomas , Lindsley Craig , Thomas Paul , Brown H.
Published: January 2023

If critical steps in the influenza infection process are identified, druggable targets could be discovered. We have uncovered the role of lipid signaling through phospholipase D (PLD) during influenza infection. Our findings suggest that influenza virus takes advantage of PLD activity to quickly and quietly enter the host cell. By inhibiting PLD with small molecules, we found that endocytosis kinetics were subdued. Slowing influenza entry allowed innate immune proteins to sense the pathogen and mount an effective defense. The molecules required for defense included Rig-I, MxA, and IRF3. When these proteins were knocked down in cells treated with PLD2 inhibitor, protection was lost. Inhibition of PLD2 protected cells from several influenza subtypes, including H5N1 and the newly emerged H7N9. Additionally, viral growth in mice treated with the PLD2 inhibitor was reduced, and mice were more likely to survive when treated with PLD2 inhibitor. Our data suggest that signaling through PLD during influenza infection plays a critical role in innate immune detection and defense mechanisms, and that drugging PLD2, allows a better chance to defend against pathogens using innate molecules. Currently, second generation small molecule inhibitors of PLD2 are proving to be more efficacious and potent. Additionally, further insights are being gained regarding the temporal-spatial regulation of innate immune molecules during the first hours of infection.
Inflammation directs waning vaccine immunity during the aging proces (IRM12P.653)
Authors: Norton Elizabeth
Published: January 2023

Aging is a risk factor for disease characterized by complex changes to the immune system and increased disease susceptibility. Vaccination remains the key strategy to prevent infections (e.g., Influenza) in adults. We hypothesized that the age-dependent increase in inflammation directs vaccination outcomes. We tested our hypothesis using commercial Fluzone or TDaP vaccines and BALB/c mice of three ages: young adult (14 weeks), middle-aged (50 weeks), and old (100 weeks). Naive mice exhibited a progressive increase in inflammation (inflammatory cytokines, cellular proliferation) with age. Vaccinated mice exhibited a progressive decrease in development of vaccine antigen-specific immunity with age. Adoptive transfer of splenocytes from young mice did not rescue vaccine responsiveness in older mice. Curiously, we found progressively higher expression of IκBζ with age and vaccination. Lastly, we confirmed our findings using human blood samples and ex vivo PBMC cultures. Overall, our results supported our hypothesis. Altered IκBζ activity (a non-classical NFκB inhibitor) was unexpected as age is linked to higher NFκB expression. However, IκBζ has been linked to cellular senescence and inflammatory cytokine secretion. This suggests that waning vaccine efficacy with age is not a “failure to respond” but a nonproductive immune response post-antigen challenge within a setting of chronic inflammation, much like the theory of Inflammaging.