bluuebunny/crossref_metadata_2025 huggingface dataset:
Shared VH1-46 antibody gene usage in pemphigus vulgaris predicts antibody cross-reactivity to desmoglein 3 and rotavirus VP6 (HUM1P.271)
Authors: Cho Michael Jeffrey , Ellebrecht Christoph , Hammers Christoph , Sapparapu Gopal , Crowe James , Payne Aimee
Published: January 2023

Pemphigus vulgaris (PV) is a potentially fatal skin blistering disease caused by autoantibodies (autoAbs) to desmoglein 3 (Dsg3). Previously, we identified shared VH1-46 gene usage in the anti-Dsg3 repertoire among 4 PV patients via high throughput B cell receptor (BCR) cloning. Somatic mutation reversion studies indicated that VH1-46 BCRs require few to no acidic amino acid mutations to bind Dsg3, which may favor their early selection in the immune response. Interestingly, shared VH1-46 gene usage also occurs in the B cell response to rotavirus protein VP6, and similarly, few somatic mutations are required to bind VP6. We thus investigated whether Dsg3-reactive VH1-46 BCRs cross-react to VP6 which may explain the tolerance of these autoreactive clones. We screened ~108 combinatorial IgM clones from a PV patient, which identified 13 heavy chain CDR3 clonotypic families that cross-react to Dsg3 and VP6. 7/13 used VH1-46, indicating enrichment by Dsg3/VP6 antigen selection. ELISA and human skin immunofluorescence using purified soluble Abs confirmed VP6 and Dsg3 reactivity. Additionally, 3 of 5 VH1-46 anti-Dsg3 IgG clonotypes previously isolated from 3 different PV patients cross-reacted to VP6 in either their somatically mutated or unmutated form. Our data suggest that a VH1-46 B cell response to rotavirus may be an initiating event that develops into an autoimmune response to Dsg3 in susceptible individuals.
Development of novel sterol-based Liver X Receptor agonists as therapeutics for inflammatory diseases (HUM1P.307)
Authors: Yu Shan , Li Shangjin , Henke Adam , Welzel Gustav , Li Sijia , Cheng Bo , Evan Muse , Glass Christopher , Chatterjee Arnab , Roland Jason , Schultz Peter , Tremblay Matthew
Published: January 2023

Liver X Receptor (LXR), a nuclear hormone receptor, is an essential regulator of immune responses. Activation of LXR by synthetic agonists, including T0901317 and GW3965, attenuates progression of inflammatory diseases of animal models. However, the adverse effects of traditional LXR agonists in elevating liver lipids have impeded exploitation of this intriguing mechanism for chronic therapy. The endogenous LXR agonist desmosterol and its sterol-based analog DMHCA activate LXR without the lipogenic action in the liver - this inspired us to develop anti-inflammatory therapies based on these analogs. Here, we show DMHCA oral treatment in mice significantly reduce DSS colitis-induced body weight loss, which is accompanied by reduced expression of inflammatory markers in the large intestine. This reduced inflammation is associated with induced LXR target gene expression, suggesting DMHCA attenuating colitis is mediated, at least partially, through LXR activation. In addition, DMHCA treatment dramatically suppresses inflammatory cytokine expression in traumatic brain injury model. Importantly, in both disease models, DMHCA does not increase liver lipid or serum ALT levels, while T0901317 results in significant liver lipid accumulation and injury. The anti-inflammatory property of DMHCA is recapitulated in vitro in CD3/CD28 activated human PBMCs. Overall, these results provide evidence for the development of sterol-based LXR agonists as novel therapeutics for inflammatory diseases.
Inflammation and gut microbiota-induced hepatic lipogenesis drives metabolic syndrome in TLR5 deficient mice (HUM1P.310)
Authors: Singh Vishal , Chassaing Benoit , Zhang Limin , Yeoh Beng San , Xiao Xia , Harvatine Kevin , Ntambi James , Patterson Andrew , Gewirtz Andrew , Vijay-Kumar Matam
Published: January 2023

The gut microbiota plays a key role in host metabolism. Toll-Like Receptor 5 (TLR5), a flagellin receptor, is required for gut microbiota homeostasis. Accordingly, TLR5 deficient (T5KO) mice are prone to develop microbiota-dependent spontaneous colitis and metabolic syndrome. Herein, we investigated whether metabolic syndrome in T5KO mice correlates with hepatic dyslipidemia and inflammation. T5KO mice displayed elevated neutral lipids with substantial enrichment of C18:1 (n9) relative to wild-type littermates. Oleate enrichment of hepatic lipids was microbiota-dependent. Analyzing cecal contents via 1H NMR-based metabolomics revealed that T5KO mice exhibited elevated short chain fatty acids (SCFA) with a concomitant increase in colonic SCFA receptors and hepatic pro-inflammatory genes and lipogenic enzymes including stearoyl-CoA desaturase1 (SCD1). SCFA treatment further aggravated metabolic syndrome in T5KO mice. Interestingly, deletion of hepatic SCD1 not only prevented hepatic neutral lipid oleate enrichment but also ameliorated hepatic inflammation and metabolic syndrome in T5KO mice. Collectively, these results underscore the key role of the gut microbiota-liver axis in the pathogenesis of metabolic diseases.
Immune-related gene polymorphisms as risk factors for the development of Factor VIII inhibitors in hemophilia A patients: a sibling-pair association analysis (HUM1P.257)
Authors: Ferrante Andrea , Mosher Bryan , Hoffman Megan
Published: January 2023

Inhibitors are one of the most feared complications in hemophilia, occurring in 15-30% of individuals with severe forms of Hemophilia A Human Leukocyte Antigen (HLA) haplotype, and polymorphism of genes regulating the immune response have been indicated as risk factors for inhibitor development. However, it is still unclear the extent to which each of these genetic variants contribute to inhibitor formation as a function of the F8 gene mutation. Here, 96 patients clustered in 45 sibling-pairs and 2 sibling-triplets of which 15 are discordant and the remaining are concordant for inhibitors were considered in a case-control analysis. Next Generation Sequencing on patients’ genomic DNA was performed for detection of FVIII mutation, genotyping of HLA-DRB1 allele and for TNF-α, IL-10, CTLA4, DOCK2 and MAPK9. Our results demonstrate a higher frequency of DRB1*15 allele in inhibitor patients [odds ratio (OR) 2.57; P = 0.022]. In TNF-α, the A allele of the -308G>A polymorphism was found with higher frequency in the inhibitor cohort. This finding was ore evident for the homozygous A/A genotype (7.7% vs 1.8% in inhibitor patient group vs. non-inhibitor respectively, p=0.028). For IL-10, the -1082G allele was observed more frequently in patients with inhibitors (59% vs. 37.5%; P = 0.0037). Our data validate the hypothesis that specific genomic haplotypes affect the development of FVIII inhibitors and can support inhibitor prediction for prognostic and therapeutic applications.
Chronic upper airway inflammation is characterized by an extrafollicular B cell response and elevated antibody production in humans (HUM1P.260)
Authors: Hulse Kathryn , Tan Bruce , Kasjanski Richard , Van Roey Griet , Zhang Chaoying , Norton James , Carter Roderick , Suh Lydia , Kato Atsushi , Kern Robert , Conley David , Peters Anju , Grammer Leslie , Harris Kathleen , Schleimer Robert
Published: January 2023

Chronic rhinosinusitis (CRS) is an upper airway disease that affects 30 million Americans and a subgroup of CRS patients also has nasal polyps (CRSwNP). In CRSwNP patients, inflamed sinus tissues can be removed as part of treatment, which provides a unique model system to investigate mechanisms of human mucosal inflammation. We have previously reported local elevations in NP of B cells, antibodies (Ab), and expression of Epstein-Barr virus-Induced protein 2 (EBI2), a marker of extrafollicular plasma cells (PC). To characterize the mechanisms mediating these responses, we assessed B cells from fresh tissue (d0) or after 4 days of culture (d4) from NP, uncinate tissue (UT) or tonsils from CRS and control subjects by flow cytometry. We measured Ab production by multiplex array and assessed formation of B cell follicles by immunohistochemistry. We assessed the Ab repertoire by DNA deep sequencing and by analysis of Ab genes from single cells. We found that NP uniquely supported the accumulation of PC (p<0.01) and Ab production (p<0.05) in vitro. Plasmablasts (PB) and PC were Ki67+, and EBI2 expression was highest on PB. We found no evidence for increased B cell follicles in NP compared to control UT (n>10/group). Ab from NP had fewer CDR3 mutations than Ab from UT (p<0.05), but more mutations than Ab from peripheral blood (p<0.05). These data suggest that there is a strong extrafollicular B cell response in NP that may drive Ab production during chronic airway inflammation.
The TNF-family ligand TL1A and its receptor DR3 promote T cell mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9 producing T cells (HYP2P.333)
Authors: Meylan Francoise , Richard Arianne , Tan Cuiyan , Hawley Eric , Gomez-Rodriguez Julio , Goswami Ritobrata , Yang Xiangping , Cruz Anthony , Penumetcha Pallavi , Hayes Erika , Pelletier Martin , Gabay Odile , Walsh Matthew , Ferdinand John , Keane-Myers Andrea , Choi Yongwon , O'Shea John , Al-Shamkhani Aymen , Kaplan Mark , Gery Igal , Siegel Richard
Published: January 2023

The TNF-family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation, and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3 deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. Here we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.
Pentraxins interact with misfolded proteins and inhibit aberrant innate immune activation (INC1P.361)
Authors: Dorta-Estremera Stephanie , Cao Wei
Published: January 2023

Amyloid deposition is linked to different diseases, including Alzheimer’s disease and systemic amyloidosis. How misfolded proteins, including amyloid and its in-process products, are controlled by the host remains obscure. Pentraxins are multimeric pattern recognition proteins that bind to diverse ligands and promote the clearance of microbes. Serum amyloid P-component (SAP), a short chain pentraxin, associates with amyloid deposits in patients. We hypothesize that pentraxins may interact with different forms of misfolded proteins and modulate their biological impact. We found that human SAP readily bound to all types of amyloids, including protein-only, DNA-containing or heparin-containing fibrils. This interaction depended on the presence of calcium. Interestingly, both SAP and C-reactive protein (CRP), another pentraxin, bound to soluble amyloid precursor (AP) protein in the absence of divalent cations. Furthermore, SAP decamer recognized AP whereas aggregated SAP preferentially associated with amyloid. The misfolded structure in AP was necessary for binding to SAP and CRP. DNA-containing amyloid (A-DNA) triggers type I interferon (IFN-I) production by plasmacytoid dendritic cells (pDCs) and promotes lupus-like disease. Binding of SAP to A-DNA inhibited the production of IFN-I by human pDCs. Altogether, our data suggest that pentraxins differentially interact with various forms of misfolded proteins and modulate their ability to stimulate aberrant innate immune activation.
The RiboPuromycylation Method Reveals Rapid IL12-Dependent Antigen-Independent Activation of Bone Marrow Virtual Memory T cells (INC1P.403)
Authors: Seedhom Mina , David Alexandre , Hickman Heather , Bennink Jack , Yewdell Jonathan
Published: January 2023

Protein translation is an essential and intricately regulated cellular process that can potentially be used to monitor immune cell activation. We extended the RiboPuromycylation Method (RPM) to measure protein translation in vivo. Unexpectedly, 24 h following either vaccinia virus (VACV) or vesicular stomatitis virus (VSV) infection, 15-30% of bone marrow (BM) CD4+ and CD8+ T cells, all CD44+, increase protein translation. This is days before protein translation is increased in T cells in the draining lymph node or spleen. Bystander BM but not splenic OT-1 T cells demonstrate increased protein translation after VACV infection. Early virus-induced BM T cell translation is absent in IL-12 -/- mice. In BM cultures from naïve mice, addition of IL-12, and especially IL-12 with IL-18 increase T cell translation within hours, with IL1-2 and IL-18 eventually leading to T cell division. Our findings indicate that the BM is a unique environment where bystander T cells are rapidly activated by innate immune response to viruses. We hypothesize that this may allow memory and memory-like T cells to be activated in the absence of cognate antigen to facilitate their functionality when they encounter the cognate pathogen.
Regulation of the gut microbiota and intestinal homeostasis by phagocyte NADPH oxdiase (INC1P.351)
Authors: Zeng Melody
Published: January 2023

The phagocyte NADPH oxidase generates reactive oxygen species (ROS) for microbial killing, and its impairment gives rise to Chronic Granulomatous Disease (CGD), a primary immunodeficiency characterized by recurrent infections and granulomatous lesions. Mutations in Nox2 (Cybb), a critical subunit of the oxidase, contribute 66% of CGD. In our study, we found spontaneous inflammation in the intestine of naïve Cybb-/- mice, indicated by higher levels of IL-1b, IL-6 and TNFα in lamina propria macrophages, as well as increased levels of IFNγ and IL-17A in CD4+ T cells. In addition, Klebsiella pneumoniae, a bacterium frequently found in granulomatous lesions in CGD patients, were detected in the spleens and mesenteric lymph nodes (MLNs) of naïve Cybb-/- mice, and IP administration of this pathobiont strain of K. pneumonia led to rapid mortality of wild-type mice. Oral administration of this K. pneumoniae induced intestinal inflammation in wild-type and Cybb-/- mice, but translocation of K. pneumoniae to MLNs was drastically enhanced in Cybb-/- mice and bone marrow chimeras with a neutrophil-specific deficiency in NOX2. Thus, our results demonstrate the importance of neutrophil-derived ROS in restricting dissemination of pathobionts from the intestine under steady-state conditions. Therefore, our study underscores a critical role for the NADPH oxidase in regulating intestinal homeostasis and provides insights into intestinal complications in CGD patients.
IL-6 regulates pro- and anti-inflammatory cytokine production and mortality of Mycobacterium tuberculosis infected type 2 diabetic mice (INC2P.418)
Authors: Cheekatla Satyanarayana , Tripathi Deepak , Venkatasubramanian Sambasivan , Paidipally Padmaja , Welch Elwyn , Tvinnereim Amy , Kornfeld Hardy , Vankayalapati Ramakrishna
Published: January 2023

In this study, to determine whether diabetic (DM) mice are susceptible to Mycobacterium tuberculosis (Mtb) infection, we developed experimentally induced diabetes in wild type C57BL/6 mice with streptozotocin & nicotinamide. We then infected non-DM and DM mice with Mtb H37Rv by aerosol. Around 50% of Mtb infected DM (p=0.05) and 25% of uninfected DM (p=0.07) mice died in 5 to 6 months compared to no deaths in infected control mice. Six months after Mtb infection, there was a statistically significant but marginal increase in bacterial burden in the lungs of Mtb infected DM mice compared to non-DM infected mice (3.1 ± 0.4 x106 vs 0.9 ± 0.1 x 106, p=0.001) suggesting increased mortality was not due to increased bacterial burden. Real time PCR analysis of Mtb infected DM lungs indicated significantly increased pro- and anti-inflammatory cytokine gene expression compared to uninfected DM and infected control mice, including increased levels of IL-6 mRNA. Anti-IL-6 antibody treatment of Mtb infected DM mice enhanced the survival (100% vs 60% survival in isotype control antibody treated, p=0.02) and reduced pro- and anti-inflammatory cytokine production. Dendritic cells (DC), but not other immune cells were the major source for IL-6 in Mtb infected DM mice. NK cells of Mtb infected DM mice further enhanced IL-6 production by autologous DCs. Our results suggest NK-DC interaction enhance IL-6 production which drives the immune pathology and mortality of Mtb infected diabetic mice
Novel anti-Mer antibodies in macrophage phenotype analysis (INC2P.419)
Authors: Stepniak Dariusz , Atteberry Brandi , Li Shourong , Baterina Oscar , Oxford Natalie , Miranda Cherlyn , Miletic Ana , Schrantz Nicolas , Huang Qiulong , Funatake Castle , Just Margaret
Published: January 2023

Mer is a member of the TAM family of tyrosine kinase receptors that also includes Axl and Tyro3. Mer is expressed on a subset of anti-inflammatory macrophages and is involved in the removal of apoptotic cells. The engulfment of apoptotic cells relies on two soluble accessory molecules Protein S and Gas6 that bind to phosphatidylserine, which is found on the outer leaflet of the plasma membrane of cells undergoing apoptosis. Upon binding these ligands, Mer undergoes autophosphorylation at multiple tyrosine residues that activate the PI3K and Akt pathways. This then leads to the phagocytosis and clearance of apoptotic cells and also results in the direct inhibition of TLR-induced production of pro-inflammatory cytokines. Deficiency of Mer causes general autoimmunity, inflammation, and accumulation of apoptotic bodies. Because Mer expression strongly correlates with macrophage function, It can be used for phenotyping macrophage subpopulations. Here we use novel anti-Mer antibodies in flow cytometric analysis of macrophages and corroborate the expression of this receptor and its importance in distinguishing functional macrophage subsets.
Essential role for CCR2+ inflammatory monocytes in early and late control of persistent Salmonella infection (INC2P.412)
Authors: Van der Velden Adrianus , Tam Jason , Bliska James
Published: January 2023

Monocytes play a critical role in innate and adaptive immunity. Monocytes originate from progenitors in the bone marrow and traffic via the bloodstream to peripheral tissues, where they can differentiate into protective cell types such as macrophages and dendritic cells. Emigration of Ly6Chi monocytes from the bone marrow is dependent on CC-chemokine receptor 2 (CCR2) and largely mediated by CC-chemokine ligand 2 (CCL2, also referred to as MCP1). We previously showed that large numbers of Ly6Chi Ly6G- CD11b+ monocytes accumulate and persist in tissues of mice infected with the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Here, we report that Ccr2 is required and Ccl2 is important for the recruitment of Ly6Chi Ly6G- CD11b+ monocytes into tissues during persistent S. Typhimurium infection of hemizygous C57BL/6J Nramp1G169 mice, which are phenotypically Nramp1+. We show that Ccr2- and Ccl2-deficient mice are more susceptible to S. Typhimurium infection than wild-type mice, with Ccr2-deficient mice being more susceptible to S. Typhimurium infection than Ccl2-deficient mice. We also show that depletion of CCR2+ cells during the first or third week of S. Typhimurium infection increases susceptibility to salmonellosis. These results indicate that CCR2+ cells, including Ly6Chi Ly6G- CD11b+ monocytes, play an essential role in early and late control of S. Typhimurium infection.
TRAF6 and TAK1 control signals for a senescence response by an endosomal NK cell receptor (INC2P.414)
Authors: Rajagopalan Sumati , Lee Elizabeth , DuPrie Matthew , Long Eric
Published: January 2023

The endosomal innate receptor CD158d (KIR2DL4) induces cellular senescence in human NK cells in response to soluble ligand (HLA-G or agonist antibody). These senescent NK cells display a senescence-associated secretory phenotype (SASP) and their secretome promotes vascular remodeling and angiogenesis. To understand how CD158d initiates signaling for a senescence response, we mapped the region in its cytoplasmic tail that controls signaling. We identified a conserved TRAF6 binding motif, which was required for CD158d-induced NF-κB activation and IL-8 secretion, for TRAF6 association with CD158d, and for TRAF6 recruitment to CD158d+ endosomes in transfected cells. The adaptor TRAF6 is known to couple proximal signals from receptors such as endosomal TLRs and CD40 through the kinase TAK1 for NF-κB-dependent pro-inflammatory responses. siRNA-mediated silencing of TRAF6 and TAK1, and inhibition of TAK1 blocked CD158d-dependent IL-8 secretion. Stimulation of primary, resting NK cells with soluble Ab to CD158d, induced TRAF6 association with CD158d, induced TAK1 phosphorylation, and inhibition of TAK1 blocked the CD158d-dependent reprogramming of NK cells that produces the SASP signature. Our results reveal that a prototypic TLR and TNF-receptor signaling pathway is used by a killer cell immunoglobulin-like receptor that promotes secretion of pro-inflammatory and pro-angiogenic mediators as part of a unique senescence phenotype in NK cells.
Mast cell-derived histamine is necessary for platelet-activating factor mediated systemic immunosuppression (IRC4P.606)
Authors: Ocana Jesus , Yi Quiaofang , Yao Yongxue , Bryce Paul , Kaplan Mark , Travers Jeffrey
Published: January 2023

Platelet-activating Factor (PAF) activates many cell types via the G-protein coupled PAF-receptor (PAF-R). Systemic PAF-R activation exerts acute pro-inflammatory, as well as delayed systemic immunosuppressive effects in vivo. PAF enzymatic synthesis and degradation is tightly regulated, but reactive oxygen species from oxidative stressors, such as UVB, chemotherapy and cigarette smoke, generate PAF-like species via the unregulated oxidation of membrane lipids. The systemic immunosuppressive effects of these pro-oxidative stressors are due to PAF-R signaling. This work establishes the role of the mast cell (MC) and the soluble mediators involved in PAF-R-mediated immunosuppression. Using a contact hypersensitivity model, we show that PAF loses its immunosuppressive effects in MC-deficient (Wsh) mice, a phenotype rescued by dermal reconstitution of Wsh mice with WT bone marrow derived MCs. Use of PAF-R-deficient Wsh mice suggests that the MC PAF-R is necessary and sufficient to mediate PAF-R systemic suppression, in a process attenuated by COX-2 and TGF-β inhibitors. Finally, by transplanting histamine (histidine decarboxylase-null) deficient MCs, we demonstrate that this pathway requires MC-derived histamine release. These studies fit with the hypothesis that MC PAF-R activation with resultant histamine release mediates PAF-R systemic immunosuppression.
ILC3 detected in intestinal mucosal tissues of mice with chronic alcohol consumption (IRC4P.608)
Authors: Ito Ichiaki , Nishiguchi Tomoki , Asai Akira , Suzuki Sumihiro , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

Bacterial translocation from microbiota causes serious sepsis in mice with chronic alcohol consumption (CAC-mice). Since innate lymphoid cells (ILCs) are cells to be involved in the prosperity and decoy of host antibacterial innate immunities, in this study, we tried to isolate ILCs from intestinal mucosal tissues of CAC-mice with various durations of alcohol consumption. CAC-mice were prepared by an oral gavage of 0.6 ml/day of 20% EtOH for 4 to 14 weeks. Lineage negative (lin-) cells were isolated from the intestinal mucosal tissue of CAC-mice. These cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) for 6 hours. Obtained cells were analyzed for ILC2 and ILC3 by flow cytometry, and culture fluids of lin- cells were assayed for IL-22 and IL-17A by ELISA. In the results, the percentage of CD45+CD90+RORγt+ cells (ILC3) in the lin- cell population was gradually increased in mice with alcohol consumption for 10-12 weeks (7.84% in average), as compared to be percentage of ILC3 in normal mice (0.67%). On the other hand, CD127+ICOS+Sca-1+ST2+ cells (ILC2) were minimally isolated from the lin- cell population of 4-12 week CAC-mice. Lin- cells from 12 week CAC-mice produced 400 pg/ml of IL-22 and 1.7 ng/ml of IL-17A. These results indicate that 10-12 week CAC-mice are carriers of ILC3 which play a role in the increased susceptibility of CAC-mice to enterococcal translocation and subsequent sepsis.
Differential gene expression following stimulation of TLRs 2, 3, 7 and 8 in rag1-/- mutant zebrafish (IRC4P.613)
Authors: Muire Preeti
Published: January 2023

Natural Killer (NK) cells have shown to mediate adaptive immune responses to chemical haptens and cytomegalovirus in T and B cell deficient mice. T and B cell deficient zebrafish mount specific protection to intracellular bacteria. We hypothesize that a sub-population of zebrafish NK cells mediates this protection, and these cells have a mechanism for enhanced discrimination of a bacterial target. However, zebrafish NK cells have not been characterized, and their response to TLR agonists has not been investigated. We utilized rag1-/- mutant zebrafish as an in vivo fish model to determine if TLR agonists (TLR2: βglucan, TLR3: Poly I:C and TLR 7/8: R-848) stimulate NK cells in liver, kidney marrow and spleen tissues. We used IFNγ expression as an indicator of NK cell activation, TNFα expression as an indicator of macrophage activation, and MxA expression as an indicator of IFNa and IFNβ production. With RT-qPCR we quantified transcript levels at 1h, 6h, 12h and 24h after injecting rag1-/- mutant zebrafish with either βglucan, POLY I:C or R-848. Our results show significant increases of IFNγ, MxA and TNFα transcripts in the three tissues with βglucan, POLY I:C or R-848 compared to PBS injected control fish. Liver had the highest expression of IFNγ in response to R-848 and βglucan. The data presented are the initial findings in an investigation to help define mechanisms involved in NK cell mediated protective immunity in rag1-/- mutant zebrafish.
Cholinergic modulation of inflammatory diseases: role in the amelioration of type 1 diabetes (IRC4P.452)
Authors: Fernandez-Cabezudo Maria , George Junu , Bashir Ghada , Al-Ramadi Basel
Published: January 2023

The cholinergic anti-inflammatory pathway connects the central nervous system with the immune system via the vagus nerve and controls the release of pro-inflammatory cytokines. We have previously shown that inhibition of acetylcholinesterase (AChE), thereby leading to an accumulation of acetylcholine (ACh), modulates the inflammatory response. Type 1 diabetes (T1D) results from a deficiency in the production of insulin as a consequence of an immune-mediated damage of pancreatic β cells. Here, we investigated the potential of regulating the development of T1D through the inhibition of AChE. Modulation of AChE activity was achieved by administration of paraoxon, a potent inhibitor of the enzyme. To induce hyperglycemia, we used a well-characterized regimen of multiple low doses of streptozotocin (MLD-STZ). Our results showed that inhibition of AChE prevented the development of MLD-STZ-induced hyperglycemia (blood glucose levels >200 mg/dl) in C57BL/6 mice. At day 18 post-STZ administration, immunohistological analysis of pancreatic tissue in paraoxon pre-treated mice revealed healthy islets with normal levels of insulin. In contrast, control mice routinely exhibited glucose levels of 300-400 mg/dl and severe loss of islet morphology by the same time point. qRT-PCR analysis demonstrated that AChE inhibition prevents the loss of insulin production induced by MLD-STZ administration. Our results provide evidence for cholinergic control of the development of T1D in mice.
IL-25 promotes the function of CD4+CD25+ T regulatory cells and prolongs skin-graft survival in murine models (IRC5P.620)
Authors: Zhou Xiaohui , Tang Jiayou , Liu Jie , Meng Qingshu , Liu Zhongmin , Fan Huimin
Published: January 2023

Interleukin (IL)-25, also known as Interleukin (IL)-17E, belonging to the IL-17 family. Unlike the other members, IL-25 has long been deemed as a pro-inflammatory cytokine favoring Th2--type immune response. Here, we show that expression of IL-25 in skin and subcutaneous tissues were constantly, while increased during allograft rejection. At same time, deficiency of IL-25 aggravates skin allograft rejection in murine models. Further experiments showed deficiency of IL-25 down-regulated the expression of Foxp3 among CD4+ cells and aggravated skin allograft rejections in murine models. While, exogenous IL-25 restored the Foxp3 expression and the function of Tregs in vitro. Adoptively transfer of IL-25 deficient Tregs failed to protect the allograft from rejection, compared to the WT Tregs. Moreover, IL-25 promoted phosphorylation of NFAT2, up-regulated the Foxp3 expression and contributed to the function of Tregs. Our findings indicate that IL-25 can promote the function of Tregs via impeding the interaction of NFAT2 and Foxp3, and prolong skin-graft survival.
TSG6 as a central mediator of macrophage M2 polarization (IRC5P.626)
Authors: Mittal Manish , Toya Sophie , Malik Asrar
Published: January 2023

TNF-α stimulated gene-6 (TSG6) is an anti-inflammatory protein, secreted by activated monocytes and endothelial cells. TSG6 binds to CD44 receptor expressed on multiple cell types including monocytes/macrophages. We tested the concept that TSG6 via binding to CD44 induces a phenotypic shift in macrophages from pro-inflammatory M1 to anti-inflammatory M2 type that protects the mice against lung inflammation and injury induced by endotoxin LPS. We show that TSG6 suppressed the induction of M1 markers (IL-6, IL-1β, CXCL1, iNOS) and activated expression of M2 marker genes (such as IL-10, Mannose receptor (CD206) and Chitinase-3-like-3). The shift was abrogated in macrophages isolated from CD44-/- mice. TSG6 also suppressed the activation of NF-ΚB signaling by inhibiting interaction of MyD88 with TLR4. In addition, TSG6 induced activation of anti-inflammatory transcription factor STAT6 in LPS exposed macrophages. Intra-tracheal instillation of TSG6 protected the mice against LPS induced mortality. Moreover, mice treated with TSG6 showed significantly suppressed lung vascular injury and neutrophil sequestration on challenge with LPS. That TSG6 binding to CD44 activates the macrophage anti-inflammatory program suggests that TSG6 is a specialized regulator of inflammation and potential therapeutic target.
C1q-based tolerance mechanism in HMGB1-mediated activation of human monocytes (IRM5P.649)
Authors: Son Myoungsun , Porat Amit , Tracey Kevin , Diamond Betty
Published: January 2023

C1q, a component of the classical complement pathway, is required for clearance of apoptotic cells. C1q also regulates activation of innate immune cells through engagement of C1q receptors present on the immune cell surface. C1q deficiency is highly associated with the development of systemic lupus erythematosus (SLE). High Mobility Group Box 1 (HMGB1), a pro-inflammatory mediator, is secreted by inflammatory cells and passively released from necrotic cells. HMGB1 interacts with TLR4 and receptor for advanced glycation end products (RAGE), to augment the production of type 1 interferon and proinflammatory cytokines. Here we show that C1q inhibits HMGB1-mediated monocyte activation through cross-linking RAGE and Leukocyte-associated Ig-like receptor-1 (LAIR-1), which is an inhibitory receptor for collagen and C1q. This crosslinking causes LAIR-1 to enter lipid rafts following HMGB1 and C1q stimulation. C1q suppress HMGB-1 mediated induction of type I-interferon and pro-inflammatory cytokines including IL-12 and TNF-α. Moreover, exposure of monocytes to HMGB1 and C1q together leads to upregulation of a number of anti-inflammatory molecules that might function to induce phagocytosis and to maintain monocyte quiescence. Our results suggest that C1q regulates the activation of innate immunity by HMGB1 and maintains immune homeostasis. These data are consistent with the established requirement for C1q in the prevention of autoimmunity.
Bcl6 mediates the regulation of the innate immune response against RNA virus (IRM5P.650)
Authors: Shi Liyun , Kang Yanhua , Zhuang Ningtong , Zhang Hang
Published: January 2023

Bcl6, a POZ transcription factor, plays an essential role in the tumorgenensis, inflammation and development, but its role in the antiviral response has never been established. Here, we demonstrate that Bcl6 unexpectedly functions to inhibit RIG-I-mediated antiviral signaling and impedes the clearance of viral infection. More importantly, we identify that miR-127 as a upstream regulator of Bcl6, and the subsequent co-repressive complex in the initiation of RIG-I-driven response. This finding extends our knowledge about mitochondrial antiviral mechanisms and proposes a novel corepressor network required for the coordination of the innate immune response.
p53 regulates expression of the early response gene tumor necrosis factor-α-induced protein 8 in macrophage (IRM6P.660)
Authors: Lowe Julie , Menendez Daniel , Nguyen Thuy-Ai , Grim Sara , Resnick Michael , Fessler Michael
Published: January 2023

The cell response to pathogens involves complex signaling cascades that affect expression of thousands of genes. Characterization of the regulation and function of genes involved in the stress response is critical since failure of this response can foster the development of chronic disorders such as autoimmune diseases and cancer. A growing body of evidence shows a role for the transcription factor p53 in the immune response. To better characterize the role of p53 in the innate immune response, we performed a microarray in primary human macrophages and identified the gene Tumor Necrosis Factor-Alpha Induced Protein-8 (TNFAIP8) as novel p53 target. ChIP-seq analysis shows strong binding of p53 to an intragenic region within TNFAIP8, which contains a functional p53 response element as confirmed with reporter assays. This region is an enhancer, since ChIP assays show enhancer-like histone modifications and 3C assays indicate chromatin looping with the promoter region. Very little is known about TNFAIP8 function; however, Tnfaip8 was previously reported to confer resistance to Staphylococcus aureus, suggesting a role in host defense. Studies are currently underway to characterize expression regulation and functions of TNFAIP8 in macrophages in response to TLR ligands. These results provide a better understanding of the role of p53 in the innate immune response and the regulation and function of the understudied TNFAIP8 gene.
A dichotomy in cellular immune responses to experimental blood stage malaria infection in humans revealed by systems immunology (IRM6P.661)
Authors: Burel Julie , Apte Simon , Mccarthy James , Doolan Denise
Published: January 2023

Despite intense research for many decades, the mechanisms of protective immunity against malaria are poorly understood. Recent technological and conceptual advances in the field of immunology, in particular Systems Immunology, offer the potential to provide new insights into the key mechanisms involved in host immunity. This study aimed to dissect at the molecular level the cellular immune responses to malaria by combining miRNA and mRNA profiling, multiparameter flow cytometry with a unique P. falciparum blood stage experimental infection model. Unexpectedly, naive subjects segregated into two distinct groups based on their molecular immune profile upon infection, classified as High and Low responders. High responders presented increased expression of circulating miRNAs involved in immune response, increased lymphocyte activation, increased P. falciparum specific antibody responses and reduced blood stage parasitemia over the course infection; no significant changes were observed in Low responders. This study is the first to demonstrate inter-individual variability in immune responses to experimental malaria infection. The molecular profile of circulating lymphocytes prior to infection in High and Low responders is currently being investigated, to shed light on the molecular mechanisms driving this dichotomy in immune responses. This may predict immune response in naive individuals and therefore would have tremendous implications for vaccine development and evaluation.
The RNA-binding protein, polypyrimidine tract-binding protein, affects multiple events in CD4 T cell activation through distinct processes (IRM7P.717)
Authors: Covey Lori , La Porta James , Matus Nicodemos Rodrigo
Published: January 2023

We have previously shown that the RNA binding protein, polypyrimidine tract-binding protein (PTB) plays a crucial role in regulating the expression of CD40L in activated CD4 T cells. Regulation occurs through stabilizing the message at late times of activation as well as changing the distribution of CD40L mRNA within distinct cellular compartments. Further experiments were designed to assess how PTB affects global CD4 T cell activation by using shRNA introduced into primary human CD4 T cells. Specifically, we found that PTB is critical for the proliferation and viability of activated T cells. The propensity to undergo cell death was evident in both infected and uninfected CD4 T cells within the same population suggesting that either soluble and/or cognate interactions were being affected by decreased PTB. Accordingly, PTB was found to be required for optimal expression of CD25, CD69, TNF-α and IL-2 but not CD38 or IFN-γ. When the decay rates were assessed in anti-CD3/anti-CD28-stimulated CD4 T cells, the pattern of IL-2, TNF-α and CD69 mRNA decay did not change over a time course of activation whereas CD25 showed a pattern of mRNA decay that was highly similar to CD40L. Evaluation of signaling pathways using phosphoflow analysis revealed PTB-specific changes in the NF-κB and MAPK-ERK pathways. Together, these findings support a role for PTB in the control of CD4 T cell activation through processes that are both dependent on and independent of PTB-regulated mRNA stability.
Nedd4 family interacting proteins activate ubiquitylation pathways to limit ERK signaling following TCR engagement (IRM7P.702)
Authors: O'Leary Claire , Riling Chris , Deng Guoping , Spruce Lynn , Seeholzer Steve , Oliver Paula
Published: January 2023

Ubiquitylation tunes signaling pathways in stimulated T cells to regulate activation and function. We have found that, in vitro, Nedd4-family interacting protein 1 (Ndfip1) and Ndfip2 activate Nedd4-family catalytic E3 ligases with known roles in T cells. Ndfip1 negatively regulates T cell activation and Th2 polarization, but the in vivo role of Ndfip2 is unknown. We generated Ndfip2-/- mice and found that Ndfip2 is not a prominent negative regulator of T cell activation or Th2 polarization. However, loss of Ndfip2 exacerbates the inflammatory Ndfip1-/- phenotype, suggesting that Ndfip2 dampens inflammation. We generated mixed fetal liver chimeras and found that Ndfip1/Ndfip2 deficiency leads to T cell intrinsic hyperactivity. To identify signaling pathways affected by loss of Ndfips, we used semiquantitative proteomics to compare stimulated control and knockout CD4+ T cells. Pathway analysis revealed increased MEK1 activity in knockout cells, which was confirmed by observing increased ERK phosphorylation in stimulated Ndfip1/Ndfip2 deficient CD4+ T cells. To identify aberrant ubqiuitylation of proteins upstream of ERK, we have taken a quantitative proteomic approach, using Tandem Ubiquitin Binding Entities (TUBEs) to enrich for polyubiquitylated proteins in isotope labeled control and Ndfip deficient CD4+ T cells. This approach has yielded several potential targets of Ndfip-dependent ubiquitylation in CD4+ T cells that are currently under investigation.
CD5 inhibits the activity of dendritic cells to activate T cells and induce immune responses (IRM7P.703)
Authors: Xu Hui , Li Hui , He Donggou , Xu Shin , Raman Chander
Published: January 2023

It is known that CD5 mediates inhibitory signals in T cells and plays important roles in immune tolerance. Little is known about whether CD5 has a role in dendritic cell (DC) induced T cell activation and immune responses. Here we show that CD5 was expressed by CD11c+ DC in the thymus, lymph node, spleen and skin of mice. A deficiency in CD5 gene (CD5-/-) did not have a significant effect on the development of DC. However, it enhanced the activity of DC to activate CD4 and CD8 T cells in vitro and in vivo. CD5-/- DC were more potent than wild type DC in the induction of anti-tumor immunity and contact hypersensitivity response in animals. CD5-/- DC induced higher levels of IL-2 and IFN-gamma production by T cells than wild type DC. Restoration of CD5 expression on CD5-/- DC inhibited their activity to activate T cells and induce immune responses in mice. Further analysis indicated that the deficiency in CD5 increased the production of IL-12 and IL-23 by DC and that the restoration of CD5 expression inhibited the cytokine production by CD5-/- DC. Collectively, the study has demonstrated that CD5 inhibits the production of inflammatory cytokines by DC and has an inhibitory effect on their activity to activate T cells and induce immune responses. It provides novel insights into mechanisms for DC induced T cell tolerance and immune suppression.
CD28 induces mitochondrial respiration through Slp-76 in long-lived plasma cells for reactive oxygen species dependent survival. (LYM2P.737)
Authors: Utley Adam , Carlson Louise , Rozanski Cheryl , Lee Kelvin
Published: January 2023

Sustained humoral immunity is dependent upon bone marrow (BM) resident long-lived plasma cells (LLPC). We have shown that CD28 signaling in LLPCs is necessary for their survival and antibody production; however, the mechanistic basis for this is unclear. In T cells, CD28 induces glycolysis at the expense of mitochondrial respiration. Surprisingly, CD28 activation drives mitochondrial respiration in LLPCs but not short-lived plasma cells (SLPC) through PLCγ1. Paradoxically, respiration dependent production of reactive oxygen species is required for CD28 mediated survival. Mechanistically, CD28 provides a pro-survival signal in LLPCs through Slp-76, an adaptor protein known to be upstream of PLCγ1 in T cells. CD28 itself drives Slp-76 transcription and subsequent protein expression. Additionally, at the mRNA level Slp-76 is highly expressed in human BM PCs (LLPCs) when compared to splenic PCs (SLPCs). Furthermore, CD28 upregulates the APRIL/BAFF receptor BCMA in LLPCs but not SLPCs. Due to the high levels of Slp-76 seen in LLPCs, but not in SLPCs, we propose a model wherein CD28 induces a longevity program through Slp-76 during plasma cell differentiation that facilitates plasma cell responsiveness to extrinsic survival signals in the bone marrow niche, allowing for a plasma cell to become long-lived. By doing so, CD28 may regulate LLPC induction, function and survival. CD28 therefore represents a novel signaling axis to exploit for the augmentation of vaccine design.
Role of IL-4Rα on myeloid dendritic cells in pathogenicity of respiratory syncytial virus infection and asthma development (MUC1P.909)
Authors: Shrestha Bishwas , You Dahui , Saravia Jordy , Siefker David , Cormier Stephania
Published: January 2023

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and severe disease is associated with asthma development in later life. We have shown that neonatal mice respond to primary RSV infection with T helper type 2 (Th2) biased immune responses which are enhanced following reinfection. Our lab has demonstrated elevated levels of IL-4Rα on neonatal mDCs compared to adults. Therefore, we investigated the role of IL-4Rα on mDCs in the RSV induced Th2 biased response in neonates. CD4+ T cells from adult mice were co-cultured with RSV infected CD11c+ cells (DCs) from neonatal wild type or IL-4Rα-/- mice and resulting CD4+ T cell sub phenotypes were analyzed. DCs from IL-4Rα-/- mice induced more Th1 and less Th2 cells compared to DCs from wild type mice. Thus, we hypothesized that IL-4Rα on neonatal mDCs is responsible for the pathogenicity in RSV infection. To test this hypothesis, we used transgenic mice in which IL-4Rα is specifically depleted on CD11c+ cells. These mice were infected with RSV as neonates and reinfected four weeks later. We then analyzed the pulmonary T cell profile, cytokines, and pathophysiological responses. Mice in which IL-4Rα was deleted on mDCs showed significant reductions in their pathological Th2 responses. Together, these data demonstrate that the asthmatic Th2 biased immune phenotype induced during neonatal RSV infection is mediated in part by IL-4Rα expression and signaling on mDCs.
Transfer factor: forgotten, but not gone (MUC2P.934)
Authors: Myles Ian , Datta Sandip
Published: January 2023

It has been over 70 years since the discovery of cellular immunity. Further investigation revealed that the even if immune cells are lysed and the molecular size limited to 8kDa, such injections retain some of the immunity-transferring properties. The term “transfer factor” (TF) was coined to denote the undiscovered substance responsibly for this phenomenon. Yet despite early interest, TF was all but abandoned in the 1990’s; in part due to unsubstantiated and fraudulent claims as well as the discovery of HIV, which suspended consideration of deriving therapy from blood. However, while TF was shelved as poorly elucidated, no clear explanation was provided for the observation of a small molecule harboring antigen-specific properties and therapeutic potential. Reopening this chapter, we discovered transfer factor is produced by memory T-cells and acts directly upon dendritic cells in an antigen-specific manner; ultimately activating mucosal immunity via Tc17 pathways. Antigen-targeted TF enhanced inflammatory response in models against Candida as well as S. aureus. Mass spec data suggests TF is hybrid compound, predicted to containing elements of peptides and nitrogenous bases. Finally, we provide ex vivo human data to demonstrate the therapeutic potential of TF and even attempt to address some of the past’s more fanciful claim. This report represents transfer factors’ resurrection and provides a modern scientific investigation into this once forgotten area of immunology.