bluuebunny/crossref_metadata_2025 huggingface dataset:
PLZF null mice have reduced type 2 pulmonary inflammation due to defective ILC2 effector function (HYP6P.265)
Authors: Verhoef Philip , Constantinides Michael , McDonald Benjamin , Sperling Anne , Bendelac Albert
Published: January 2023

Rationale. Pulmonary ILC2s have been recently identified as a source of IL-5 and IL-13 during allergic inflammation and helminth infection. Fate-mapping experiments in our lab revealed that the transcription factor PLZF is upregulated during ILC2 development. We hypothesized that the PLZF null mouse would have an impaired innate type 2 response. Methods. Type 2 pulmonary inflammation was induced by intranasal treatment of PLZF null mice and wild-type littermates with either the protease papain, the cytokine IL-25, or saline for 3 days, followed by bronchoalveolar lavage, cellular isolation, and cytokine assays. PLZF null-wild type mixed bone marrow chimeras were generated to determine if the defects in the type 2 response were cell-intrinsic. Nippostrongylus brasilensis infection was used as a physiologic type 2 inflammatory stimulus. Results. While ILC2s were present in PLZF null mice, pulmonary eosinophilia and type 2 cytokine levels were both significantly reduced in response to papain, IL-25, or Nippostrongylus infection. Mixed chimera experiments indicated that these defects were intrinsic to ILC2s: fewer PLZF-null-derived pulmonary ILC2s were reconstituted and they were less able to produce type 2 cytokines, while eosinophils from wild-type or PLZF null mice responded comparably. Conclusion. Mice lacking PLZF have a defective type 2 immune response in response to various stimuli, due to impaired ILC2 effector function.
Doxycycline exerts multiple anti-allergic effects in vitro and in vivo though modulating PI3K/Akt pathway (HYP6P.269)
Authors: Zheng Song Guo , Su Wenru , Chen Sangxia , Liang Dan
Published: January 2023

Tetracyclines, belonging to an old antibiotic drug family that possesses a striking variety of non-antibiotic properties, have been successfully applied in a wide range of diseases, including asthma. However, their roles in allergic diseases and underlying anti-allergic mechanisms remain elusive.To explore the roles of doxycycline in allergic pathogenesis, we have focused on its role on B cells, mast cells (MCs) and histamine function using and experimental allergic conjunctivitis (EAC) and passive systemic anaphylaxis (PSA) animal models. Our results demonstrated that treatment with doxycycline significantly reduced IgE release of B cells and IgE-mediated activation of MCs. Furthermore, doxycycline treatment significantly inhibited histamine-stimulated vascular hyperpermeability. Mechanistically, doxycycline-mediated inhibition of B cells, mast cells and histamine may be through modulating PI3K/Akt pathway. Using mouse models of EAC, PSA and histamine-induced systemic anaphylaxis like reaction (HISA), we demonstrated that treatment with doxycycline significantly attenuated allergic symptoms. Doxycycline-mediated anti-allergic effects on EAC, PSA and HISA were abrogated when an Akt activator, SC79 was administrated. Our study has suggested that doxycycline inhibits B cell, mast cell and histamine function and attenuates experimental allergic conjunctivitis and systemic anaphylaxis by modulating PI3K/Akt pathway.
Responsiveness to anaphylatoxin C5a delineates hematopoietic lineage of pulmonary dendritic cells (HYP6P.257)
Authors: Nakano Hideki , Moran Timothy , Bortner Carl , Cook Donald
Published: January 2023

The complement subunit, C5a (anaphylatoxin), can either promote or suppress allergic responses through its effects on pulmonary dendritic cells (DCs). However, DCs are heterogeneous, and responsiveness of specific DC subsets to anaphylatoxins has not been well characterized. Two distinct precursors can give rise to DCs, including fms-like tyrosine kinase receptor 3 (FLT3)-dependent preDCs and FLT3-independent monocytes. The integrins CD11b and CD103 are widely used to identify DC subsets in the lung, but CD11bhi DCs are composed of both preDC-derived conventional DCs (cDCs) and monocyte-derived DCs (moDCs), including Ly-6Chi inflammatory DCs and Ly-6CloCD64hi (also CD14hi) resident moDCs. Microarray analysis of FLT3-dependent CD11bhi cDCs and FLT3-independent CD11bhi moDCs revealed that C5aR/CD88 is highly expressed by moDCs. Flow cytometric analysis confirmed that CD88 is displayed on Ly-6Chi inflammatory DCs and CD64hi resident moDCs, but not on CD103+ and CD64lo cDCs. CD88hi lung DCs were FLT3-independent, and adoptively transferred monocytes differentiated exclusively to CD88hi DCs. Calcium-influx analysis of purified lung DCs upon C5a stimulation confirmed that only Ly-6Chi inflammatory DCs and CD64hi moDCs, but not CD103+ DCs and CD64lo cDCs respond to C5a. Our findings demonstrate that CD88 can reliably distinguish moDC from cDCs, and that only moDCs are directly stimulated by anaphylatoxin C5a.
Changes of cell surface markers CD3, CD4, CD44, CD62L on mouse spleen lymphocytes after culture in vitro (HYP7P.303)
Authors: Li Yanchun , Hua Shucheng , Gao Hang , Li Wenxue , Zhu Yaping
Published: January 2023

Object To Study the changes of cell surface markers CD3, CD4, CD44 and CD62L on mouse spleen lymphocytes after culture. Method Spleen cells were separated from Balb/c mice and asthmatic mice and lymphocytes were isolated by using lymphocyte separation liquid. We cultured lymphocytes with RPMI 1640 medium(10% FBS) in cell incubator for 3 days. Then the four cell surface markers were detected by using flow cytometry. Results In normal mice, 19.09% of lymphocytes stained positively for CD3 and CD4, among these cells 98.61% was CD44+ T cell and 68.71% was CD62L+ T cell before the culture. After culture with ConA for 3 days, CD3+CD4+ and CD3+CD4+CD44+ T cell population was down regulated to 8.96% and 71.82%, respectively. While CD62L almost disappeared, accounting for 11.27% only. The similar results were obtained even cells cultured without ConA present. For asthmatic mice, CD3+CD4+ T cell accounted for 20.33% in total lymphocytes, among which 97.72% was CD44+ and 75.74% was CD62L+ before the culture. After culture, CD3+CD4+T cell accounted for 10.4%, among which CD44+ only 55.5% and CD62L+ only 2.63%. The similar results were obtained when cells were cultured with OVA. Conclusion In both normal and asthmatic mice, CD3,CD4,CD44 and CD62L on spleen lymphocytes were down regulated after culture in vitro regardless with or without stimulating factors, especially CD62L, almost disappeared. This should be given full consideration when these markers are detected after cell culture.
Effect of extrusion processing on in vivo allergenicity of hazelnut in an adjuvant-free mouse model (HYP7P.313)
Authors: Ortiz Tina , Para Radhakrishna , Gonipeta Babu , Ravi Ramasamy , Reitmeyer Mike , He Yingli , Srkalovic Ines , Ng Perry , Gangur Venugopal
Published: January 2023

Life-threatening nut allergy is a growing public health problem in many countries including the USA. Causes underlying this alarming trend are incompletely understood and methods are urgently needed to prevent such immune reactions. Here, we tested the hypothesis that extrusion processing of hazelnuts will reduce in vivo allergenicity using an adjuvant-free mouse model of hazelnut allergy established in our lab. Groups of mice received transdermal exposure (TDE) to extrusion processed hazelnut protein (EHNP) versus raw hazelnut protein (RHNP) extract preparations. Mice were evaluated for systemic IgE antibody responses (sIgER), hypothermia shock response (HSR), and mucosal mast cell responses by measuring plasma mouse mast cell protease-1 (mMCP-1) levels. Results showed that EHNP elicited a reduced sIgER compared to RHNP after 4 TDE but similar responses after 5 TDE. However, EHNP induced less HSR compared to RHNP upon oral challenge. Both protein preparations induced similar HSR upon i.p., challenge. Finally, we measured plasma mMCP-1 levels after oral and i.p., injection challenges. We found that EHNP elicited reduced mMCP-1 response compared to RHNP upon both oral as well as i.p., challenges. This is the first study demonstrating the utility of a mouse model to evaluate the effect of food processing on in vivo food allergenicity in general and hazelnut in particular.
Role of α-melanocyte stimulating hormone in the induction of regulatory T cells in ocular allergy (HYP7P.318)
Authors: Galicia-Carreón Jorge , Rodríguez-Lemus Karen Alejandra , Ayala Balboa Julio , Pérez-Tapia Sonia Mayra , Jimenez-Martinez Maria
Published: January 2023

Allergic conjunctivitis (AC) is one of the most common eye disorders in ophthalmology. AC is a disorder mediated by Th2 cells, where the eye inflammation is caused by a type I hypersensitivity. It has been suggested that some neuropeptides are increased (CGRP, SP and VIP) in AC contributing to local damage. In other hand, α-MSH plays an immunomodulatory role in ocular tissues; however, its role in ocular allergic disease is unknown, thus it was the aim of this study. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from blood samples of healthy donors (HD) and AC-patients skin reactive to Dermatophagoides pteronyssinus (Der p), then PBMC were cultured with different stimuli: Der p, α-MSH, or Der p plus α-MSH. After 7 days, PBMC were harvested, labeled with mAbs against CD4-PECy5, CD25-PE, FOXP3-FITC and analyzed by flow cytometry. Results. Der p or α-MSH stimulation did not induced significant changes in the frequency of Tregs; interestingly, after α-MSH plus Der p stimulation a significant higher frequency of Tregs cells was observed than Der p alone. Conclusions: Tregs differentiation in patients with AC is dependent of Ag and addition of α-MSH, if α-MSH is involved in local Treg differentiation is not known and needs further investigation.
Alpha-tocopherol inhibits the development of select dendritic cell subsets and lung inflammation during allergic responses in neonates (HYP7P.282)
Authors: Cook-Mills Joan , Abdala-Valencia Hiam , Berdnikovs Sergejs , Soveg Frank
Published: January 2023

Offspring from allergic mothers develop allergic responses to suboptimal allergen challenge. This is mediated by dendritic cell responses in the offspring. Dendritic cell development and responses are dependent on protein kinase Cα (PKCα) and we reported that recombinant PKCα is inhibited by binding of the vitamin E isoform α-tocopherol (αT). However, it is not known whether αT regulates development of dendritic cell subsets or development of allergic responses. Therefore, we determined whether αT supplementation inhibited development of dendritic cell subsets and allergic responses in offspring of allergic female mice. αT supplementation of allergic female mice during pregnancy/lactation resulted in a dose-dependent reduction in allergic lung inflammation in the pups challenged with the antigen OVA. Interestingly, in these pups, there was a reduction in lung CD11b+ but not CD11b- subsets of dendritic cells. There was also a reduction in allergen-induced lung mRNA expression of indoleamine 2,3-dioxygenase, IL-4, IL-33, TSLP, CCL11 and CCL24. In conclusion, supplementation with αT reduced development of CD11b+ dendritic cells and allergic responses in offspring from allergic mothers. These results have implications for supplementation of allergic mothers with αT.
In vivo bisphenol-A exposure in combination with high-fat diet modulates murine innate immune responses (INC6P.345)
Authors: Walser-Kuntz Debby , Cho John , Rab Rashed , Wu Ivy , Iroz Cassandra , Kansakar Shamir
Published: January 2023

The recent obesity epidemic has led to an expansion in our understanding of the link between adipose tissue and inflammation. The prevalent use of bisphenol-A (BPA) in food containers and other products provides an additional environmental challenge to the immune system. BPA disrupts the action of physiologic estrogens by binding to estrogen receptors and has previously been shown to impact immune responses. We explored the combined effects of high-fat diet and in vivo BPA exposure on spleen innate immune mediators. BPA treatment (2.5 or 25.0 ug/kg body weight per day; 4 weeks) reduced the LPS-induced secretion of KC chemokine, a neutrophil chemotaxin, by 25% and 40% respectively in normal diet mice, with a more dramatic BPA-induced decrease in KC secretion observed in high-fat fed mice. High-fat diet increased TNF-α mRNA expression overall compared to normal diet, and high dose-BPA reduced LPS-stimulated TNF-α mRNA expression in both normal (19%) and high-fat fed (34%) mice. High-fat fed mice also had a greater increase in LPS-induced COX-2 mRNA expression than normal diet mice, with BPA modulating expression in both groups in a dose dependent manner. High-fat diet and low dose BPA exposure increased non-fasting blood glucose levels by 30% on average when compared to control normal diet mice. These data demonstrate that peripubertal BPA exposure dampens aspects of innate immune function and that BPA combined with high-fat diet may disrupt glucose metabolism.
Differential regulation of CD8 T cell responses by surface and endosomal TLRs (INC6P.347)
Authors: Mandraju Rajakumar , Murray Sean , Forman James , Pasare Chandrashekhar
Published: January 2023

Sensing of pathogen/pathogen derived products through Toll like receptors (TLRs) induces dendritic cell (DC) maturation characterized by the expression of co-stimulatory molecules and secretion of pro-inflammatory cytokines. These two signals play a critical role in activation and differentiation of CD4 T cells. However, since the role of TLRs in the regulation of CD8 T cell responses is not clear we sought to address their role in this process. Our results show that although most TLRs induce the CD8 T cell proliferation in-vitro, there are significant differences in the CD8 T cell priming in-vivo to pathogenic infections. Endosomal TLRs (TLR3 and TLR9) that sense nucleic acids show a remarkable ability to induce CD8 T cell responses. However, surface TLRs (TLR2 and TLR4) that sense bacterial ligands were incapable of inducing CD8 T cell responses. Moreover, surface TLRs dominantly suppress the CD8 T cell activation induced by endosomal TLRs. Cell migration experiments show that surface TLR activation skewed lymphoid (CD11c+, CD8+) to myeloid (CD11c+, CD11b+ CD8-) DC populations in the draining lymph nodes. We also found that DC intrinsic TLR2 and TLR4, acting in a MyD88-dependent manner, altered the ability of DCs to prime CD8 T cells. In summary, our results demonstrate that bacterial co-infection dampens the ability of DCs to induce efficient anti-viral CD8 T cell responses.
Tetramer identification of functional mouse mucosal associated invariant T cells (INC6P.352)
Authors: Sakala Isaac , Kjer-Nielsen Lars , Eickhoff Christopher , Wang Xiaoli , Liu Ligong , Fairlie David , Rossjohn Jamie , Hoft Daniel , Hansen Ted
Published: January 2023

Mucosal associated invariant T (MAIT) cells have a semi-invariant TCR Vα chain, and their development is dependent upon the commensal flora and the expression of MR1. MAIT cells are activated in vitro in an MR1-restricted manner by cells infected with diverse strains of bacteria suggesting a widely shared antigen. Such an antigen was recently defined by the observation that human MR1 binds bacterial riboflavin metabolites (ribityllumazines, RL antigens) that are capable of activating MAIT cells. Recently MR1/RL tetramers were constructed allowing us to compare MR1-dependency and function of mouse MAIT cell subsets in invariant TCR Vα19-Jα33 (Vα19i) transgenic (Tg) mice. Although significant numbers of tetramer+ cells were detected in both MR1+/+ and MR1-/- Vα19i Tg mice, only tetramer+ cells from MR1+/+ and not MR1-/- Vα19i Tg mice had predominant expression of CD69, NK1.1 and Vβ6/8 and displayed robust in vitro responses to IL-12+IL-18 or RL Ag. In addition, tetramer+ MAIT cells expressing CD4, CD8 or neither developing in MR1+/+ Vα19i Tg mice had disparate cytokine profiles in response to RL Ag. Most notably, after mycobacterial infection all MAIT cell subsets decreased in the blood regardless of whether they express NK1.1 and/or Vβ6/8; however, only the NK1.1+Vβ6/8+ subset predominated in the lung airways of infected MR1+/+ Vα19i Tg mice. These findings present evidence for the functional diversity of MAIT cell responses to innate cytokines, RL Ag and bacterial infection.
IFNγ drives human primary skin mast cells to function as antigen presenting cells. (INC6P.341)
Authors: Lotfi-Emran Sahar , Ward Brant , Woodfolk Judith , Manjili Masoud , Schwartz Lawrence
Published: January 2023

We assessed the antigen presenting ability of mature primary human skin mast cells (MCs) in vitro. MCs do not express HLA II at baseline, but do so when stimulated with IFNγ. IFNγ-primed, but not un-primed, MCs facilitated activation of Jurkat T cells by superantigen. Unlike polyclonal primary CD4+ T cells, which harbor a subset of IFNγ producing cells, Jurkat cells do not spontaneously produce IFNγ. Co-culture of un-primed MCs with CD4+ T cells for 2 days upregulated HLA II expression on MCs and subsequent superantigen mediated T cell activation. MC HLA II upregulation is primarily due to the Th1 cell subset and is prevented by IFNγ neutralizing and/or receptor blocking antibody. Superantigen bypasses HLA II-loaded peptide:T cell receptor interactions; therefore, we assessed the ability of MCs to present cytomegalovirus antigen. MCs primed ±cytomegalovirus antigen exposure for 3 days then ±IFNγ for the last 2 of those days were washed thoroughly and co-cultured with HLA-matched CD4+ cells. After one week, CD4+ T cells from cytomegalovirus seropositive, but not seronegative, individuals showed proliferation (CFSE dilution), activation (CD25high and CD69high expression), and appropriate intracellular cytokine production (IFNγ and IL-4, but not IL-17). Thus, MCs can take up, process, and present antigen in an HLA II-restricted manner, causing antigen specific activation and proliferation of CD4+ T cells.
NLRP12 suppresses host innate immune signaling to limit inflammation during emergency hematopoiesis (INM6P.404)
Authors: Linz Brandon , Neely Crystal , Malik Julia , Kartchner Laurel , Brickey Willie , Ting Jenny , Cairns Bruce , Maile Robert
Published: January 2023

The hematopoietic system is capable of rapidly increasing myeloid cell production in response to tissue damage. NLRP12 is expressed in peripheral leukocytes and by osteoclasts and marrow progenitor cells. This NLR family member has been controversial but has been implicated in regulation of innate immune responses. We hypothesized that NLRP12 is a central regulator of innate immune reconstitution following an emergency hematopoietic event. To test this, wild type and Nlrp12-/- C57BL/6 female mice underwent a radiation-thermal combined injury (RCI) consisting of a 20% total body surface area, full thickness contact burn in addition to receiving a single whole body dose of 5 Gy γ-radiation. Following RCI, NLRP12-deficient mice display a lack of peripheral innate immune cell reconstitution as seen in wild type mice; specifically a ten-fold decrease in total numbers of splenic and pulmonary neutrophils and immature monocytes compared to wild type, and a three-fold increase in apoptosis of bone marrow granulocyte/monocyte progenitor cells resulting in a three-fold reduction of total bone marrow cells. Lack of NLRP12 results in a sustained increase in inflammation after injury, with significant increase in serum levels of TNFα, IL-6, IL-12, and type I interferons compared to injured, wild type mice that is inflammasome-independent. These data suggest that NLRP12 acts as an important regulator of inflammation during events that trigger immune hematopoeisis and reconstitution.
Inhibition of synergy between NOD2 & TLR signaling pathways underlies the profound efficacy of RIP2 kinase inhibitors in models of inflammatory bowel disease (INM6P.415)
Authors: Hanning Charles , Yu Jong , Haile Pamela , Cassillas Linda , Foley Kevin , Gough Peter , Bertin John , Votta Bart
Published: January 2023

Loss of intestinal epithelial barrier integrity in inflammatory bowel disease (IBD) leads to aberrant interaction between commensal bacteria and mucosal immune cells. This triggers inflammation via activation of pattern recognition receptor (PRR) signaling pathways. Which PRR signaling complexes are critical in mediating disease pathogenesis remains to be elucidated. Our recent studies have shown that selective and potent inhibitors of RIP2 kinase, the signaling partner of NOD1 and NOD2, can dramatically reduce murine TNBS-induced colitis and the spontaneous release of proinflammatory cytokines in cultured intestinal biopsies from IBD patients. The striking magnitude of the inhibition observed led us to interrogate the effect of these inhibitors on the modulation of crosstalk between NOD1/2 and other PRRs. Using human PBMCs we defined conditions for observing synergistic cytokine release (TNF-α, IL-6) in response to combinations of NOD2 and either TLR2, TLR4, or TLR5 ligands. In each instance, selective inhibition of RIP2 kinase activity robustly blocked synergistic cytokine release, but had little or no effect on TLR ligand alone induced cytokine production. These results suggest that RIP2 kinase inhibitors are likely to have profound anti-inflammatory effects in situations where multiple PRRs are being stimulated simultaneously, and illustrate the potential of RIP2 inhibitors as therapeutics for the treatment of human inflammatory diseases.
Negative regulation of Nod2-mediated NF-κB activation (INM6P.417)
Authors: Sukumaran Bindu , Bist Pradeep
Published: January 2023

The cytoplasmic innate immune receptor NOD2 is required for innate immune defense against Gram-positive and Gram-negative intracellular bacterial pathogens. Dysregulation of NOD2 signaling is implicated in several inflammatory disorders (e.g., Crohn’s disease). However, a mechanistic global understanding of the negative regulation of this pathway needs further studies. Through a genome-scale RNA-interference screen, we identified 75 negative regulatory proteins of NOD1/2-mediated NF-κB activation. We characterized the role of several newly identified negative regulators in the regulation of NOD2-mediated NF-κB signaling, focusing on E3 ubiquitin ligases. Depletion or over expression of these ubiquitin ligases respectively enhanced or suppressed NOD2-mediated NF-κB activation, cytokine secretion and defense against intracellular bacteria. We identified and characterized a novel ubiquitin ligase that degrades Rip2, the adaptor of NOD2, via K48-linked ubiquitination, leading to negative feedback regulation. We also identified that ubiquitin ligase-mediated RIP2 degradation was essential for the tolerance in human immune cells observed following chronic NOD2 stimulation, providing a novel mechanism by which cells establish MDP tolerance. Our findings provide new insights into the mechanisms by which NOD2 pathway attenuated, and provide new directions to understand the role of NOD2 pathway in both infectious and inflammatory diseases.
Essential role for the inositol pyrophosphates in type-I interferon response (INM6P.418)
Authors: Krishnan Manoj , Pulloor Niyas
Published: January 2023

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified that the cellular pathway synthesizing inositol pyrophosphates as a novel positive regulator of interferon production. Gene expression and catalytic activities of several involved in the synthesis of the inositol pyrophosphates were critical for interferon induction. In contrast, inositol pyrophosphate-hydrolases negatively regulated interferon transcription. Mechanistically, inositol pyrophosphate synthesis pathway was needed for the phosphorylation and activation of IRF3, a transcription factor of interferon. The inositol pyrophosphatesynthesis was essential for the control of cellular infection by Sendai and influenza A viruses. This study thus identified several novel regulators of RIG-I, a new cellular role and potential therapeutic interferon response modulating application for inositol pyrophosphates.
cGAS is activated by DNA-induced oligomerization and has pivotal roles in antiviral defense and immune adjuvant effects (INM6P.412)
Authors: Wu Jiaxi , Zhang Xu , Li Xiao-Dong , Sun Lijun , Chen Zhijian
Published: January 2023

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that alerts the host to microbial infection. We have recently identified cyclic GMP-AMP synthase (cGAS) as a cytosolic DNA sensor that triggers innate immune responses. Here, we further investigate the mechanism of cGAS activation by DNA and the function of cGAS in vivo through a combination of structural, biochemical and genetic approaches. Surprisingly, in contrast to several recent structural studies, which described the formation of a 1:1 complex between cGAS and DNA through a single interaction site, our results reveal that cGAS interacts with DNA through two distinct binding sites, forming a complex composed of dimeric cGAS bound to two molecules of DNA. Functional analyses of cGAS mutants demonstrate that both the dimeric interface and the two DNA binding surfaces are critical for cGAS activation. We also show that multiple cell types from cGAS-deficient mice failed to produce type I interferons and other cytokines in response to DNA virus infection. cGas-/- mice were more susceptible to lethal infection with herpes simplex virus 1 (HSV1) than wild-type mice. In addition, cGAS product cGAMP is an adjuvant that boosts antigen-specific T cell activation and antibody production in mice. These results not only provide new insights into the mechanism of DNA sensing by cGAS, but also demonstrate that cGAS is a nonredundant and general cytosolic DNA sensor that activates type I interferon pathway.
Innate immune responses play a role in heterogeneity of EAE. (INM8P.445)
Authors: Shinohara Mari , Inoue Makoto
Published: January 2023

Multiple sclerosis (MS) is a heterogeneous disease, which makes treatment difficult. For example, responsiveness to IFNβ treatment among MS patients varies to a great extent. Using EAE as an animal model of MS, we found that IFNβ treatment is not effective when EAE develops in the absence of the NLRP3 inflammasome with alternative EAE induction methods. Importantly, innate immune responses triggered by alternative EAE induction methods played a role to generate different subtype of EAE. This led us to hypothesize EAE have subtypes (although they are not mutually exclusive), at least based on the dependency of the disease pathology on the NLRP3 inflammasome and on the response to IFNβ. In NLRP3 inflammasome-dependent IFNβ-responsive EAE (termed type-A EAE), the NLRP3 inflammasome plays a key role as a pathogenic factor of EAE by enhancing immune cell migration and the resulting immune cell recruitment in the CNS. On the other hand, the NLRP3 inflammasome is not essential for EAE induction, and IFNβ treatment is not effective in NLRP3 inflammasome-independent EAE (termed Type-B EAE). Type-A and Type-B EAE show clear difference in their phenotypes and pathological mechanisms, mediated by differential activation of innate immune responses. M.I. performed all the experiments and M.L.S. is the corresponding author of this study.
Distinct sensitivity of the corneal P. aeruginosa clinical isolates to neutrophil extracellular trap-mediated immunity (INM8P.449)
Authors: Gadjeva Mihaela , Shan Qiang
Published: January 2023

Ocular bacterial keratitis is a frequent sight-threatening condition usually associated with P. aeruginosa infection. In this study we analyzed the contribution of the neutrophil extracellular traps (NETs) in mediating protection during ocular keratitis. Both, invasive and cytotoxic, P. aeruginosa clinical isolates induced NET release by neutrophils. NETs shared a common protein signature regardless of the inducing stimuli and were decorated with histones, elastase, lysozyme, myeloperoxidase, and metabolic enzymes. While the cytotoxic P. aeruginosa strains 6077 and 6206 strains (serogroup O11) and PA14 (serogroup 010) were resistant to NET capture, the invasive P. aeruginosa strains PAO1 (serogroup O5), 6294 (serogroup O6) were trapped by NETs. The mechanism of escape of the cytotoxic strains from adhesion to NETs depended on shedding of outer membrane vesicles (OMV) that competed with P. aeruginosa for NET binding. Unlike cytotoxic strains, NETs adherent invasive strains were weakly sensitive to NET-mediated killing. When ocular infection was caused by invasive strain, NETs were released at the ocular surface to capture bacteria, limiting it’s spread. Treatment with DNase I facilitated the clearance of NET-captured bacteria, decreasing ocular bacterial burden and disease pathology. Cumulatively, these data suggest that current therapeutic strategies might utilize DNAse I to promote recovery from acute keratitis.
MAP kinase phosphatase 2 (MKP-2) regulates the innate immune response to influenza infection (INM8P.436)
Authors: Jiao Huipeng , Zhang Yongliang
Published: January 2023

MAP kinase phosphatases (MKP) are major negative regulators of MAP kinases and are pivotal in the regulation of immune responses. As one member of MKP protein family, MKP-2 has been shown critically regulate p38 and JNK MAP Kinases in response to LPS stimulation in macrophages. While MKP-2 KO mice are more susceptible to intracellular parasite challenge, little is known about the role of MKP-2 in the innate immune response to viral infections. To investigate the role of MKP-2 in innate immune response against influenza, wild type (WT) and MKP-2 knockout (KO) mice were intranasally inoculated with sub-lethal dosage of A/Putero Rico/8/34 (PR8, H1N1) viruses. We found that viral titers in the lung of MKP-2 KO mice are significantly lower on day 3 and day 5 post-infection than those in the lung of WT mice. Correspondingly, the anti-viral cytokines IFN-α and IFN-β were higher in bronchoalveolar lavage fluid from MKP-2 KO mice compared with WT mice. Primary macrophages and dendritic cells from MKP-2 KO mice produced significantly higher amount of IFN-α and IFN-β in response to PR8 virus infection than cells from WT mice. Mechanistically, ERK and IRF-3 activation was prolonged in MKP-2 KO cells infected with PR8 virus. The regulation of type I IFNs by MKP-2 was further investigated. Together, these data support the model that MKP-2 plays an important role in the regulation of type-I IFNs production, thus altering the innate immune responses during pulmonary influenza infection.
Fpr2 and its endogenous ligand CRAMP promote dendritic cell maturation (IRC5P.461)
Authors: Wang Ji Ming , Xiang Yi , Huang Jiaqiang , Gong Wanghua , Yoshimura Teizo , Tessarollo Lino , Li Yingying , Chen Keqiang
Published: January 2023

Mouse Fpr2 is a chemoattractant receptor interacting with pathogen and host-derived agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-/- mice in association with diminished DC recruitment into the airway. These defects prompted us to investigate the potential changes in the differentiation of DCs in Fpr2-/- mice. Immature DCs from Fpr2-/- mice showed normal expression of iDC markers. However, upon stimulation with LPS, Fpr2-/- DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2-/- DCs also failed to induce normal allogeneic T-cell proliferation in vitro and their recruitment into the T cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, since neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We further observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation, and DCs from CRAMP-/- mice showed lower levels of co-stimulatory molecules, MHC II, and poor chemotaxis in response to CCL21 after LPS stimulation. Thus, Fpr2 and CRAMP play a non-redundant role in DC maturation in immune responses.
Myeloid DC-specific mTORC2 deficiency enhances Th1 and Th17 immunologic responses (IRC5P.472)
Authors: Raich-Regue Dalia , Rosborough Brian , Turnquist Heth , Thomson Angus
Published: January 2023

The mechanistic/mammalian target of rapamycin (RAPA) (mTOR) is a key integrative kinase that functions in at least two independent complexes: RAPA-sensitive mTOR complex (C)1 and RAPA-insensitive mTORC2. While mTORC2 in T cells promotes Th2 and Th17 differentiation, its immunologic function in dendritic cells (DC) is largely unknown. We defined the role of rictor, a key mTORC2 component, in myeloid DC and their ability to polarize interacting allogeneic Th cells. When stimulated with TLR ligands, bone marrow-derived DC generated from conditional rictor KOs displayed lower co-inhibitory B7-H1 (CD274) molecule expression, enhanced IL-12p70, IL-23, IL-6 and TNFα production and augmented allogeneic T cell stimulatory ability. Analysis of allopeptide-specific CD4+ T cells after activation by rictor KO DC in vivo revealed significant expansion of IFN-γ+ and IL-17+, but not IL-10+ or CD4+Foxp3+ T cells. These data establish that mTORC2 activity restrains pro-inflammatory responses of conventional DC and their ability to differentiate Th1/Th17 cells.
Biofunctions of three new B7 family members (IRM7P.486)
Authors: Wang Jinghua , Manick Brian , Wu Guoping , Hao Ruyi
Published: January 2023

Recently, three new B7 family members, B7-H5 (VISTA, Gi24, or Dies1), B7-H6, and B7-H7 (HHLA2) were identified. However, the functions of the three new B7 family members remain uncertain. We expressed the human B7-H5, B7-H6, and B7-H7 extracellular domain human IgG1 Fc Chimera in mammalian cell line and investigated the functions of the purified recombinant B7-H5/Fc, B7-H6/Fc, and B7-H7/Fc in vitro. To determine the functions of B7-H5 and B7-H7, human T cells were treated with plate-bound anti-CD3 and either B7-H5/Fc, B7-H7/Fc or control IgG1 Fc. The cytokine levels of supernatant were measured using the Proteome Profiler Human Cytokine Array and Quantikine ELISA kit. B7-H5 significantly reduced production of seven cytokines from T cells including IFN-γ, IL-2, IL-4, IL-8, IL-13, IL-16, and TNF-α. B7-H7 markedly decreased four cytokines including IFN-γ, IL-2, IL-8, and TNF-α. To investigate the bioactivity of B7-H6, NK92 cells were treated with plate-bound B7-H6/Fc or control IgG1 Fc. B7-H6/Fc induced IFN-γ production in NK cells in a dose dependent manner. Furthermore, B7-H6/Fc showed binding to recombinant human NKp30/Fc. Taken together, our data suggest that B7-H5 and B7-H7 are co-inhibitory molecules for T cell activation. Our data also indicate that B7-H6, a major NKp30 ligand, triggers NKp30-dependent NK cell activation.
Rad52 deficiency Increases immunoglobulin class switch DNA recombination (IRM8P.714)
Authors: Tat Connie , Hayama Ken , Zan Hong , Casali Paolo
Published: January 2023

Class switch DNA recombination (CSR) plays a central role in the maturation of the antibody response. CSR entails the generation of double-strand DNA breaks (DSBs) mediated by activation-induced cytidine deaminase (AID). Resolution of these DSBs can occur through Ku70/Ku86 dependent classical non-homologous end-joining (C-NHEJ), or through an undefined alternative-end joining (A-EJ) that introduces microhomologies in the S-S junctions. Here we investigate the role of Rad52, an important DSB repair factor in CSR. We found that Rad52 deficiency elicits an increase in NP-specific IgG1 and IgG3 responses to T-dependent and T-independent antigens. The increased antibody response is not due to alterations in lymphoid differentiation, B cell proliferation or cell division but rather a direct effect on the CSR machinery. This phenomenon is B cell intrinsic, as CSR to IgG1, IgG2a, IgG3 and IgA virtually double in Rad52-/- B cells stimulated with LPS and respective cytokines. In CSR, both Rad52 and Ku70/Ku86 are specifically recruited to S region DNA in an AID-dependent manner. In the absence of Rad52, the recruitment of Ku70/Ku86 to the S regions was significantly increased. Finally, the frequency and length of microhomologies in the S-S junctions was significantly reduced in Rad52-/- B cells. Altogether, our findings suggest that Rad52 competes with Ku70/Ku86 in binding S region to repair DSBs in CSR possibly utilizing an alternative end-joining pathway.
Dissect the function of PKCε in the formation of central supramolecular activation clusters and B cell activation (IRM8P.707)
Authors: Liu Wanli , Xu YinSheng
Published: January 2023

Protein kinase C (PKC) is a family of kinases encoded by nine genes with redundant and meanwhile distinct biological functions. One of such member, PKCε, is abundantly expressed in B lymphocytes. However its roles in the formation of B cell immunological synapse (IS) and B cell activation are still unclear. Using a series of optical imaging based cell biological approaches, we showed that PKCε was efficiently recruited to the B cell IS after antigen and B cell receptor (BCR) engagement. The proper distribution of PKCε in IS is dependent on the bioactivity of PKCε as mutant PKCε in either constitutively activated or kinase dead format are unexpectedly extruded to peripheral region of IS. Over expression of WT PKCε but not its mutants enhanced the contraction of BCR toward the central supramolecular activation clusters (cSMACs) as determined by the contact area and the BCR fluorescence intensity and the percentage of cSMAC formation. We also found that the strength of the initiation of BCR signaling cascade was potently influenced by PKCε. The precise molecular mechanism of PKCε to enhance the cSMAC formation and B cell activation will also be discussed.
Identification of emetine as a therapeutic agent that targets pigment epithelium derived factor - mediated inflammation and improves hyperglycemia in murine models of type 1 diabetes (IRM9P.728)
Authors: Hudson LaQueta , Chavan Sangeeta , Dancho Meghan , Bruchfeld Johanna , Li Jianhua , Ragab Ahmed , Tssava Tea , Hanes William , Fritz Jason , Silverman Harold , Tanzi Matthew , Pavlov Valentin , Olofsson Peder , Al-Abed Yousef , Tracey Kevin
Published: January 2023

Pigment epithelium derived factor (PEDF) is a 50kDa secreted protein with inflammatory, antiangiogenic and neurotrophic properties. Elevated levels of PEDF are significantly associated with metabolic diseases including type 1 and type 2 diabetes, metabolic syndrome, and insulin resistance. PEDF administration to naïve animals result in the development of insulin resistance, and neutralization of PEDF with anti-PEDF antibodies improves the clinical outcome. Here we have identified a clinical drug candidate that attenuates PEDF mediated inflammation. Using a high-throughput screening approach, murine macrophages were stimulated with PEDF in the presence or absence of clinical drug candidates, and levels of secreted TNF analyzed. Following screening of 1,600 clinical candidate compounds, three lead compounds were identified, one being emetine, that dose dependently reduce PEDF induced macrophage activation without toxicity. Surface plasmon resonance analysis revealed that emetine binds to PEDF with high affinity (kD<25nM), and inhibits PEDF binding to its receptor adipose triglyceride lipase. Additionally, emetine reduces endocytosis of PEDF into macrophages. Administration of emetine to murine models of type 1 diabetes improves hyperglycemia, suppresses circulating levels of anti-insulin antibodies, and reduces pancreatic TNF and PEDF. Our studies suggest that emetine attenuates PEDF mediated inflammation, and has a therapeutic potential for treating type 1 diabetes.
Resolvin D1 inhibits multiple pro-inflammatory signaling pathways in primary human lung cells exposed to diverse inflammatory stimuli (IRM9P.721)
Authors: Hsiao HsiMin , Thatcher Thomas , Phipps Richard , Serhan Charles , Sime Patricia
Published: January 2023

Resolution of inflammation is a bioactive process involving a new genus of lipid molecules called “specialized pro-resolving lipid mediators” (SPMs), mainly derived from dietary ω-3 polyunsaturated fatty acids, which actively antagonize inflammatory signaling pathways and promote resolution. Although it is broadly known that SPMs inhibit inflammation, the specific pathways and targets of many SPMs remained to be discovered. Here, we investigated the mechanism by which the SPM resolvin D1 (RvD1) inhibits pro-inflammatory signaling pathways using two distinct stimuli; IL-1β, which acts primarily via its own receptor, and poly(I:C), a double-stranded viral RNA mimetic that acts primarily through toll-like receptor (TLR)3. RvD1 inhibited production of pro-inflammatory mediators including IL-6 and IL-8, both primary human lung fibroblast and epithelial cell strains, when stimulated with either IL-1β or poly(I:C). RvD1 also blocked phosphorylation of ERK1/2 and translocation of NF-κB p65 in cells treated with both stimuli. Importantly, we found that RvD1 inhibited phosphorylation of TAK1, an upstream effector protein that regulates both the MAP-kinase and NF-κB pathways. These results demonstrate that RvD1 targets multiple pro-inflammatory signaling pathways and may have significant therapeutic potential in complex inflammatory diseases including pulmonary fibrosis, asthma and chronic obstructive pulmonary disease.
VISTA deficiency synergizes with a non-redundant immune checkpoint pathway and leads to enhanced immune activation (IRM10P.744)
Authors: Wang Li , LeMercier Isabelle , Suriawinata Arief , Chen Wenna , Li Jiannan , Noelle Randolph
Published: January 2023

V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that suppresses T-cell mediated immune responses. Previous studies using VISTA-neutralizing monoclonal antibody show that VISTA-blockade enhances T cell-activation in an inflammatory disease model EAE, as well as in murine tumor models. Current study describes a comprehensive characterization of VISTA knockout (KO) mice. We show that despite the apparent normal hematopoietic development in young ko mice, VISTA genetic deficiency leads to a pro-inflammatory phenotype in aged animals, as well as enhanced T-cell activation in response to acute antigen immunization. In addition, we show that VISTA deficiency significantly enhanced disease development in a spontaneous model of autoimmune disease, which is correlated with the spontaneous activation of auto-antigen specific CD4+ T cells. Lastly, when combined with the genetic deficiency of another checkpoint molecule, synergistic or additive immune activation was observed.
Regulation of E3 ubiquitin ligase Cbl-b expression by protein tyrosine phosphatase SHP-1 (IRM10P.753)
Authors: Xiao Yun , Qiao Guilin , Zhang Jian
Published: January 2023

Casitas B cell lymphoma protein-b (Cbl-b) is a RING finger family E3 ubiquitin ligase. It has been well documented that Cbl-b plays a crucial role in T cell activation and tolerance induction. Our recent study also indicates that Cbl-b regulates proallergic T cell development and allergic airway inflammation. Therefore, the regulation of Cbl-b expression in T cells is important for its biological functions. Our previous studies demonstrate that CD28 costimulation potentiates Cbl-b sel-ubiquitination, CTLA-4-B7 interaction induces Cbl-b expression. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b tyrosine phosphorylation in primary mouse T cells requires CD28 costimulation. The failure to detect Cbl-b tyrosine phosphorylation upon CD3 stimulation is not due to defective PTK activation, but rather interestingly SHP-1 is recruited to Cbl-b upon CD3 stimulation, while CD28 costimulation abrogates the interaction between Cbl-b and SHP-1. Furthermore, we identified Cbl-b N-terminal tyrosine residues within TKB domain and linker region (Y106, Y133, and Y363) are required for its E3 ubiquitin ligase activity. The aberrant Th2 phenotype observed in T cell-specific SHP-1-/- (tSHP-1-/-) mice is reminiscent of our recent observation that Th2 responses are heightened in Cbl-b-/- mice. In support of this notion, Cbl-b expression is down-regulated in naïve T cells of tSHP-1-/- mice.
MALT1 has both protease activity-dependent and -independent roles in innate and adaptive immunity (IRM10P.739)
Authors: Foley Kevin , Yu Jong , Hoffman Sandy , Finger Joshua , Kasparcova Viera , Rickard David , Dare Lauren , Dykon Angela , Ramanjulu Joshi , Emery John , Gough Peter , Bertin John
Published: January 2023

Hetero-trimeric CARMA/BCL10/MALT1 CBM complexes play important roles in B and T cell antigen receptor signaling and other pathways. Previous studies have suggested that MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a cysteine protease to cleave and inactivate downstream signaling molecules such as A20 and CYLD. However, little is known about the relative importance of these two distinct activities in orchestrating immune responses. Utilizing mice homozygous for either null or protease-dead C461A mutations in MALT1, we found that some but not all MALT1 functions were dependent upon its protease activity. Although protease-dead mice had normal follicular B cell and T cell populations in the spleen, defects were observed in the generation of splenic marginal zone and peritoneal B1 B cells. Ex vivo, B cell receptor-stimulated proliferation was normal, but CD4+ and CD8+ T cells displayed decreased activation-induced proliferation and IL-2 production. C-type lectin receptor stimulation of cytokine production by dendritic cells was also found to be partially dependent upon protease activity. In vivo, protease-dead mice displayed severely reduced basal immunoglobulin levels, and defective T-dependent and T-independent immune responses. Our findings suggest that pharmacologic inhibition of MALT1 protease activity may be a useful therapeutic approach for diseases linked to uncontrolled immune cell activation.
Molecular mechanism of transcription and epigenetic alteration at immunoglobulin variable gene heavy chain locus induced by GANP (IRM11P.761)
Authors: Singh Shailendra , Maeda Kazuhiko , Eid Mohammed , Almofty Sarah , Shimoda Mayuko , Kawabata Teruya , Sakaguchi Nobuo
Published: January 2023

Epigenetic modification at IgV-region gene is the key base to generate somatic hypermutation (SHM) mediated by activation-induced cytidine deaminase (AID). However, the details how AID-targeting to the rearranged IgV-region locus have remained unclear. As a key player, we demonstrate that the histone-acetyl transferase activity of the germinal center-associated protein GANP is necessary for efficient AID-accession. GANP is upregulated in germinal center B-cells upon T-cell dependent antigen responses. GANP forms a complex with AID and accompanies into the nucleus. GANP co-precipitates with RNA polymerase II (Pol-II) and its stalling factor Spt5 that also interacts with AID, indicating that the recruitment of GANP to IgV-region locus undergoes transcription-coupled manner. GANP augments the transcription elongation toward the downstream of the IgV-region gene with active RNA Pol-II. The histone acetyltransferase (HAT) domain of GANP is involved in the chromatin modification selectively at the rearranged IgV-region segment with the interference of nucleosome occupancy, controlling the accessibility of AID positioning at IgV-region gene. This suggests a specific role for the GANP associated complex in antigen specific high affinity B-cells.