bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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&lt;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

Abstract
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.