bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA miR-146a is a critical suppressor of arthritis in the mouse model of Lyme Disease (P1275)
Authors: Lochhead Robert , O'Connell Ryan , Ma Ying , Zachary James , Weis John , Weis Janis
Published: January 2023

Lyme Disease is caused by infection with the spirochete Borrelia burgdorferi, and is the most common vector-borne disease in the United States. Often, infection leads to acute arthritis in humans. Our lab and others have exploited the mouse model system to elucidate the mechanical mechanisms of Lyme Disease development. Because miRNAs have been shown to be important regulators of inflammatory and immune responses, we performed a microarray screen for expression of miRNAs in joint tissue from three infected mouse strains. This screen identified upregulation of miR-146a, a key negative regulator of NF-κB signaling. Others and we have previously demonstrated that Toll-like receptor 2/MyD88 dependent activation of NF-κB is a major regulator of immune response in mice and humans, but determining its role in arthritis development has remained elusive. We show that miR-146a plays a critical nonreduntant role in modulating Borrelia-induced NF- κB response to infection, and mice lacking this miRNA develop more severe arthritis without compromising immune response. Furthermore, miR-146a-/- mice have elevated transcript levels of NF-κB-regulated cytokines and chemokines in joint tissue, as well as increased myeloid cell infiltration, linking NF-κB dysregulation to arthritis development. Together, these data show that miR-146a is required for suppression of Lyme Arthritis, and provides a much-needed model for studying the role of NF-κB activation in arthritis development.
Pathogen clearance by engineering novel host innate immunity (P1251)
Authors: Gupta Goutam , Dandekar Abhaya , Gouran Hossein , Aguero Cecilia , Bruening George , A. Feldstein Paul , Nascimento Rafael , Goulart Luiz , Norvell Meghan , Pardington Paige , Chaudhary Anu , Civerolo Edwin
Published: January 2023

A few years ago, we introduced a concept that a protein chimera of pathogen recognition and lysis domains would be able to rapidly clear a broad-spectrum of pathogens [Crit Rev Immunol 2007;27(3):233-245]. For a wide variety of viral, bacterial, and fungal pathogens, appropriate recognition and lysis domains can be chosen from the host innate immune repertoire. A chimera of the recognition and lysis domains would be designed with the aid of a flexible linker to ensure synergy of the two functions and therefore, the rapid clearance of the targeted pathogen. In this work, we demonstrate the design of such a chimera and the efficacy of this chimera in clearing a plant pathogen Xylella fastidiosa (Xf) that causes diseases in multiple plants of economic importance. The most notable ones are Pierce’s disease (PD) in grape and variegated chlorosis (CVC) in citrus. Specifically, we show the construction of the transgenic grapevines expressing a protein chimera of recognition and lysis domains specific for Xf. This chimera clears Xf from the xylem (the site of colonization) and blocks the development of PD [Proc Natl Acad Sci U S A. 2012;109(10):3721-3725]. The same chimera can be applied to block CVC. Finally, we indicate how such chimeras of recognition and lysis domains can be developed to target multiple human pathogens.
Memory CD8 T cells are programmed to rapidly synthesize core 2 O-glycans for tissue-specific trafficking in response to inflammatory cues (P5112)
Authors: Nolz Jeffrey , Harty John
Published: January 2023

Immune cell trafficking from the circulation into peripheral tissues is a highly orchestrated process involving a number of receptor-ligand interactions. Previously, it has been demonstrated that memory CD8 T cells localize to areas of infection/inflammation in an antigen-independent manner and that these recruited CD8 T cells are highly cytolytic, thus providing rapid protection against pathogens expressing cognate antigen. However, the molecular mechanisms regulating memory T cell recruitment to inflamed tissue remain ill defined. Herein, we demonstrate that memory CD8 T cells utilize core 2 O-glycans, which mediate interactions with P- and E-selectin, for trafficking to inflamed tissues. Following infection, antigen-specific effector CD8 T-cells strongly express core 2 O-glycans, but this glycosylation pattern is lost by almost all memory CD8 T cells. However, following an inflammatory challenge, de novo generation of core 2 O-glycans occurs on memory, but importantly, not naïve CD8 T cells. Mechanistically, memory and naive CD8 T-cells exhibit the opposite pattern of histone H3 modifications at the Gcnt1 locus, a critical enzyme for core 2 O-glycan synthesis and its expression is regulated by IL-15. Thus, initial antigen encounter epigenetically programs memory CD8 T cells to rapidly generate functional selectin ligands in response to inflammatory cues, a previously unknown mechanism controlling differential localization of naïve and memory CD8 T cells.
Sumoylation is crucial for the stability of Leukocyte-specific protein 1 (P5118)
Authors: Hossain Md Mokarram , Huang Jiannan , Cayabyab Francisco , Liu Lixin
Published: January 2023

Leukocyte-specific protein 1 (LSP1) is an intracellular protein expressed in leukocytes and endothelial cells and regulates several leukocyte and endothelial cell functions including transmigration and chemotaxis of leukocytes in inflammation. There is a discrepancy between its predicted molecular weight (≈37KDa) and that detected in SDS-PAGE (52KDa). To solve this discrepancy, we tested the hypothesis that LSP1 is sumoylated immediately after its translation. Here we demonstrate that, in transfected cells, SUMO1 is conjugated to LSP1 and Ubc9 acts as a SUMO1 ligase for LSP1. Moreover, the sumoylated LSP1 can be desumoylated by the over-expression of SENP1, a SUMO-specific protease. Single lysine to alanine mutation at lysine residue 270 or 318 of recombinant mouse LSP1 prevented its sumoylation in vitro. These LSP1 mutants were degraded rapidly compared to their wild type counterpart. Our results show that sumoylation enhances LSP1 stability, which may ensure its functions in leukocytes and endothelial cells.
Adipose-derived mesenchymal stem cells restore impaired mucosal immune responses in aged mice (P3162)
Authors: Aso Kazuyoshi , Kimura Koh , Tokuhara Daisuke , Kobayashi Taira , Takagaki Kentaro , Sugimoto Shinya , Oki Katsuyuki , Tanemura Hideki , Urushihara Naoki , Lu Shen , Mcghee Jerry , Fujihashi Kohtaro
Published: January 2023

It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into red blood cells and epithelial cells as well as mesenchymal cells such as adipocytes, chondrocytes and osteoblasts. Various clinical trials have shown the ability of AMSCs to regenerate these cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier than is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>2yr) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. When cytokine production was examined, OVA-stimulated Peyer’s patch CD4+ T cells produced increased levels of IL-4. Further, CD4+ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA-specific mucosal SIgA or plasma IgG Ab or cytokine responses. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.
Food allergenic proteins in lactose as the pharmaceutical excipient (P6222)
Authors: Sakai Shinobu , Nakamaura Ryosuke , Nakamaura Rika , Adachi Reiko , Teshima Reiko
Published: January 2023

Most drugs contain pharmaceutical excipients which are pharmacologically inactive substances used as vehicles to deliver the active ingredients of a medication. Some of these pharmaceutical excipients are produced from foods containing allergenic proteins (e.g., milk, egg, peanut, soybean, and sesame) and removing these completely from the excipients is difficult. Therefore, if individuals with food allergies consume drugs containing allergenic food-derived excipients, they are at risk for developing specific allergic symptoms. We determined the levels of total protein in lactose found in pharmaceutical excipients and ethical drugs known to contain lactose by spectrophotometric analyses. The level of total protein in lactose was approximately 1 mg/g. We also determined the levels of allergenic proteins in lactose using commercial enzyme-linked immunosorbent assay systems. The milk proteins in lactose were detected in the range of 1.4-13.1 μg/g. Furthermore, we performed a luciferase-based in vitro elicitation test (EXiLE test) in addition to western-blot analysis using the sera from patients with milk allergies in order to examine the allergenicity of protein residues in lactose. The result of the EXiLE test indicated that the protein residues in lactose have the ability to crosslink human FcϵRI on mast cells.
Complement protein C3 exacerbates disease in a mouse model of chronic wasting disease. (P1343)
Authors: Michel Brady , Zabel Mark , Ferguson Adam
Published: January 2023

Accumulating evidence shows a critical role of the complement system in facilitating attachment of prions to both B cells and FDCs and assisting in prion replication. Complement activation intensifies disease in prion-infected animals, and elimination of complement components inhibits prion accumulation, replication, and pathogenesis. Chronic wasting disease is a highly infectious prion disease of captive and fee-ranging cervid populations that has utilized the complement system for efficient peripheral prion replication and most likely efficient horizontal transmission. Here we show that complete genetic or transient pharmacological depletion of C3 prolongs incubation times and significantly delays splenic accumulation in a CWD transgenic mouse model. Using a semiquantitative prion amplification scoring system we show that C3 impacts disease progression in the early stages of disease by slowing the kinetic rate of accumulation and/or replication of PrPRES. The delayed kinetics in PrPRES replication correlates with delayed disease kinetics in mice deficient in C3. Taken together, these data support a critical role of C3 in peripheral CWD prion pathogenesis.
NICIP, an inhibitory molecule for lamprey variable lymphocyte receptor to signal transduction (P1291)
Authors: Wu Fenfang
Published: January 2023

Lamprey and hagfish are the most ancient jawless vertebrates, which are serving as a “living fossil” of immunological origin and evolution. NICIP is a novel gene that we cloned from lamprey’s lymphocyte-like cells. Structural analysis indicates that NICIP contains two Ig-like domains, a PKD domain, a TM region and a cytoplasm immunoreceptor tyrosine-based activation motifs (ITAM) region. It belongs to IgSF family and may involve in immune response, cell signaling and other functions. Our present research showed that there are interaction between NICIP and VLR, and this interaction can effectively inhibit VLR to transduce signal to intracellular.
Validating cathepsins as selective therapeutic targets for the treatment of particulate-mediated sterile inflammatory disease. (P1373)
Authors: Orlowski Gregory , Kataoka Hiroshi , Rock Kenneth
Published: January 2023

IL-1β mediates a multitude of debilitating, and sometimes fatal, acute and chronic inflammatory diseases. However, existing therapies for these diseases target IL-1β directly, suppressing both its pathologic and protective activities. We propose that cathepsins are therapeutic targets specific to IL-1β-mediated inflammatory diseases caused by sterile particulates. These particulates, such as cholesterol crystals (atherosclerosis), silica (silicosis) and monosodium urate (gout), activate the NLRP3 inflammasome in immune cells, leading to active IL-1β secretion. Rather than conferring protection, this response causes disease. Here, we show that a bioavailable inhibitor of cathepsins B, L & C, called K777, specifically suppresses particulate-mediated IL-1β production by primary macrophages. Using primary and immortalized macrophages deficient in cathepsins B & L, we demonstrate these proteases are critical targets likely responsible for much of K777’s effect. Moreover, we show for the first time that, unlike other NLRP3 stimuli, particulate-mediated inflammatory cell death is inflammasome-independent and cathepsin-dependent. Finally, in a murine model of acute peritonitis, both subcutaneous and intravenous K777 treatment substantially and preferentially reduced IL-1β-dependent neutrophil influx caused by particulate stimuli. Together, these data are the first to translate cathepsin inhibition in vitro into an in vivo model, validating cathepsins as potentially selective therapeutic targets in particulate-mediated inflammatory disease.
The enigma of B cell memory: the scaffolding protein synapse-associated protein 97 is required for enhanced activation signaling through isotype-switched IgG memory B cell receptors (P1124)
Authors: Liu Wanli , Xu ChenGuang , Zhao XingWang , Chen XiangJun
Published: January 2023

The acquired immunological memory was probably first described by the Greek historian Thucydides who observed during a plague in Athens in 430 BCE that “the same man was never attacked twice”. Now 2442 years later, the mechanisms by which the IgG-BCRs function to enhance B cell responses are not completely understood. We showed earlier that the membrane proximal region of the mIgG1 cytoplasmic tail is necessary and sufficient to dramatically enhance the initiation of BCR signaling. We identified that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ-binding motif, associated with SAP97, a PDZ domain containing scaffolding molecule that is involved in controlling receptor density and signal strength at neuronal synapses. SAP97 accumulated and bound to IgG BCRs in the immunological synapses (IS) that formed in response to B cell engagement with antigen. Knocking down SAP97 in IgG+ B cells or mutating the putative PDZ-binding motif on mIgG impaired formation of the IS, initiation of IgG BCR signaling, and downstream activation of the mitogen-activated protein kinase p38. Our subsequent studies showed that SAP97 binding protein, ERM family member ezrin, actively involved in the burst-enhanced memory B cell activation through isotype-switched IgG memory B Cell Receptors. Thus, our studies propose that SAP97 serves as a scaffold protein stabilizing the antigen-driven formation of IgG BCR oligomers and facilitating assembly of the BCR signalosome.
Erk2 is a critical regulator of spontaneous osteochondromas (P1016)
Authors: Wehenkel Marie , Edwards Benjamin , Calabrese Christopher , Pagès Giles , Pouysségur Jacques , Hedrick Stephen , Corr Maripat , Vogel Peter , McGargill Maureen
Published: January 2023

Osteochondromatosis is the most frequent tumor of the skeletal system. Although these bone tumors are benign, they create deformities resulting in impaired use of the affected joints, severe pain, and potential paralysis due to nerve compression. Patients often have a single tumor, although a disorder characterized by the presence of multiple osteochondromas is well established. Surprisingly, mice with a germline deficiency in Erk1 bred to mice with a conditional deletion of Erk2 in the T cell lineage (Erk1-/-.Erk2fl/fl.CD4cre) [DKO] developed multiple osteochondromas by 28 weeks of age. Gross protrusions of the spine and carpus were visible in the DKO mice, and radiological imaging revealed boney abnormalities in the spine, carpus, and sternum. Histological analysis of these protrusions showed an excessive proliferation of cartilage originating from the growth plates, but no mononuclear infiltrate indicative of inflammation. Moreover, we did not find autoantibodies in the serum of the DKO mice. Flow cytometric analysis indicated that an innate-like population of cells was more prominent in the DKO mice compared to littermate controls. These data suggest that the presence of Erk2 in a CD4-expressing, non-T cell population may play a central role in preventing the development of osteochondromas.
GPI-GM-CSF protein transferred onto H5 influenza VLPs remains stably expressed and functionally active (P6168)
Authors: Patel Jaina , He Sara , Amaram Nikhil , Yamanaka Satoshi , Vartabedian Vincent , Radhakrishnan Vijayaraghavan , Song Jae Min , Shashidharamurthy Rangaiah , Compans Richard , Kang Sang-moo , Selvaraj Periasamy
Published: January 2023

Pathogenic H5N1, a lipid-enveloped influenza virus, is a pandemic threat. The use of virus-like particles (VLPs) as an alternative to current influenza vaccines is highly promising. VLPs are similar in structure to their live viral counterparts but do not contain viral genome that is required for replication; hence VLPs provide for a safe and immunogenic vaccine. Although the particulate nature of VLPs allows them to be highly immunogenic, the need for protection against heterotypic viruses still remains. Therefore, inclusion of immunostimulatory molecules (ISMs) onto the VLP surface can help to induce cross-protection against strains and provide for stronger immunity. We were able to show that GPI-anchored-GM-CSF can incorporate stably onto H5 influenza VLP surfaces after a simple and quick protein transfer method. This method allows for the incorporation of the GPI-anchor onto the surface of lipid-bilayered VLPs within a matter of hours and allows for the incorporation of multiple GPI-ISMs in a concentration-dependent manner. Furthermore, protein transferred VLPs were functional in leading to bone marrow derived cell proliferation compared to unmodified VLPs, and incorporated GM-CSF was as functional as equal concentrations of commercially available recombinant soluble GM-CSF. Therefore, VLPs expressing GPI-GM-CSF could lead to enhanced immunogenicity and antiviral immune responses compared to unmodified VLPs.
CD4+ but not CD8+ memory T cells escape DN Tregs-mediated regulation via expression of Serpin Protease Inhibitor 6 (P2160)
Authors: Zhang Zhu Xu , Su Ye , Huang Xuyan , Lian Dameng , Jevnikar Anthony
Published: January 2023

Memory T cell (Tm) homeostasis is poorly understood and is a critical challenge to prevent transplant rejection. At present there are no effective clinical strategies to control Tm and contradictory reports exist as to their control by regulatory T cell (Treg). We previously found that TCRab+CD3+CD4-CD8-NK1.1- (double-negative, DN) Treg could effectively suppress CD4+ and CD8+ effector T cells and thus prevent transplant rejection. We utilized this model to test whether DN Tregs could similarly suppress Tm. Interestingly DN Tregs suppressed CD8+ Tm but had no effect on CD4+ Tm cells. As well DN Tregs express very high levels of granzyme B (GrB), suggesting a potential control mechanism and indeed no suppression was observed using perforin null DN Tregs. In BALB/c (H-2d) to B6-Rag1-/- mice (H-2b) skin allograft transplantation, DN Tregs significantly reduced total CD44high CD8+ Tm in recipients and significantly prolonged graft survival. In contrast, co-transfer of DN Tregs had no effect on CD4+CD44high Tm mediated rejection. There was no effect with co-transferred CD4+ Tm. CD4+ Tm cells express higher levels of GrB inhibitory Serpin Protease Inhibitor 6 (SPI-6) mRNA and protein than CD8+ Tm. Furthermore, SPI-6 null CD4+ Tm is not resistant to DN Treg-mediated regulation. We show for the first time, that DN Treg can control CD8+ Tm and suggest that DN Treg along with targeting SPI-6 may be useful to limit the expansion of both CD4+ and CD8+ Tm cells in transplantation.
Generation of secondary TCRs is critical for T cell development and the formation of an alloreactive T cell repertoire (P2132)
Authors: Ni Peggy , Allen Paul , Morris Gerald
Published: January 2023

A small number of peripheral T cells in humans and mice express two TCRs. Secondary TCRs arise from a lack of allelic exclusion at the TCRα locus. A functional role for secondary TCRs has been demonstrated in several immune responses, including a particularly important contribution to alloreactivity, the molecular basis for transplant rejection. Genetic elimination of secondary TCRs in mice resulted in a 50% reduction in the naive alloreactive T cell frequency. Recently, studies have elucidated a high degree of peptide specificity in alloreactivity, but the role of secondary TCRs is unknown. We hypothesize that secondary TCRs are important for the formation of the naive T cell repertoire capable of responding to individual allopeptides. To address this, we analyzed T cells in mice heterozygous for Trac (Cα+/-), which can only generate a single functional TCRα rearrangement, to look at T cell selection and alloantigen specificity. In TCR Cα+/- mice, there is a defect in the generation of mature T cells attributable to altered thymic selection. The effect on T cell development does not impact peripheral T cell numbers. Despite this, there are functional consequences for the inability to express secondary TCRs: binding to individual allopeptide-MHC tetramers was significantly diminished in naive T cells from TCR Cα+/- mice. Our work reveals a fundamental role for secondary TCRs in thymic selection and in the formation of complete allopeptide-binding T cell populations.
Impaired selectin-dependent leukocyte trafficking induces T cell exhaustion and prevents chronic allograft vasculopathy and rejection (P2140)
Authors: Sarraj Bara , Ye Junsheng , Zhang Zheng , Miller Stephen , Kansas Geoffrey , Ansari M. Javeed
Published: January 2023

Selectin-selectin ligand interactions mediate the initial steps in leukocyte emigration, an integral part of immune response. Fucosyltransferase-VII (FucT-VII) enzyme, encoded by Fut7 gene, is essential for post-translational modification and function of selectin ligands. In an established model of cardiac allograft vasculopathy and chronic rejection, Fut7-/- recipients, with impaired selectin-dependent leukocyte trafficking, enjoyed long-term graft survival with minimal vasculopathy. This was associated with T cell exhaustion in the periphery characterized by impaired effector cytokine production, proliferative defect, increased expression of inhibitory receptors PD-1 and Tim-3 and low levels of IL-7Ra on CD4 T cells and reduced trafficking of polyfunctional CD4 memory T cells to the allograft. Blocking PD-1, triggered rejection only in Fut7-/- recipients, whereas depleting regulatory T cells triggered rejection in WT recipients. Adoptive transfer experiments confirmed that the exhausted phenotype is seen primarily in CD4 T cells with impaired trafficking ability. In summary, these data suggest that impaired leukocyte trafficking is a novel mechanism of CD4 T cell exhaustion and our experimental system serves as an excellent model to study CD4 T cell exhaustion as a dominant mechanism of transplant tolerance. Further, targeting FucT-VII may serve as a promising strategy to prevent chronic allograft rejection and promote tolerance.
Modulation of CD8 expression represents a novel mechanism of peripheral tolerance (BA8P.120)
Authors: Rodriguez-Rodriguez Noe , Apostolidis Sokratis , Nguyen Tran , Martin Villa Jose , Tsokos George , Crispin Jose
Published: January 2023

The ontogeny and raison d'être of peripheral TCR-αβ+CD3+CD4-CD8- T cells, henceforth “DN T cells”, remain controversial. They represent a small population within the bulk of peripheral T cells that expands in certain inflammatory conditions such as systemic lupus erythematosus (SLE) and autoimmune lymphoproliferative syndrome. In fate-mapping experiments, we demonstrate that DN T cells are thymic emigrants. In peripheral lymphoid organs they display an activated phenotype and their TCR repertoire is similar to that of CD8+ T cells. In unmanipulated mice, DN T cells express PD-1 and Helios, molecules induced by TCR activation. Using adoptive transfer experiments with OT-I and HY TCR T cells, we prove that CD8+ but no CD4+ T cells downregulate the expression of CD8 and become DN T cells after encountering their cognate antigen as a self-antigen. After an initial activation and expansion phase where CD8+ T cells upregulate PD-1, Sca-1, and Helios, CD8 is downregulated. Importantly, this phenomenon is not observed during productive immune responses induced by foreign cognate antigens. Notably, the phenotype of DN T cells induced by self-antigen exposure is identical to that of naturally occurring DN T cells from unmanipulated mice. In conclusion, CD8 silencing represents a mechanism that controls activation of self-reactive CD8+ T cells. The presence of small numbers of DN T cells in unmanipulated mice suggests that this mechanism is constantly active.
The age-related loss of FoxN1 and associated thymic involution leads to increased susceptibility to autoimmunity by impairing negative selection rather than influencing the generation of Tregs. (HEM4P.235)
Authors: Coder Brandon , Wang Hongjun , Su Dong-Ming
Published: January 2023

The thymus maintains immunotolerance to self-antigen by deleting self-reactive T cells and generating nTregs. The thymus involutes with age, which is driven by the loss of FoxN1. Thymic involution is thought to be linked with increased susceptibility to autoimmune disease. However, how the age-related loss of FoxN1 induced thymic involution impacts autoimmunity remains unclear. We used a FoxN1 cKO mouse model that mimics natural thymic involution. We found impaired negative selection in the FoxN1 cKO thymus, evidenced by an increase in the frequency of CD4 and CD8 SP thymocytes and a decrease in Aire+ mTECs. Recent thymic emigrants from the FoxN1 cKO thymus have increased proliferation and are more often CD44+, indicating that they may be self-reactive T cells. Additionally, we found that nTreg frequency was increased in the thymus, but not in the spleen. We adoptively transferred aged wild-type splenocytes, in which there are a higher proportion of Treg cells into young Rag2-/- mice. We found that the young periphery was able to reverse Treg accumulation. Additionally, the adoptive transfer led to an increase in salivary infiltration independent of peripheral age. We conclude that loss of FoxN1 impairs negative selection, which may lead to an escape of self-reactive T cells. However, the age-related accumulation of Tregs depends on the age of the microenvironment in which they stay.
NKT cell-dependent differential effects of OPN on functional modulation of macrophages and T lymphocytes in RSV infection (HYP6P.273)
Authors: Durre Kainath , Qureshi Nagib , Beal Brooks , Qureshi Mahboob
Published: January 2023

RSV bronchiolitis is an important cause of pediatric hospitalization, which often leads to developing asthma in later life. Studies have indicated a Th2 skewed polarization of the lung environment in these children, which can be modulated by exogenous administration of osteopontin (OPN). In this study, using an NKT cell KO (CD1d-/-) mouse model, we have examined the effects of OPN on macrophages (MQ) and T lymphocytes (Tc) during RSV infection and identified the role of NKT cells in this process. Flowcytometry analysis determined that OPN treatment in RSV infection had reduced MQ and Tc recruitment in the lung parenchyma and alveolar spaces. However, OPN induced activation of the CD4 and CD8 Tcs in an NKT- dependent fashion, as the magnitude of activation was reduced to only 20% in the CD1d-/- mice as compared to the wild type (WT) mice. Also, expression of MHCII on MQs was significantly increased in the CD1d-/- mice, indicating a downregulatory effect of NKT cells. Altogether, these data suggest that OPN differentially affects MQ and Tc response and their functional modulations in an NKT cell-dependent fashion. A better understanding of these inverse effects of OPN on different immune effector cells during RSV infection may help establish OPN as a potential therapeutic for prevention of RSV-associated asthma in children.
Silver nanoparticles induce silver nanoparticle-specific allergic responses (HYP6P.274)
Authors: Hirai Toshiro , Yoshioka Yasuo , Ichihashi Ko-ichi , Mori Takahide , Nishijima Nobuo , Handa Takayuki , Takahashi Hideki , Tsunoda Shin-ichi , Higashisaka Kazuma , Tsutsumi Yasuo
Published: January 2023

Metals, such as Au, Ag, and Ni cause various types of allergic reactions. Several models have been proposed to explain the activation of metal-allergy, for example, analogous to typical haptens such as TNP, T cell recognize a complex of metal ions with MHC-embedded self-peptides. In general, metal-allergy means metal-ion allergy. On the other hand, with recent development of nanotechnology, nanomaterials (≤ 100 nm) including metal nanoparticles have been used in variety of fields, such as cosmetics and food. Due to their ultrafine size, nanoparticles have distinct property including their in vivo dynamics compared with both molecules and bulk particles. Here, we evaluated the sensitization potential of silver nanoparticles (nAg). The BALB/c mice were immunized with various size of nAg (10-100 nm) with LPS as adjuvant. One week after weekly immunization for 4 weeks, we challenged their immunized nAg on their ears and evaluated the ear swelling for 4 days. nAg with diameter of 10 nm, but not more than 10 nm, enhanced nAg-mediated ear swelling. Thus nAg less than 10 nm might have sensitization potential. Because nAg-immunization did not enhance Ag ion (Ag+)-mediated ear swelling, nAg-elicited immune responses did not have cross-reactivity to Ag+. Therefore, nAg-elicited immune responses were nAg-specific immune responses. We are now analyzing which cells and how the cells enable the specific recognition of nAg to reveal new perspective of acquired immune systems.
Mechanism of food allergy (FA) induction by medium chain triglycerides (MCT). (HYP7P.314)
Authors: Khodoun Marat , Gudimetla Vishnu , Krishnamurthy Durga , Strait Richard , Finkelman Fred
Published: January 2023

Food ingestion generally induces antigen (Ag)-specific tolerance rather than immunity. Consistent with this, induction of IgE-mediated intestinal FA in mice has required either Ag priming through a parenteral route or ingestion of Ag with a potent toxin. In contrast, we recently reported (JACI, 131:442-50, 2013) that Ag-specific, IgE-mediated FA is induced when a protein Ag is ingested with the widely used nutrient, MCT. We also showed that MCT ingestion induces increased epithelial cell expression of the pro-Th2 cytokines TSLP, IL-25 and IL-33. We now report that MCT ingestion, by itself, can induce shock by damaging intestinal epithelium, with a consequent increase in intestinal permeability. In contrast, ingestion of the anionic detergent, sodium dodecyl sulfate, damages intestinal epithelium but does not promote a Th2 response. Both direct MCT induction of intestinal epithelial damage and MCT promotion of a Th2 response and FA to co-ingested egg white are suppressed in mice treated with the lipase inhibitor orlistat, which blocks intestinal triglyceride absorption. Consistent with MCT induction of TSLP, IL-25 and IL-33, treatment with a cocktail of mAbs to these cytokines or their receptors blocks FA induction by ingested MCT plus egg white. Taken together, these observations suggest that MCT must be absorbed to damage epithelial cells and induce the pro-Th2 cytokines that promote FA development; they also provide a potential paradigm for natural FA development.
Regulation of respiratory mucosal immunity by epithelial cell secreted antimicrobial molecules (INC5P.330)
Authors: Di Peter , Liu Yanyan , Di Marissa
Published: January 2023

The ability of the host to avoid infection depends largely on mechanisms of innate immunity. Several secreted epithelial cell-secreted proteins including MUC5AC, SPLUNC1, SP-A, and LL37 form an antimicrobial milieu to clear inhaled pathogens and to participate in immune response. In this study, we try to determine the underlying mechanisms of pulmonary antimicrobial activity following Pseudomonas aeruginosa infection. Splunc1 KO mice showed accelerated mortality and increased susceptibility to microbial infection with significantly decreased survival rates, increased bacterial burdens, exaggerated tissue injuries, and elevated proinflammatory cytokine levels, compared with those of their wild-type littermates. Increased neutrophil infiltration in Splunc1 KO mice was accompanied by elevated levels of chemokines including Cxcl1, Cxcl2, and Ccl20. Moreover, the expression of several epithelial secretory proteins such as Muc5ac, Muc5b, and CCSP, as well as antimicrobial molecules including LL37, lysozyme, and lactoferrin, was considerably suppressed in the lungs of Splunc1 KO mice. The deficiency of Splunc1 in mouse airway epithelium also resulted in increased biofilm formation. Using in vitro and in vivo approaches, we demonstrated the role of secreted antimicrobial molecules in protecting the host from Pseudomonas-induced respiratory infections and supported the notion that host antimicrobial milieu plays a critical role in determining infectious susceptibility.
Neural control of innate immunity by beta2-adrenergic receptor (INC5P.323)
Authors: Agac Didem , Estrada Leonardo , Murray Sean , Farrar David
Published: January 2023

The nervous system controls a variety of physiological responses through the release of several chemical messengers. Although the immune system has been thought to be a self-regulated system, there is growing evidence that the immune system and the nervous system are capable of reciprocally regulating their function. We recently identified a unique role for adrenergic signaling in modulating the innate response to pathogen-associated molecular patterns. First, we found that norepinephrine (NE) can suppress pro-inflammatory cytokine secretion from bone marrow-derived macrophages and dendritic cells in response to TLR signaling in vitro. This suppressive effect depends on the early induction of the anti-inflammatory cytokine IL-10, where neutralizing the autocrine IL-10 signaling restores the cytokine secretion. NE exerts its effects by signaling through the beta2-adrenergic receptor (ADRB2). Second, upon challenging mice with a sub-lethal dose of Listeria monocytogenes, ADRB2 knockout mice present with splenomegaly and enhanced bacterial clearance. Additionally, treatment of whole human blood with TLR ligands in the presence of the ADRB2 agonist albuterol suppresses TNFα secretion, demonstrating the presence of this pathway in humans. This supports the hypothesis that ADRB2 signaling is a negative regulator of TLR signaling and suggests that this pathway plays an important role in immune activation and homeostasis.
GM-CSF and IL-4 produced by NKT cells inversely regulate IL-1β production by macrophages (INC6P.402)
Authors: Jeong Dongjin , Ahn Sehee , Oh Sae Jin , Ahn Ji Ye , Lee Seung Hyo , Chung Doo Hyun
Published: January 2023

NKT cell is a distinct T cell subset that links innate and adaptive immune responses. IL-1β, produced by various immune cells, plays critical role in the regulation of innate immunity in vivo. However, it is unclear whether NKT cells regulate IL-1β production in vivo. Here, we demonstrate that NKT cells regulate production of IL-1β by macrophages via producing GM-CSF and IL-4. Among cytokines secreted from NKT cells, GM-CSF enhanced IL-1β production by macrophages through regulating pro-IL-1β expression and NLRP3-dependent inflammasome activation, whereas IL-4 enhanced M2-differentiation of macrophages, thereby decreasing IL-1β production. Furthermore, NKT cells aggravated IL-1β-mediated peritonitis by producing GM-CSF, but attenuated peritonitis via producing IL-4. Taken together, our findings suggest the NKT cells exert double-sided effects on IL-1β-mediated innate immune responses by producing IL-4 and IL-1β.
Tyrosine phosphorylation of IFITM3 blocks an endocytic motif necessary for its complete influenza virus restriction (INM8P.438)
Authors: Chesarino Nicholas , McMichael Temet , Hach Jocelyn , Yount Jacob
Published: January 2023

Influenza viruses are a major health concern, with seasonal influenza afflicting an average of 5% to 20% of the US population annually. Interferon-induced transmembrane protein 3 (IFITM3) is important for the inhibition of influenza infection by blocking entry of endocytosed viruses into the cytoplasm. However, the exact mechanisms of IFITM3 regulation and antiviral activity are not clearly defined. Tyrosine phosphorylation by Fyn kinase at the N-terminus of IFITM3 has been previously identified, but the effects of this modification are unknown. We observe IFITM3 cell surface retention for phosphorylation-deficient mutants, minimal cell surface localization in Fyn-deficient cells, and cell surface retention as result of Fyn kinase overexpression. These findings are reproduced for both mouse and human IFITM3, and suggest that phosphorylation of tyrosine 20 in both species blocks an endocytosis motif. Accordingly, tyrosine 20 mutants lose anti-influenza virus activity, confirming the role of endosomal localization in the antiviral function of IFITM3. Our findings classify phosphorylation as a negative regulator of IFITM3, and provide important insights towards delineating the mechanisms by which IFITM3 traffics and restricts viral infection.
Cobalt-chrome micro-particles induce a robust MyD88-independent type 2 immune response (IRM7P.495)
Authors: Mishra Pankaj , Palma Mark , Millman Ariel , Hallab Nadim , Beebe Kathleen , Benevenia Joseph , Kanneganti Thirumala-Devi , Gause William
Published: January 2023

Orthopaedic wear debris may contribute to harmful inflammation and implant failure. In this study, we examined whether Cobalt Chrome (CoCr) can trigger inflammatory responses under aseptic conditions. Intra-peritoneal injection of mice with CoCr microparticles induced a type 2 innate immune response with marked increases in M2 macrophages, neutrophils and eosinophils in WT and MYD88/TRIF-/- mice. In contrast, although Th2 cytokine MRNA elevations were intact, a marked decrease in neutrophil and M2 macrophages occurred in Caspase-1-/- mice compared to wild type mice. SYK pathway blockade with the inhibitor BAY 61-3606 caused a marked general decrease in type 2 inflammation. Adjuvant properties of CoCr particles were assessed by immunizing mice with CoCr+OVA peptide after transfer of CFSE-labeled OVA specific DO11.10 CD4+ T cells. After 7 days, marked increases in OVA-specific T cell proliferation and IL-4 production were observed compared to mice immunized with OVA alone. To more closely approximate effects of wear debris in joint replacements, localized immune cell infiltrates were examined after intra-articular knee injections of CoCr particles. Infiltration of specific immune cells and mRNA expression were similar to peritoneal inoculation. These studies indicate that the type 2 immune response is triggered by solid particles, independent of toll like receptor signalling, and differentially modulated by Inflammasome and SYK signaling pathways
Rab7 plays a B cell-intrinsic and critical role in T-dependent and T-independent antibody responses by mediating AID expression and class switch DNA recombination (IRM8P.706)
Authors: Xu Zhenming , Pone Egest , Lam Tonika , Edinger Aimee , Casali Paolo
Published: January 2023

Rab7 is a small GTPase that mediates endosomal functions and regulates T cell homeostasis. Here, we have addressed the B cell-inherent role of Rab7 in antibody responses and underlying mechanisms by constructing Igh+/Cγ1-cre Rab7fl/fl mice. These mice displayed normal B cell and T cell development and survival, and were knocked-out of the Rab7 gene specifically in B cells activated to undergo IgH germline Iγ1-Sγ1-Cγ1 transcription. This transcription process is induced by IL-4 plus a primary CSR-inducing stimulus, and is required for antibody switching from IgM to IgG1, but not to other Ig isotypes. Igh+/Cγ1-cre Rab7fl/fl mice had virtually abrogated levels of IgG1, but normal levels of IgM and other switched Ig isotypes, and failed to mount specific IgG1 responses to T-dependent or T-independent antigens. They showed markedly reduced IgG1+ B cells and antibody-forming cells, with unaltered B cell proliferation, survival and differentiation into germinal center B cells and plasma cells. Purified naïve B cells from these mice were defective in CSR to IgG1, despite normal proliferation and plasma cell differentiation. These together with the unaltered germline transcription but significantly decreased expression of AID (essential for CSR and SHM) demonstrate that B cell Rab7 mediates CSR and, therefore, the antibody response by upregulating AID, likely by regulating intracellular membranes that facilitate signal transduction for activation of specific transcription factors.
Biochemical and structural studies reveal a canonical mode of molecular recognition between TIGIT and nectin-2 (IRM10P.735)
Authors: Samanta Dibyendu , Udupi Ramagopal , Almo Steven
Published: January 2023

Besides antigen-specific signal from the interaction between T-cell receptor and peptide-MHC complex expressed on antigen presenting cells (APCs), the process of optimal T-cell activation and differentiation requires antigen-independent costimulatory signals. Costimulatory signals are provided by interaction between a number of activating as well as inhibitory receptors expressed on T cells with specific ligands expressed on APCs. The balance between activating and inhibitory signals critically controls the mammalian immune responses. Therefore, understanding the mechanisms and designing corrective therapeutics involving T-cell costimulation are imperative in both infectious as well as autoimmune diseases. TIGIT (T cell immunoglobulin and ITIM domain), a recently identified immune receptor expressed on T and NK cells, upon interaction with either of its two ligands, nectin-2 or PVR, that are over-expressed on certain tumors, inhibits activation of T and NK cells. Several biophysical studies, including X-ray crystallography suggest that TIGIT may exist in its monomeric as well as dimeric forms. Based on the structural and biochemical studies, we mapped the nectin-binding interface on TIGIT. Interestingly, structure-guided mutations that disrupt the interaction between nectin-2 and TIGIT also limit the homodimerization of TIGIT in solution. Our data provide structural insights into how TIGIT recognizes a cell-adhesion molecule nectin-2 to execute immunoregulatory function.
Modification of proteins by O-linked N-acetylglucosamine in activated T cells (IRM10P.756)
Authors: Lund Peder , Elias Joshua , Davis Mark
Published: January 2023

The process of T cell activation is mediated by signal transduction cascades involving changes in protein post-translational modifications. While the importance of some modifications, such as phosphorylation, is well appreciated, relatively little is known about the modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc). Given that O-GlcNAc appears to regulate signaling pathways akin to phosphorylation, we are investigating how O-GlcNAc may be involved in T cell activation. We have observed that T cell receptor engagement leads to changes in protein O-GlcNAc levels. Furthermore, we have found that the enzyme catalyzing the addition of O-GlcNAc to proteins accumulates at the immunological synapse and differs in the relative expression of its splice isoforms in activated versus resting T cells. To identify proteins that may be regulated by O-GlcNAc, we have employed a chemical labeling strategy on resting and activated human T cells to identify potential O-GlcNAc sites on a number of proteins, such as the transcriptional regulator host cell factor 1. Future experiments will focus on how O-GlcNAc sites responsive to T cell receptor stimulation influence molecular and cellular activity during T cell activation. Beyond its significance to basic cell biology, our work may have clinical implications since abnormal O-GlcNAc levels have been associated with human diseases such as cancer and diabetes.
Potentially neutralizing B cell clonotypes are eliminated at peripheral selection checkpoints (LYM6P.768)
Authors: Scholz Jean , Soldemo Martina , Dosenovic Pia , Naradikian Martin , Zhou Lin , Wilson Richard , Wyatt Richard , Lackner Andrew , Karlsson Hedestam Gunilla , Cancro Michael
Published: January 2023

B lymphocyte stimulator (BLyS) signals via BR3 mediate selection and survival in the transitional, mature, and germinal center B cell pools. We have previously shown that treatment of mice with exogenous BLyS prior to immunization with HIV-1 envelope (Env) trimers improves neutralizing antibody breadth and potency. We are therefore investigating the hypothesis that broadly neutralizing B cell clonotypes are rare because of counter-selection at the transitional or germinal center checkpoints. Here we show that Env-specific murine B cells in pre-immune, germinal center, and memory B cell subsets can be visualized and tracked by flow cytometry using a biotinylated Env probe. Initial results indicate loss of Env+ B cells at both the transitional developmental stage and in the germinal center following immunization. As a first step in testing the feasibility of a BLyS pre-immunization treatment approach in nonhuman primates, we show that BLyS enhances survival of quiescent and dividing rhesus macaque B cells in vitro.
Eosinophils enhance immune pathology and are increased with cell-wall chitin exposure in Aspergillus fumigatus infection. (MPF3P.802)
Authors: Templeton Steven , O'Dea Evan , Li Hongtao , Amarsaikhan Nansalmaa
Published: January 2023

In humans and experimental animals infected with the opportunistic fungal pathogen Aspergillus fumigatus, Th1 responses are considered protective, while Th2 responses are associated with increased morbidity and mortality. How host-pathogen interactions influence the development of these protective or detrimental immune responses is not well understood. We compared lung immune responses to conidia from two fungal isolates that expressed different levels of the fungal cell wall component chitin. We observed that repeated aspirations of the high-chitin expressing isolate, Af5517, induced increased airway eosinophil and Th2 cell recruitment in the lungs of recipient mice when compared to the isolate Af293. In addition, lung transcription of Th2 cytokines/chemokines was increased while IFN-γ was decreased in response to Af5517. Eosinophil recruitment in response to conidia aspiration was correlated with the level of chitin exposure during germination, and was decreased by constitutive lung chitinase expression. Furthermore, eosinophil-deficient mice subjected to multiple aspirations of Af5517 prior to neutrophil depletion and infection exhibited decreased morbidity, mortality, and lung fungal burden when compared to wild-type mice. These results suggest that chitin exposure in germinating conidia promotes Th2-skewed airway immune responses and that recruited eosinophils may be considered as a therapeutic target in patients that respond poorly to A. fumigatus infection .