bluuebunny/crossref_metadata_2025_split huggingface dataset:
TRPM2 links oxidative stress to the NLRP3 inflammasome activation (P1268)
Authors: Zhong Zhenyu , Zhai Yougang , Liang Shuang , Mori Yasuo , Han Renzhi , Sutterwala Fayyaz , Qiao Liang 
Published: January 2023

Abstract
Exposure to particulate crystals, either environmental irritants or endogenous metabolites, can induce oxidative stress in phagocytes, which in turn triggers NLRP3-inflammasome mediated interleukin 1β (IL-1β) secretion to initiate undesired inflammatory responses associated with many autoinflammatory and metabolic diseases. Although mitochondrial reactive oxygen species (ROS), accumulated under oxidative stress, has been shown essential for NLRP3 inflammasome activation, little is known how ROS signals the assembly of the NLRP3 inflammasome. Here, we first identified liposomes as a novel group of particulate activators for NLRP3 inflammasome, and further demonstrated that this immune recognition process also requires mitochondrial ROS. Moreover, we found that stimulation of particulate substances (liposomes and crystals) induced a ROS-dependent calcium influx via the TRPM2 ion channel that directs the assembly of NLRP3 inflammasome. Macrophages deficient in TRPM2 displayed drastically impaired NLRP3 inflammasome activation and IL-1β secretion in response to liposomes/crystals. Consistently, Trpm2−/− mice were resistant to crystals/liposomes-induced, IL-1β-mediated peritonitis in vivo. Taken together, these results identify TRPM2 as a novel player that links particulates-induced oxidative stress to the NLRP3 inflammasome activation. Thus, targeting TRPM2 may be beneficial for inflammatory disorders associated with deranged NLRP3 inflammasome activation.
Kinetics of NLRP3 inflammasome independent and dependent mitochondrial damage during inflammasome activation (P1276)
Authors: Heid Michelle , Watkins Simon , Salter Russell 
Published: January 2023

Abstract
The NLRP3 inflammasome is a protein complex that plays a central role in innate immunity and is involved with caspase-1 activation. In murine bone marrow derived macrophages (BMDM), NLRP3 inflammasome activation can lead to the processing and secretion of IL-1 beta, as well as pyroptotic cell death. Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. In response to treatment with nigericin, we have observed that mitochondrial depolarization occurs independent of the presence of the NLRP3 inflammasome, however the loss of mitochondrial membrane integrity is NLRP3 dependent. This loss of mitochondrial integrity is preceded by NLRP3 dependent lysosomal membrane permeability. Hence, we have shown kinetically that NLRP3 inflammasome independent damage to the mitochondria precedes NLRP3 inflammasome dependent organelle damage and eventual cell death. The kinetic information provided via these data suggests a multi-step process involving both NLRP3 dependent and independent events during inflammasome activation.
Bacterial RNA:DNA hybrids: novel activators of the NLRP3 inflammasome (P1250)
Authors: Kailasan Vanaja Sivapriya , Rathinam Vijay , Kalantari Parisa , Fitzgerald Katherine , Leong John 
Published: January 2023

Abstract
The NLRP3 inflammasome is essential for IL-1β production in response to Enterohemorrhagic Escherichia coli (EHEC). To determine the mechanism of NLRP3 inflammasome activation by EHEC, we screened EHEC mutants lacking various virulence genes and found that none of them are essential for inflammasome activation. However, lysosomal acidification following infection was found to be essential for IL-1β production implying that bacterial lysis and its products such as nucleic acids play a role. Consistent with this, pretreating macrophages with RNase A specifically reduced EHEC-induced IL-1β response. Also, EHEC RNA was found to be co-localizing with NLRP3 in the cytosol, revealing a potential mechanism for NLRP3 activation. Interestingly, treating cells with RNase H, which degrades RNA of RNA:DNA hybrids, also markedly reduced IL-1β activation indicating that RNA:DNA hybrids formed as replication and transcription intermediates in bacteria also play a role in NLRP3 activation. Supporting this idea, synthetic EHEC RNA:DNA hybrids potently induced IL-1β production in a NLRP3-dependent manner. Importantly, an E. coli rnhA mutant incapable of producing RNase H induced greater than wild type levels of IL-1β and complementing this mutant with rnhA reduced IL-1β to wild type levels. Altogether these findings identify bacterial RNA:DNA hybrids as novel NLRP3 activators and demonstrate that multiple nucleic acid species such as RNA and RNA:DNA hybrid activate NLRP3 in bacterial infections.
The timing of stimulation of primary memory CD8 T cells impacts the rate of secondary memory generation and differentiation (P1402)
Authors: Khan Shaniya , Martin Matthew , Zhou Xinyuan , Xue Hai Hui , Badovinac Vladimir 
Published: January 2023

Abstract
Memory CD8 T cell responses are essential in establishing long-lasting immunity, as these responses confer protection from repeated infections. Various factors have shown to influence the generation of memory CD8 T cells, suggesting that the process of memory differentiation is not fixed and can be manipulated. Here, we asked whether the timing of stimulation (or recruitment) of primary memory CD8 T cells impacts the generation and differentiation of secondary memory CD8 T cell responses after bacterial and viral infections. We show that despite decreased secondary expansion, the differentiation of primary memory CD8 T cells into populations with the long-term secondary memory phenotype (ex. CD62Lhi, CD27hi) and function (ex. tissue distribution, IL-2 production, antigen-driven proliferative expansion) is facilitated by late recruitment into the immune response. Additionally, we show that the expression of survival factors and transcription factors known to be involved in memory CD8 T cell differentiation (ex. Bcl2, Eomes, Tcf1) is increased in late-stimulated secondary effector CD8 T cells. Thus, these data suggest that the timing of antigen-stimulation modulates the program of secondary memory CD8 T cell differentiation, which may have relevance for the design of future prime-boost vaccine protocols.
Requirements for tissue resident T cell migration in the salivary gland following LCMV infection (P1428)
Authors: Stolp Bettina , Pieczyk Markus , Kreutzfeldt Mario , Merkler Doron , Fukui Yoshinori , Stein Jens 
Published: January 2023

Abstract
Following clearance of viral infection memory T cells are retained to protect the host from reinfection. Central memory (CM) T cells efficiently migrate within the loose network of fibroblastic reticular cells inside the lymph node (LN) parenchyma and monitor antigen presenting cells for cognate antigen. Tissue-resident effector memory (EM) T cells cope with the denser environment within peripheral organs, migrating between endo- and epithelial cells and dense extracellular matrix. Little is known about migration behavior and mechanisms required for EM T cell tissue surveillance. In this study we compare the migratory behavior of CM and EM T cells within the same mouse by consecutive multiphoton intravital imaging of the popliteal LN and the salivary gland (SG), respectively. We establish intravital imaging of the SG as a model for a peripheral organ harboring tissue-resident effector memory cells in between the tightly packaged endo- and epithelial cells. Comparison of the migratory behavior of cognate T cells during the acute and the memory phase of a LCMV infection revealed efficient migration of effector T cells within the SG at all timepoints, although with reduced velocity compared to the LN parenchyma. During the memory phase of infection EM T cells show a probing phenotype, projecting dendrites in between the epithelial cells. Using the ROCK-inhibitor Y27632 and DOCK8-deficient T cells we refine the picture of EM T cell migration within peripheral organs.
Characterization of IL-15 independent memory CD8 T cell subsets (P1438)
Authors: Fraser Kathryn , Schenkel Jason , Vezys Viava , Masopust David 
Published: January 2023

Abstract
IL-15 is required for the maintenance and homeostatic proliferation of conventionally characterized memory CD8 T cells, and thus sets a carrying capacity for the size of the memory CD8 T cell compartment. However, we recently demonstrated that heterologous prime boost (HPB) vaccine regimens that establish very abundant memory CD8 T cell populations result in an enlargement of the memory CD8 compartment, which is almost entirely due to the addition of vaccine-specific memory T cells that lack CD62L expression. Here, we test the hypothesis that memory CD8 T cells resulting from HPB are less dependent on IL-15. We demonstrate that boosted memory cells express decreased levels of IL15Rβ and undergo little homeostatic proliferation. When co-transferring primary and boosted memory CD8 T cells into IL15KOs, boosted memory CD8 T cells that lacked CD62L expression were better able to persist compared to primary memory CD8 T cells (regardless of CD62L expression). We extended these studies by demonstrating that primary memory CD8 T cells that are resident in several peripheral tissues were also maintained in the absence of IL-15, and in some cases, exhibited IL-15 independent homeostatic proliferation. These observations point to a distinct lineage, not wholly defined by lack of CD62L expression, that harbors less dependence on IL-15 for maintenance. This has ramifications for regulation of memory CD8 T cell quantity following certain vaccine modalities and at front line tissues.
TLR7/9 stimulation by viral RNA is not a major driver of immune activation in pathogenic SIV infection (P6191)
Authors: Barratt-Boyes Simon , Kader Muhamuda , Smith Amanda , Watkins Simon , Guiducci Cristiana , Barrat Franck 
Published: January 2023

Abstract
Chronic immune activation is a fundamental component of AIDS pathogenesis but the mechanism responsible for this process is not understood. Emphasis has been placed on plasmacytoid dendritic cells (pDC), given their capacity to produce interferon (IFN)-α in virus infections and the link between persistent IFN-stimulated gene (ISG) expression and immune activation. We used a dual antagonist of TLR7 and 9 to selectively inhibit pDC responses in SIV infection. Administration of TLR7/9 antagonist blocked blood pDC production of IFN-α and TNF-α following SIVmac251 infection of rhesus macaques, but infection alone induced pDC hyporesponsiveness, whereas blood mDC gained the capacity to secrete IFN-α. In lymph nodes pDC were major but not exclusive producers of IFN-α, which was transient, and inhibited by TLR7/9 blockade. In contrast, TNF-α production in SIV-infected lymph nodes derived from macrophages and T cells, was sustained and was not impacted by TLR7/9 blockade. TLR7/9 blockade had no impact on plasma IFN-α production or ISG expression and did not diminish activation and proliferation of CD4+ and CD8+ memory T cells in either blood or lymph node. These experiments reveal that virus-mediated TLR7/9 activation is not a major contributor to immune activation and suggest that other pathways such as persistent TNF-α production may be significant factors in this process.
Impact of IFNγ on HIV-specific CD4 T cell and antigen presenting cell function (P6163)
Authors: Porichis Filippos , Hart Meghan , Barblu Lucie , Zupkosky Jennifer , Kavanagh Daniel , Kaufmann Daniel 
Published: January 2023

Abstract
One of the major impediments of HIV infection is the abnormally high levels of immune activation that contributes to immune dysregulation and impairs viral clearance. While IFNγ secretion is used as a marker of the functional state of T lymphocytes and NK cells, its role in regulating immune responses is poorly understood. We isolated CD8-depleted PBMCs from HIV infected individuals (n=13). We measured proliferation and cytokine production by HIV-specific CD4 T cells stimulated with HIV Gag in the presence of isotype control, anti-IFNγ and/or anti-PD-L1. We performed apoptosis measurements using Annexing-V binding assays. We also determined the modulatory impact of IFNγ on APCs by measuring expression of PD-1 ligands, HLA-DR, HLA-I and IL-12 secretion. Neutralization of IFNγ produced by Gag stimulated CD4 T cells enhanced proliferation (p=0.0161) and reduced apoptosis of HIV-specific CD4 T cells. IFNγ blockade enhanced IL-13 secretion (p=0.0039) but had no effect on IL-2, IL-10 and TNFα levels. IFNγ induced strong up-regulation of PD-L1, HLA-DR and HLA-I on monocytes but not on B, T and NK cells. IFNγ also induced IL-12 secretion by APCs that acted in a positive feedback loop to further enhance IFNγ secretion. Concurrent blockade of IFNγ and PD-L1 led to a more prominent increase in proliferation of HIV-specific CD4 T cells than blockade of individual pathways. These data provide mechanistic insight on the causal role of IFNγ in T-helper dysregulation in HIV infection.
Rapid Galectin-9 release occurs following HIV-1 acquisition in acute HIV-1 infection (P6165)
Authors: Tandon Ravi , Fujita Tsuyoshi , Chew Glen , Byron Mary Margaret , Niki Toshiro , Hirashima Mitsuomi , Barbour Jason , Ostrowski Mario , Clayton Kiera , Lanteri Marion , Ishii Naoto , Borrow Persephone , Norris Philip , Hecht Frederick , Ndhlovu Lishomwa 
Published: January 2023

Abstract
Despite successes with antiretroviral therapy, HIV infection continues to be a significant health burden with patients remaining at risk of non-AIDS defining events. The earliest immunological responses induced during HIV infection are key events in the control of HIV disease progression. Soluble biomarkers may aid in understanding HIV-immunopathogenesis, inflammation and prophylactic vaccine design. Galectin-9 (Gal-9) is a bidirectional immune regulator that promotes tissue inflammation and cell death but induces immune tolerance in part through engagement with the Tim-3 receptor. We measured circulating levels of Gal-9 in the plasma of 10 acute HIV-infected subjects at multiple time points over a 42-day period, before and after detectable HIV RNA. Plasma HIV RNA levels increased rapidly in all subjects. We found that Gal-9 levels rose with the first median elevation time of 5 days after detectable viremia and a median peak of 57.4 pg/ml. Overall Gal-9 levels generally tracked with viremia. In vitro, recombinant Gal-9 significantly increased IFN-γ production by CD8+ T cells compared to untreated cells (0.02% vs 1.2%) in cultures from early HIV-infected subjects and this was reversed by α-lactose blockade. Our findings reveal that Gal-9 is a novel plasma factor that is rapidly elevated during the first wave of the cytokine storm after HIV emergence and may have important roles in CD4+ T cell loss or the response to tissue damage.
Peroxisome proliferator-activated receptor-gamma in CD11c+ lung dendritic cells programs peripheral CD4+ T cells for Foxp3 induction during airway tolerance. (P6001)
Authors: Khare Anupriya , Krishnamoorthy Nandini , Raundhal Mahesh , Morse Christina , Ray Prabir , Ray Anuradha 
Published: January 2023

Abstract
Peroxisome proliferator-activated receptor-gamma (PPARγ) is a widely expressed molecular regulator with anti-inflammatory functions. However, whether it has an anti-inflammatory role in dendritic cells (DCs) that influence the Treg/Teffector balance has not been experimentally addressed. In this study, we investigated the role of PPARγ in DCs regulating the delicate balance between tolerance versus inflammation in the periphery using a model of airway induced tolerance by inhaled allergen. Using mice with selective deletion of PPARγ in CD11c+ cells, we observed that PPARγ expression in CD11chi lung DCs is essential for de novo Foxp3 expression in CD4+ T cells. We demonstrate that PPARγ plays a crucial role in enhancing aldh1a2 expression in lung CD103+ DCs under tolerogenic conditions with significantly less expression of the enzyme in these cells when mice are immunized for airway inflammation. When tolerized CD11ccre/+ PPARγfl/fl mice were antigen-challenged, a complete loss of immune tolerance with increased neutrophil-dominated airway inflammation was observed. Taken together, our study identifies PPARγ as a central regulator in DC-mediated programming of Foxp3 expression in naive CD4+ T cells dictating tolerance versus inflammation upon antigen provocation.
Novel grass pollen antigens are the major inducers of Th2 cytokine producing T cells in allergic individuals (P6039)
Authors: Schulten Veronique , Greenbaum Jason , Hauser Michael , Baker Denise , Sidney John , Kolla Ravi , Lindestam Arlehamn Cecilia , Oseroff Carla , Alam Rafeul , Broide David , Ferreira Fatima , Grey Howard , Sette Alessandro , Peters Bjoern 
Published: January 2023

Abstract
T cells play an important role in the pathogenesis of allergic diseases. However, potential immunogens of allergenic T cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is an inhaled allergen for which major IgE-reactive allergens have also been shown to trigger Th2 responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE-reactivity. A TG pollen extract was analyzed by 2D-gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry in combination with de-novo sequencing of the whole TG pollen transcriptome identified 93 novel proteins for further study, 64 of which were not targeted by IgE. Peptides from the novel TG proteins were screened for T cell reactivity in PBMCs from allergic donors. Strong IL-5 production was detected in response to peptides from several novel proteins, most of which were not targeted by IgE. Responses against the dominant novel epitopes were associated with memory T cells and could be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic-, proteomic-, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.
Effect of NADPH oxidase 2 deficiency on allergic lung inflammation and differentiation of helper T cells (P6045)
Authors: Kwon Bo In , Lee Seung-Hyo 
Published: January 2023

Abstract
Nox2 has been originally identified in phagocytes, which kills engulfed pathogens by producing reactive oxygen species (ROS). It has been also revealed that activated T cells produce ROS that is an important second messenger for the functions of effector T cells. Recent studies have shown that Nox2 dificient CD4 T cells contain increased IL-17 producing capability and Nox2 knock-out mice have a tendency an enhanced susceptibility to spontaneous arthritis with age. To investigate whether Nox2 or ROS deficiency in T cells affect differentiation and function of CD4 T cells in T cell intrinsic manner. Specifically we adopted allergic asthma model to clarify the function of Th2 cells without Nox2 or ROS. Asthma phenotypes were analyzed in fungal protease mixed with chicken egg ovalbumin (OVA) allergen (APO) induced experimental asthma. To understand mechanisms of exaggerated effector T cell functions, we investigated the degree of T cell activation and differentiation of Treg cells with Nox2 deficient and wild type CD4 T cells. All experimental asthma phenotypes including airway hyper-responsiveness, lung inflammation along with eosinophilia and mucus production were increased in Nox2 deficient mice. Nox2 deficient T cells also showed enhanced hyper-activation, reduced AICD and decreased Treg cell differentiation. Our results indicate that Nox2/ROS deficiency showed exaggerated experimental asthma, which was caused by enhanced function of Th2 cells.
Respiratory infection with rhinovirus interferes with the induction of tolerance to aeroallergens (P6007)
Authors: Mehta Amit , Duan Wei , Soroosh Pejman , Doerner Astrid , Zuraw Bruce , Croft Michael 
Published: January 2023

Abstract
Respiratory viral infections can influence the course of asthma at different time points. Rhinovirus (RV) infections during early age associate with a higher frequency of asthma in later childhood. Because airway exposure to innocuous antigen normally engenders T cell tolerance and prevents the development of airway inflammation, we examined the effect of RV infection on airway tolerance induced by exposure to intranasal OVA. We found that simultaneous infection with RV1B, a minor serotype, that binds LDL receptors in mice, abrogated tolerance induced by exposure to OVA, suppressing the generation of antigen-specific CD4+forkhead box protein 3 (FOXP3)+ regulatory T (Treg) cells while at the same time promoting IL-4-secreting effector CD4+ T cells. Rhinovirus infection in-vivo resulted in selective lung expression of the innate cytokines thymic stromal lymphopoietin (TSLP) and IL-33, and the T cell costimulatory TNF family molecule OX40L that is regulated by TSLP. Furthermore, RV infection subsequently resulted in full susceptibility to develop asthmatic disease, which was blocked by targeting TSLPR, IL-33R, or OX40L. These results suggest that RV infection of the respiratory tract can prevent the induction of tolerance to inhaled antigen through a mechanism involving TSLP, IL-33 and OX40L that results in susceptibility to developing acute allergic lung inflammation.
IL-4- or IL-13-mediated modification of structural molecules in murine and human epidermal keratinocytes (P6015)
Authors: Omori-Miyake Miyuki , Yamashita Masakatsu , Yagi Junji 
Published: January 2023

Abstract
Th2 cytokines including IL-4 and IL-13 are strongly associated with development of atopic dermatitis. IL-4 and IL-13 have been demonstrated to play a regulatory role in structural molecules and antimicrobial peptides expressed by keratinocytes at the final stage of differentiation in the stratum corneum. Since a regulatory role of IL-4 and IL-13 in structural molecules of keratinocytes at earlier stages has remained unknown, we investigated the role of Th2 cytokines using primary murine and HaCaT human keratinocytes. Our study revealed that IL-4 or IL-13 was capable of downregulating keratins and desmosomal cadherins expressed in the spinous layer of murine keratinocytes but not in the basal layer. The identical results were obtained in differentiating HaCaT human keratinocytes. The attenuated expression of these molecules did not occurred in Stat6- or Il4ra-deficient murine keratinocytes, and the overexpression of an active form of STAT6 in keratinocytes without stimulation did not change the expression of keratins in the spinous layer. All suggest that STAT6 and unknown IL-4- or IL-13-induced transcription factor(s), possibly p44/42 MAPK signaling molecules, might synergistically downregulate the expression of structural molecules of keratinocytes in the spinous layer. The attenuated expression of the structural molecules of keratinocytes in both the spinous layer and the stratum corneum in the presence of IL-4 or IL-13 may thus cause the instability of the epidermis.