bluuebunny/crossref_metadata_2025 huggingface dataset:
Exudate suction blister: a novel in vivo model of acute infllammation and resolution in humans. (CAM1P.145)
Authors: Motwani Madhur , De Maeyer Roel , Fullerton James , Marks Daniel , Smith Andrew , GIlroy Derek
Published: January 2023

Resolution of acute inflammation is an active process and its dysregulation may contribute to chronic inflammatory disorders. While animal models have been used to study resolution their appropriateness has been questioned. We describe a novel model of resolving inflammation in humans triggered by intradermal injection of UV killed E. coli into the forearm of healthy volunteers (n=30). Inflammatory exudate was drawn into a skin blister induced by negative pressure applied over the injection site. Blister cells were analyzed by flow cytometry while the cell free exudates were assayed for cytokines. To study the different phases of inflammation, blisters were raised at multiple time points up to day 14 post-injection. Vascular response was monitored by laser doppler. Onset (8hr) was characterised by peak erythema and neutrophilia (HLA-DR-/CD16++). By 24hr, neutrophil numbers had fallen by 80% while CD14+ monocytes and CD1c+ dendritic cell numbers increased, indicating resolution. At 48hr blood flow declined along with mononuclear cell numbers. Lymphocytes (CD4+, CD8+ and CD56+ cells) dominated the site at day 3 and persisted up to day 14. Levels of TNF-α, IL-8 and IL-1β were maximal at 8hr, IFN-γ at 24hr. This safe and reproducible model allows, for the first time, exploration of cells and soluble mediators in parallel with the clinical signs of resolution in humans. Potential applications include testing the efficacy of novel anti-inflammatory and pro-resolving drugs.
The flavonoid, rutin, ameliorates neurodegeneration in the retina of diabetic rats (CAM1P.164)
Authors: AlSharari Shakir , Fatani Amal , Ola Mohammad , Alrojayee Fatima , A. Al-Hosaini Abdulaziz
Published: January 2023

Diabetic retinopathy remains the leading cause of blindness worldwide. Dietary flavonoid, rutin may protect the diabetes induced retinal damage. The purpose of this study was to examine the ameliorative effect of rutin on diabetic rat retina through neurotrophic, inflammatory and immunological parameters. Diabetes was induced in male Wistar rats by streptozotocin (65mg/kg). Rutin (100mg/kg) was orally administered to normal and diabetic rats after two weeks of diabetes induction and continued for five weeks. After treatments, retinas were isolated and analyzed for potential neuroinflammatory and immunological markers. Our results indicate that the reduced level of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the diabetic retina were significantly improved by rutin. Increased levels of soluble intracellular adhesion molecule (sICAM-1) and monocyte chemotactic protein (MCP-1) in the diabetic retinas were significantly attenuated to normal level by rutin. Reduced level of GSH in diabetic retina was increased and the increased level of TBARS in the diabetic retina was significantly decreased by rutin administration. The increased level of caspase-3, and decreased level of Bcl-2 in diabetic retina were significantly ameliorated to normal levels by rutin. These results suggest the effectiveness of rutin in protection of diabetic induced damage to the retina by maintaining neurotrophic support, and antioxidants levels.
T cell egress via the afferent lymph modulates the course of local tissue inflammation (CAM1P.149)
Authors: Debes Gudrun , Gómez Atria Daniela , Crosby Erika , Weinkopff Tiffany
Published: January 2023

T cells recirculate through extralymphoid tissues by entering from the blood and egressing via the afferent lymph. While T cell migration into effector sites is key to inflammation, the influence of T cell egress in this process is not known. In a mouse model, antigen recognition at the effector site reduced the egress of antigen-specific pro-inflammatory Th1 cells. Transgenic expression of ‘tissue exit receptor’ CCR7 enhanced egress of antigen-sequestered Th1 cells from the inflamed site and ameliorated inflammation. In contrast, lack of CCR7 on Th1 cells diminished their tissue egress while enhancing inflammation. Lymph-borne Th1 and Th17 cells draining the inflamed skin of sheep chemotaxed to the CCR7 ligand CCL21, suggesting the CCR7-CCL21 axis as a physiological target in regulating inflammation. In conclusion, exit receptors can be targeted to modulate T cell dwell time and inflammation at effector sites, revealing T cell tissue egress via lymph as a novel parameter of the pathophysiology of inflammation.
HSC-independent lymphopoiesis (HEM1P.228)
Authors: Ghosn Eliver , Yamamoto Ryo , Phillips Megan , Waters Jeffrey , Yang Yang , Nakauchi Hiromitsu , Herzenberg Leonore
Published: January 2023

The mammalian immune system develops progressively during ontogeny. In both mice and humans, the types of immune cells that develop in fetuses and neonates differ from those that develop in adulthood. Among lymphocytes, B-1, γδT, and iNKT cells are the earliest to emerge and persist at low frequencies thereafter. In contrast, B-2, marginal zone B (MZB) and αβT emerge later but rapidly rise to predominance in adults. The accepted dogma has been that a single hematopoietic stem cell (HSC) can give rise to all of these developmentally distinct immune cell subsets. However, our in vivo single-cell transfers show that bone marrow-derived long-term HSC give rise to B-2, MZB and B-1b but do not detectably reconstitute B-1a, providing definitive proof that B-1a develop prior to and independent of the HSC. These findings place B-1a in a separate lineage derived from HSC that are rare or missing in adults. Here, continuing with this study, we show that HSC harvested from fetal mice similarly fail to fully reconstitute B-1a in adoptive recipients, while unsorted E12 yolk sac cells fully reconstitute all B cell subsets. Thus, we have demonstrated that B-1a develop via an unsuspected pathway that is independent of the classical HSC development pathway. This finding introduces dramatic changes into the current understanding of the developmental landscape of the immune system. Further, it raises questions about human regenerative therapies in which HSC are used to restore hematopoiesis.
Aryl hydrocarbon receptor modulates the development and activity of innate-like B cells (HEM1P.226)
Authors: Yeramilli Venkata , Tanner Scott , Martin Colin , Kearney John
Published: January 2023

The Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that recognizes polyaromatic hydrocarbons. While the contributions of AhR ligands to the differentiation and functions of Th17/Treg cells are relatively well understood, little is known about AhR in B cells. Microarray and quantitative PCR analysis of B cell subsets revealed that B1 and marginal zone (MZ) B cells expressed higher levels of AhR compared to follicular (FO) B cells. We found ~ two-fold more B1 B cells, but fewer MZ B cells in AhR-/- mice compared to AhR+/+ mice. Following immunization with Streptococcus pneumonia, AhR-/- mice had significantly higher titers of anti-phosphorylcholine IgM antibodies. In contrast, after immunization with a model T cell-dependent antigen, Trinitrophenyl Keyhole Limpet Hemocyanin (TNP-KLH), AhR-/- mice had anti-TNP titers that were comparable to those in AhR+/+ mice. Further, we found significantly fewer B cells in C57BL/6 mice raised on AhR ligand free diet, and an increase in titers of IgM and IgG3 in mice following a switch to an AhR ligand free diet when compared to littermates raised and maintained on conventional diet. Taken together, these findings suggests that AhR plays a role in the development of B1 and MZ B cells and regulates T cell-independent but not T cell-dependent humoral response. Further studies using mice with a B cell specific deletion of AhR are underway to elucidate the mechanism by which AhR ligands influence humoral immunity.
Human umbilical cord blood γδ T cells expanded ex vivo with zoledronic acid are distinct from human peripheral blood γδ T cells (HEM2P.244)
Authors: Tomchuck Suzanne , Evans Scarlett , Perko Ross , He Jin , McKenna Amy , Dash Pradyot , Thomas Paul , Dallas Mari
Published: January 2023

Ex vivo expansion of human peripheral blood (PB) γδ T cells using aminobisphosphonates has been extensively studied. However, as human umbilical cord blood (CB) γδ T cells are not as well described, we conducted a detailed comparison of proliferation, phenotype, TCR repertoire and function of CB and PB γδ T cells cultured with zoledronic acid. The percent of γδ T cells within the lymphocyte subset significantly increased in both samples after culture, but CB γδ T cells proliferated more than PB. Phenotypically, Vγ9Vδ2 was a common clone in fresh PB γδ T cells, which predominated after culture, yet the number of CB Vγ9Vδ2 cells remained low. The memory subsets of fresh γδ T cells were similar, but after culture, PB γδ T cells became traditional memory cells while CB γδ T cells were intermediate CD45RA+CD45RO+ memory cells. Memory status corresponded with TCRγδ variant; more Vγ9 and Vδ2 cells were memory while Vδ1 cells were naïve. Functionally, PB and CB γδ T cells had distinct cytokine secretion profiles. PB γδ T cells secreted more IFN-γ and TNF-α after culture, while CB γδ T cells secreted more IL-10. Using a single cell multiplex PCR assay, we found the TCRγδ repertoire of fresh cells to be diverse in both samples, yet CB γδ T cells maintained diversity after culture. Our results suggest that zoledronic acid may not stimulate γδ T cells in CB the same as PB and analysis of the TCRγδ variants may give insight into the difference in γδ T cell function of these samples.
Regulation of c-Myc/survivin from notch signaling modulates T cell differentiation (HEM2P.250)
Authors: Song Jianxun , Fino Krisitn , Haque Mohammad
Published: January 2023

Notch signaling is essential for T cell lineage commitment and is further required during early phases of thymocyte differentiation. Active Notch signaling during these early stages of T cell development inhibits other lineage potentials, such as B cell and myeloid cell (e.g., dendritic cell) potential. However, the intracellular signaling pathways by which Notch signaling regulates T cell differentiation remain unknown. Here we show that the transcriptional factor c-Myc is controlled by Notch signaling which regulates T cell differentiation. In a well-established in vitro differentiation of T lymphocytes from stem cells, Notch signaling directly controls c-Myc expression. Overexpression of active c-Myc promotes while dominant-negative (dn) c-Myc inhibits early cell differentiation. Moreover, c-Myc expression mediated by Notch signaling modulates survivin, an inhibitor of apoptosis (IAP) protein, which is crucial for T cell development. Overexpression of active c-Myc increases while dn c-Myc reduces survivin expression, which is correlated with T cell differentiation within the in vitro differentiation system. These data identify c-Myc, together with survivin, as regulators of the differentiation of T cells from Notch signaling.
Pre-operative immune signatures correlate with recovery from surgical trauma (HUM1P.267)
Authors: Gaudilliere Brice , Fragiadakis Gabriela , Ganio Edward , Tingle Martha , Lancero Hope , Nolan Garry , Angst Martin
Published: January 2023

Traumatic injury produces a profound immune response that mobilizes both innate and adaptive branches of the immune system. Patient recovery after a major trauma is highly variable. Protracted recovery after surgery -a major trauma- causes significant societal and economic costs. However, the mechanistic underpinning of recovery after surgery remains poorly understood. We recently applied high-parameter mass cytometry at the bedside for the deep immune profiling of patients undergoing orthopedic surgery1. The data revealed patient-specific immune responses in monocyte subsets that contained strong correlates of clinical recovery. In this study, we asked whether differences in patients’ pre-surgical immune states determined recovery. Activation of canonical signaling pathways was elicited in vitro, in pre-surgical patient samples. Single-cell evoked immune responses were analyzed in cell subsets spanning the entire immune system using mass cytometry. Surprisingly, the magnitude of these responses varied significantly across patients, thereby defining patient-specific “immune states”. Among these responses, signaling downstream of the TLR4 receptor in CD14+ monocytes strongly correlated with surgical recovery (R=0.64-0.69). The findings highlight a fundamental role for monocytes in the recovery process and provide the mechanistic basis for a pre-operative diagnostic test to predict the course of surgical recovery. 1.Gaudilliere et al., Sci Transl Med. 2014
Decline in relative plasma cell-free mitochondrial DNA levels in response to prolonged moderate aerobic and eccentric exercise (HUM1P.306)
Authors: Shockett Penny , Khanal Januka , Sitaula Alina , Kraemer Robert
Published: January 2023

Elevated plasma levels of cell-free mitochondrial DNA (cf-mDNA), a cell damage-associated molecular pattern (DAMP), contribute to neutrophil activation and inflammation in trauma patients, and may occur in cancer and autoimmunity. To further understand relationships between cf-mDNA released by tissue injury, inflammation, and health benefits of exercise, we conducted trial by time experiments for plasma cf-mDNA response to prolonged moderate aerobic exercise, and to eccentric exercise. Seven healthy moderately-trained young men (n=7) completed treadmill exercise trials for 90 min at 60% VO2 max, and resting control trials, and blood was sampled immediately prior to (Pre), during (at +18, +54, and +90 min), and after (R40) trials. A significant difference in cf-mDNA response was observed between exercise and control trials. Specifically, cf-mDNA levels differed at +54 and +90 (with or without plasma volume (PV) shift correction). A significant time effect was observed, with relative cf-mDNA levels declining at +54 and +90 during exercise. These results suggest increased clearance or reduced release of plasma cf-mDNA with exercise. Declines in cf-mDNA were also seen after eccentric exercise. Our finding of cf-mDNA decline with prolonged moderate treadmill and eccentric exercise contrasts with studies by others involving exhaustive short-term treadmill exercise, where cf-mDNA levels were unchanged, and highlights differences between exercise- and trauma-induced inflammation.
CD4+ T cells from fibrotic livers direct aberrant autoantibody production from B cells (HUM1P.308)
Authors: Tedesco Dana , Thapa Manoj , Mendel Justin , Best Victoria , Ibegbu Chris , Grakoui Arash
Published: January 2023

Two of the most common causes of end-stage liver disease (ESLD) are hepatitis C virus (HCV) infection and alcoholic liver disease (ALD); the only therapeutic option for liver failure is a liver transplant. Extrahepatic manifestations of ESLD include antibody-mediated autoimmunity; an indication of B cell and CD4+ T cell dysfunction. To investigate the contribution of CD4+ T cells during liver disease, we induced liver fibrosis in mice by a conventional weekly carbon-tetrachloride treatment regimen. Fibrotic animals show elevated serum IgG, positive ANA titers and spontaneous IgG production from hepatic B cells. However, this was not evident in CD4+ T cell depleted fibrotic animals despite comparable fibrosis by pathology. Despite significant expression of Foxp3 on fibrotic liver CD4+ T cells, concomitant expression of CD27, PD-1 and CD40L may account for their ability to promote B cell activation. To understand the implications during ESLD in humans, we performed parallel analysis from human liver explant samples. In accordance with our fibrotic mouse model, we saw an enrichment of Foxp3+ CD4+ T cells as well as spontaneous IgG production that was not evident in healthy liver or PBMC. Taken together, these data suggest that altered hepatic CD4+ T cells aberrantly instruct B cells to drive extrahepatic sequelae of ESLD.
Maresin-1 regulates type 2 innate lymphoid cells and promotes generation of regulatory T cells to limit allergic inflammation (HYP2P.336)
Authors: Krishnamoorthy Nandini , Burkett Patrick , Dalli Jessmond , Abdulnour Raja-Elie , Colas Romain , Ramon Sesquile , Phipps Richard , Petasis Nicos , Kuchroo Vijay , Serhan Charles , Levy Bruce
Published: January 2023

Asthma is a chronic inflammatory disease that fails to resolve and is classically considered to be mediated by an overzealous CD4+Th2 immune response. Recently, a key role for type 2 innate lymphoid cells (ILC2s) was shown to have an important role in the initiation and amplification of allergic inflammation; however, mechanisms for ILC2 regulation remain to be determined. In this study, metabololipidomics of murine lungs identified temporal changes in the endogenous levels of docosahexaenoic acid (DHA)-derived pro-resolving mediator, maresin 1, (MaR1) during self-limited allergic inflammation to ovalbumin (OVA). MaR1 decreased after allergen challenge relative to levels found during sensitization, and subsequently increased during the resolution of inflammation. Exogenous administration of MaR1 reduced lung inflammation and ILC2 expression of IL-5 and IL-13 and increased amphiregulin. MaR1 augmented de novo generation of regulatory T cells (Tregs), which interacted with ILC2s to markedly suppress cytokine production in a TGF-β-dependent manner. Antibody-mediated depletion of Tregs interrupted MaR1 control of ILC2 expression of IL-13 in vivo. These results point to a pivotal role for Tregs in the control of ILC2s in a new proresolving mechanism engaged by MaR1.
Bee venom phospholipase A2 suppresses allergic airway inflammation in an ovalbumin-induced asthma model through the recruitment of regulatory T cells (HYP2P.325)
Authors: Bae Hyunsu , Baek Hyunjung , Park Soojin , Jung Kyung-Hwa , Shin Dasom
Published: January 2023

Bee venom (BV) is one of the alternative medicines that have been widely used in the treatment of chronic inflammatory diseases. We previously demonstrated that BV induces immune tolerance by increasing the population of regulatory T cells (Tregs) in immune disorders. However, the major component and how it regulates the immune response have not been elucidated. Here, we investigated whether bee venom phospholipase A2 (bvPLA2) exerts protective effects that are mediated via Tregs in OVA-induced asthma model. The bvPLA2 treatment induced the Tregs and reduced the infiltration of inflammatory cells into the lung in the OVA-challenged mice. Th2 cytokines in the bronchoalveolar lavage fluid (BALF) were reduced in bvPLA2-treated mice. Although bvPLA2 suppressed the number of inflammatory cells after OVA challenge, these effects were not observed in Treg-depleted mice. In addition, we investigated the involvement of CD206 in bvPLA2-mediated immune tolerance in OVA-induced asthma model. We observed a significant reduction in the levels of Th2 cytokines and inflammatory cells in the BALF of bvPLA2-treated OVA-induced mice but not in bvPLA2-treated OVA-induced CD206-/- mice. These results demonstrated that bvPLA2 can mitigate airway inflammation by the recruitment of Tregs in an OVA-induced asthma model.
Novel mechanisms of conferring broad protection against influenza by Lactobacillus casei lactic acid bacteria by mobilizing innate and adaptive immune cells (INC1P.354)
Authors: Jung Yu-Jin , Lee Young-tae , Ko Eun-Ju , Jang Ji-Hun , Park Min-Kyung , Kim Cheol-Hyun , Kang Sang
Published: January 2023

Heat-killed Lactobacillus casei strain lactic acid bacteria DK128 was investigated as potential probiotics to determine its antiviral effects on influenza virus. Intranasal pretreatment of mice with heat-killed DK128 resulted in the recruitment of various innate immune cells into the airways, conferring broad protection against lethal infection with H1, H3, and H5 subtype influenza viruses, preventing body weight loss, and lowering lung viral loads. The protection against lethal infection as a result of killed-DK128 pretreatment was correlated with significantly reduced infiltrates of natural killer cells, neutrophils, and monocytes as well as with increased alveolar macrophage populations. Mice with killed-DK128-mediated protection against H3N2 primary infection effectively developed heterosubtypic adaptive immunity to H1N1 pandemic virus. Further mechanistic studies using various knockout mouse models discovered that B cells but not CD4 and CD8 T cells were absolutely required for heat-killed DK128-mediated protection. Therefore, this study provides evidence that heat-killed DK128 can be developed as beneficial probiotics and that the cross-talk between innate and adaptive immune cells is playing an essential role in conferring influenza antiviral immunity.
Angiopoietins and angiopoietin-like proteins bind to LILRA/B receptors and activate innate immune responses in human monocytes (INC1P.405)
Authors: Person Anthony , Xiong Wendy , Erickson Christian , Shields Kate , Li Jun , BI Ming , Wu Guoping , Kalabokis Vassili
Published: January 2023

Angiopoietins and Angiopoietin-like (ANGPTL) proteins are secreted glycoproteins that play multiple roles in angiogenesis, lipid metabolism, hematopoietic stem cell expansion, and inflammation. Unlike Angiopoietins, which are known to signal through the tyrosine kinase Tie1 or Tie2 receptors, ANGPTL proteins have long been considered orphan ligands. Recently, ANGPTL1, 2, 5, and 7 were shown to bind to human leukocyte immunoglobulin like receptor B2 (LILRB2), resulting in enhancement of human haematopoietic stem cell proliferation and expansion. Leukocyte immunoglobulin-like receptors (LILRs) also named ILTs, LIRs and CD85s, are a family of immunomodulatory molecules that are expressed on professional APCs and regulate their functional properties to influence immune activation and inhibition. While the MHC class I complexes have been identified as the physiological ligands for many LILR members, the direct interaction between LILRs and ANGPTLs has suggested a new signaling mechanism for those two groups of molecules. We have tested Angiopoietins and ANGPTL proteins in binding studies with the LILRA and LILRB family members, and identified additional LILR family members as potential receptors of Angiopoietin-1 and several ANGPTL proteins. We find correlations between these newly described interactions of Angiopoietins and ANGPTL proteins with LILR family members and their abilities to activate innate immune responses in human monocytes.
Immunophenotyping of bone marrow-derived eosinophils in respiratory pathogen infections (INC2P.421)
Authors: Samarasinghe Amali , LeMessurier Kim
Published: January 2023

Asthmatic individuals are considered at high risk of developing viral and bacterial infections. Since eosinophils are abundant in the airways of allergic asthmatics, we hypothesized eosinophils were susceptible to infection by common respiratory pathogens associated with pneumonia. We used bone marrow-derived eosinophils (BMdEos) to simulate exposure to Influenza A Virus (IAV) or Streptococcus pneumoniae. A subset of BMdEos and lung eosinophils exposed to IAV possessed surface-bound nucleoprotein when examined by flow cytometry, indicating both sources of eosinophils were infected with virus. BMdEos also upregulated MHCI and CD86, while MHCII and CD80 remained unchanged. Although microscopically BMdEos appeared homogeneous, two distinct populations based on Siglec-F expression were observed when phenotyped by flow cytometry. MHCI was upregulated almost exclusively by the Siglec-Fhi cells after IAV exposure. Conversely, BMdEos pulsed with S. pneumoniae upregulated MHCII, while downregulating MHCI. Although elevated MHCII was observed for both Siglec-F populations, Siglec-Fint cells upregulated MHCII levels when pulsed with fewer bacteria. Here, we showed that BMdEos can interact with both IAV and S. pneumoniae, responding divergently by modulating surface levels of MHC molecules. Furthermore, we propose the use of BMdEos as a viable in vitro system for examining eosinophils responses to respiratory pathogens.
Effects of nicotine on expression of membrane cofactor protein CD46 in human ARPE-19 cells (INC2P.409)
Authors: Rudolf Xeniya , Lyzogubov Valeriy , Bora Puran , Bora Nalini
Published: January 2023

Age-related macular degeneration (AMD) causes vision loss in humans and its development is often associated with malfunction of the complement system. The complement system is regulated by various proteins including a cofactor protein CD46. Cigarette smoke is a major risk factor for AMD but the impact of nicotine, main component of smoke, on retinal pigment epithelium (RPE) cells is not well studied. We hypothesized that nicotine decreases expression of CD46 in human adult RPE (hARPE-19) cells in vitro. The effects of nicotine were evaluated in two experiments using hARPE-19 cells. In the first experiment, hARPE-19 cells were treated with either 1 µM or 100 µM nicotine. In the second experiment hARPE-19 cells were treated with combinations of 100 µM nicotine and complement depleted or sufficient 10% normal human serum. Western blot and immunofluorescent analyses were used to evaluate the level of CD46 and its cellular localization in hARPE-19. Three isoforms of CD46 occurred in all experimental groups. We suggest that hARPE-19 cells produce full isoform (52 kDa) and two soluble isoforms (38 kDa and 45 kDa). The level of total CD46 and each of the isoforms were similar in all experimental groups. We did not detect any effects of nicotine on the expression of CD46 isoforms with or without active complement system in the medium. Our findings suggest that expression and localization of CD46 on hARPE-19 cells is independent of complement activity, nicotine, or their combination.
Genetic deletion of AU-rich region in the tristetraprolin (TTP) transcript increases TTP stability and expression and provides protection against inflammatory diseases (INM1P.433)
Authors: Patial Sonika , Stumpo Deborah , Huang Weichun , Flake Gordon , Blackshear Perry
Published: January 2023

Tristetraprolin (TTP) is an mRNA binding protein that binds to AU-rich elements (AREs) in the 3’-untranslated regions (3’UTR) of specific mRNAs, such as that encoding tumor necrosis factor (TNF), and increases their rate of mRNA turnover. TTP mRNA also contains AREs within its own 3’UTR that are partly responsible for its own rapid turnover. In the present study, we generated a novel TTP mouse line (TTPΔARE) with a 136 base deletion in the 3’UTR AU-rich region of TTP mRNA, and studied the effect of this genetic modification on the levels of TTP mRNA and protein, and susceptibility of the mice to experimentally-induced inflammatory conditions. TTP mRNA stability was found to be enhanced in TTPΔARE bone marrow-derived macrophages (BMDMs) and mouse embryonic fibroblasts (MEFs). TTP protein expression was increased, both in the basal state and upon LPS stimulation, in BMDMs, and in selected mouse tissues. LPS-induced expression of TTP target mRNAs, particularly Tnf and Il-1β, was significantly reduced in TTPΔARE BMDMs. Most importantly, TTPΔARE mice were almost completely protected from collagen antibody-induced arthritis, and exhibited significantly reduced inflammation in an imiquimod-induced model of psoriasis. These data suggest that increased levels of TTP can protect against some experimentally induced inflammatory diseases. They also raise the possibility that increasing TTP levels in man might be an attractive therapeutic approach in some inflammatory conditions.
Translation inhibition by bacterial pathogens: links to inflammation (INM1P.428)
Authors: Barry Kevin , Fontana Mary , Portman Jonathan , Ingolia Nicholas , Vance Russell
Published: January 2023

Legionella pneumophila is an intracellular bacterial pathogen that is the cause of a severe pneumonia in humans called Legionnaires’ Disease. L. pneumophila infects alveolar macrophages and its virulence requires a type IV secretion system (T4SS) that injects bacterial effector proteins into the host cytosol. Macrophages distinguish infection with T4SS+ (virulent) and T4SS- (avirulent) bacteria, and mount several distinct immune responses to virulent bacteria. Virulent L. pneumophila induces a block in host protein synthesis that is partially induced by 7 bacterial effectors that directly block host translation. L. pneumophila infection also induces the inflammatory cytokine IL-1α. We link translation inhibition to the production of IL-1α, and show that IL-1α signaling, through the interleukin-1 receptor, protects the host during L. pneumophila infection. We show that translation inhibition leads to a sustained production of Il1a transcript and we suggest that this overcomes the block in translation and allows for preferential production of inflammatory cytokines in infected macrophages. To gain insight into the L. pneumophila induced block in host translation, we used ribosome profiling and RNAseq of L. pneumophila infected macrophages to analyze global and mRNA-specific translation rates. Our data suggest that translation inhibition induced by pathogenic infection may be an important pathway that induces inflammation and protects animals from multiple bacterial pathogens.
Function of HCMV derived chemokines on NK cells (IRC4P.614)
Authors: Yamin Rachel , Mandelboim Ofer
Published: January 2023

Natural killer (NK) cells belong to the innate immune system and are able to control various microbial infections as well as tumor cell burden. NK cells can be divided into two different subsets based on the expression of the two surface markers: CD16 and CD56. Two major NK cell populations are present in the blood; one that express high levels of CD56 and low levels of CD16; named CD56Bright cells and a bigger population (90%) that express CD56 in inermediate levels and express high levels of CD16, named CD56Dim. The latter poultion is more mature and more cytotoxic, while the CD56Bright cells produce high levels of cytokines. The human Cytomegalovirus (HCMV) is a β herpesvirus with a double-stranded DNA genome which encodes a broad range of genes that involved in immune evasion and regulation. Among these, are 3 viral chemokines named vCXCL1, vCXCL2 and UL128. The function of these 3 viral chemokines is not completely understood. Using fusion and recombinant viral proteins we showed that all three chemokines are capable of binding to the CD56Dim CD16Pos NK cell population. We demonstrate that only one of them, the vCXCL1, can surprisingly cause NK cells migration and we identified the chemokine receptors involved in these migrations.
Expression of CD40L on CD8 T cells and its role in influenza A infection (IRC5P.618)
Authors: Tay Neil , Prabhu Padubidhri Nayana , Wong Hok Sum , Chua Yen , Kemeny David
Published: January 2023

CD8 T cells play an important role in protective immunity against a wide range of pathogens and the factors that control their activation are clearly important. CD40L-mediated CD40 licensing of dendritic cells by CD4 T cells is known to be necessary for the generation of a robust CD8 T cell response. Less clear is the contribution of CD40L on CD8 T cells to their activation. We have previously shown that CD8 T cells are able to induce the production of IL-12 p70 by dendritic cells in a CD40L-dependent manner. To better understand the role of CD40L on CD8 T cells responses, we generated and characterized CD40L-expressing CD8 T cells both in vitro and in vivo. We found that CD40L is expressed on 30-50% of activated effector CD8 T cells and the differentiation of CD8 T cells into CD40L-expressing cells is strongly induced by IL-12, suggesting the presence of a positive feedback mechanism mediated by CD40L and IL-12. Using an influenza A mouse model, we examined whether this mechanism is involved in the primary expansion of CD8 T cells during an infection and whether CD40L-expressing CD8 T cells are able to license dendritic cells for optimal memory CD8 T cell programming. In the absence of CD40L signaling, the ability to upregulate CD25 and downregulate CD62L is diminished in CD8 T cells, causing an impairment in their primary expansion and also their ability to accumulate at the site of infection.
Pulmonary vascular heterogeneity: microvascular vs. arterial generation of angiopoietin-2 (IRC5P.633)
Authors: Lomas-Neira Joanne , Heffernan Daithi , Ayala Alfred , Chung Chun-Shiang
Published: January 2023

Endothelial cell (EC) growth factors, Angiopoietin (Ang)-1 and Ang-2, are important mediators of EC function. Bound to a shared receptor, Tie2, expressed on ECs, they have opposing effects on EC activation. Ang-1/Tie2 promotes a quiescent while Ang-2/Tie2 promotes an activated phenotype and increased vascular permeability. Ang-2, stored preformed in EC Weibel Palade bodies (WPbs), is rapidly released from activated ECs. We have reported increased Ang-2 in the blood of trauma patients with ARDS, representing a potential therapeutic target. To assess lung EC activation and release of Ang-2, we used human pulmonary arterial ECs (hPAECs) and microvascular ECs (hPMECs) grown on electric cell-substrate impedance sensing arrays. These arrays measure changes in EC monolayer integrity in response to inflammatory stimulus. We show that, in response to TNF-a stimulation, hPMECs exhibited increased loss of barrier function compared to hPAECs. Also, Ang-2 in culture supernatant increased in both hPAECs and hPMECs compared to untreated controls. While ECs in larger vessels (arteries), contain WPbs, they are not observed in microvascular ECs (<10um diam). To further investigate, we inhibited WPb vesicle exocytosis. Our findings show that inhibition of WPb release decreased Ang-2 in supernatant from hPAECs, but not significantly from hPMECs, suggesting that the microvascular is a region with distinct responses potentially associated with their location and anatomic structure.
Role of tumor-infiltrating regulatory T cells according to the presence of tertiary lymphoid structures in human lung cancer (IRC5P.619)
Authors: Dieu-Nosjean Marie-Caroline , Devi Priyanka , Goc Jeremy , Germain Claire , Knockaert Samantha , Validire Pierre , Damotte Diane , Fridman Wolf Herve , Sautes-Fridman Catherine
Published: January 2023

Regulatory T cells (Tregs) exert various suppressive mechanisms to dampen the host immune response which can help tumor cells to escape immune surveillance. However, the prognostic value of tumor-infiltrating Tregs (Ti-Tregs) is controversial based on the expression of FoxP3 marker in cancer patients. Here, we decipher the phenotype of Tregs infiltrating human lung cancer to determine their role in shaping the immune response against tumor cells. Our aim was to study the differentiation, activation, and immunosuppression status of Ti-Tregs in different areas of human lung tumors. Immunohistochemistry was performed on paraffin-embedded tissue sections for CD3 and FoxP3 stainings, and phenotypic analysis was carried out on fresh human lung cancer specimens (n=50) by flow cytometry. In lung cancer patients, Ti-Tregs localize in the different part of the tumor i.e. tumor nests, stroma, and tertiary lymphoid structures (TLS). Ti-Tregs are CD4+ and show central-memory and effector-memory phenotype. Interestingly, Ti-Tregs exhibit different phenotypes based on the differential expression of activation and immunosuppression molecules. In conclusion, the presence of several Ti-Treg subsets in lung tumor suggests that they may play different roles in distinct tumor areas. Thus, the next step will be to decipher the mechanisms and functions used by these Treg populations in order to manipulate them for therapeutic intervention.
PHLDA1 is a negative regulator for TLR2-mediated inflammatory response (IRC5P.623)
Authors: Baek Suk-Hwan , Park Dae-Weon
Published: January 2023

Atherosclerosis is a slow and complex disease that causes cardiovascular complications and stroke. toll like receptor 2 (TLR2)-mediated signaling pathways and gene regulation are mainly involved in the early development and progression of this complex disease. We found that pleckstrin homology-like domain family A member 1 (PHLDA1) is a unique gene upregulated by TLR2 stimulation and determined the detail mechanisms the signaling pathway for expression in macrophages. Treatment with Pam3CSK4, a TLR2 agonist, increased PHLDA1 mRNA and protein expression levels. Gain- and loss-of-function analyses revealed that PHLDA1 modulates foam cell formation by TLR2 stimulation and foam cell formation-related molecule such as MCP-1. In sequence analysis revealed the presence of typical g-activated sequence (STAT3) element in the promoters of mice PHLDA1 and confirmed the STAT3 binding sites. Subsequently, we also confirmed the detailed mechanism of PHLDA1 expression through the JAK2-ERK1/2-STAT3 pathway. These findings suggest that PHLDA1 is a novel negative regulator aimed at TLR2 and may play a vital role eliciting TLR2-mediated responses in atherosclerosis.
Loss of NADPH oxidase-derived superoxide induces CD4 T cell antigen-specific hyporesponsiveness by modulating IL-12 signaling (IRM5P.637)
Authors: Padgett Lindsey , Tse Hubert
Published: January 2023

Efficient T cell adaptive immune activation requires innate immune-derived reactive oxygen species (ROS) and pro-inflammatory cytokines. Our laboratory previously demonstrated blunted CD4 T cell autoreactivity in the absence of NADPH oxidase (NOX)-derived superoxide, as NOX-deficient Non-obese diabetic (NOD.Ncf1m1J) mice exhibited spontaneous Type 1 diabetes-resistance, partly due to decreased Th1 cytokine responses. To examine the role of ROS deficiency on antigen-specific T cells in a non-autoimmune setting and further test the hypothesis that ROS exacerbates Th1 T cell responses, we generated the OT-II.Ncf1m1J mouse containing superoxide-deficient OVA323-339-specific CD4 T cells. Stimulated OT-II.Ncf1m1J splenocytes displayed decreased Th1 T cell cytokines IFN-γ and TNF-α (p<0.05) and diminished expression of the Th1 transcription factor T-bet and Th1 signal transducer IL-12Rβ2, compared to OT-II CD4 T cells. IFN-γ synthesis and IL-12 signaling by OT-II.Ncf1m1J T cells were rescued by addition of exogenous superoxide and co-culture with superoxide-sufficient antigen-presenting cells; conversely, Th1 responses were blunted in wild type OT-II T cells via free radical dissipation with a superoxide dismutase mimetic. This data highlights the importance of NOX-derived ROS in providing a third signal for adaptive immune maturation by modulating IL-12 signaling and implicates that targeting ROS may represent a promising therapeutic strategy in curbing CD4 T cell autoreactivity.
Ethanol induced expression of forkhead box O3 and associated tolerance is amplified in female derived dendritic cells. (IRM5P.642)
Authors: Thompson Matthew , Vidrine Amy , Watkins Stephanie
Published: January 2023

Dendritic cells (DCs) are often referred to as the bridge between innate and adaptive immunity and are a critical player in inducing a robust immune response to antigen. However they also can act to dampen the immune response by a process called tolerance. Tolerance is critical in preventing autoimmune disorders but can be manipulated by tumors to prevent anti-tumor immunity. Ethanol has previously been shown to reduce the ability of DCs to present antigen and to induce a pro-inflammatory response but the mediator of this reduced DC function is not yet known. One of the key players in DC tolerance is the transcription factor Forkhead Box O3 (FOXO3). FOXO3 is a transcriptional regulator of a variety of processes associated with cell cycle progression as well as regulation of nuclear receptors, including the androgen and estrogen receptors. In these studies we investigated the effects of ethanol on FOXO3 expression in dendritic cells. We also examined the ability of dendritic cells to uptake antigen and mature into functional APCs in the presence of ethanol. and on the ability of ethanol treated dendritic cells to become mature antigen presenting cells. Additionally we examined if the effects of ethanol on DCs were increased due to gender specific differences. Our results indicate that exposure of dendritic cells to ethanol increases the expression of FOXO3 and decreases the expression of co-stimulatory and pro-inflammatory molecules.
T helper subset distribution changes with age and is influenced by IL-2 (LYM2P.724)
Authors: Hopkins Jacob , Masters April , Haynes Laura
Published: January 2023

To better understand how aging impacts the immune response to influenza infection, we have examined CD4+ T helper (Th) subsets and how they are impacted by aging. Th cells are important for helping Ab production by B cells and are required for the generation of cytotoxic and memory CD8+ T cells. Influenza nucleoprotein (NP)-specific Th subsets can be distinguished by PSGL1 and Ly6C expression: PSGL1hiLy6Chi cells are terminally differentiated Th1 effectors; PSGL1hiLy6Clo cells are more memory-like; PSGL1loLy6Clo cells are mostly T follicular helper (Tfh)-like. With age, the proportion of PSGL1hiLy6Chi Th1 effectors is reduced and Tfh-like PSGL1loLy6Clo cells are increased both in naïve mice and during infection. Additionally, Tfh cells (defined as CXCR5+PD-1+) are also increased with age. This change in subset distribution with age is also associated with a decrease in ex vivo IL-2 levels in aged animals. In order to further examine how IL-2 can impact subset distribution, an in vitro system was used in which CD4 T cells from young, middle aged and aged mice were cultured with anti-CD3/anti-CD28. Addition of exogenous IL-2 to Th cells from all age groups resulted in reduction of the PSGL1loLy6Clo Tfh population. Furthermore, blocking IL-2 with anti-CD25 Ab increased the PSGL1loLy6Clo Tfh population, regardless of age. Thus, the availability of IL-2, independent of aging, influences Th subset distribution and can account for the increase in Tfh with aging.
Examining pathogen specific T cell responses during antibiotic intervention (MPF2P.743)
Authors: Benoun Joseph , Fogassy Zachary
Published: January 2023

Salmonella enterica serovar Typhi can cause recurrent and relapsing infection in antibiotic-treated individuals, suggesting that bacterial clearance may hinder the development of adaptive immunity. We have developed an antibiotic-treatment model in mice to examine this issue using antigen-specific reagents. Infection with Salmonella Typhimurium strain BRD509-2W1S caused expansion of 2W1S-specific CD4 T cells that were easily detected in peripheral blood and lymphoid tissues. In contrast, antibiotic-treated mice displayed lower 2W1S-specific CD4 clonal expansion in blood and lymphoid tissues that persisted for months after infection. This reduced clonal frequency correlated with diminished protective immunity to re-challenge infection. We are currently examining the phenotype and functional differences in CD4 T cell memory populations to determine if central, effector, and tissue-resident memory cells are equally impacted by antibiotic-intervention. Greater understanding of how antibiotics affect CD4 memory development may allow for therapeutics to boost protective immunity to secondary or relapsing Salmonella infections.
A novel role for Irgm1 in immune homeostasis (MUC1P.906)
Authors: Azzam Kathleen , Madenspacher Jennifer , Lai Lihua , Gowdy Kimberly , Cain Derek , Clayton Natasha , Cunningham Willie , Janardhan Kyathanahalli , Taylor Gregory , Fessler Michael
Published: January 2023

Irgm1 is an IFN-inducible GTPase that mediates host resistance to intracellular pathogens. We sought to define the role of Irgm1 in pulmonary immune cell homeostasis by profiling steady state and pathogen-induced immunity in the lungs of Irgm1-/- mice. The lungs of naïve Irgm1-/- mice displayed abnormal bronchiolocentric cellular infiltration composed predominantly of B cells with focal collections of T cells, associated with increased lung and airway cytokines. Compared to WT controls, naïve Irgm1-/- mice had elevated serum and airway levels of immunoglobulins, suggesting dysregulated B cell function. B cells were found to be reduced in the blood of Irgm1-/- mice, and B cell precursors were also decreased in the bone marrow. Whereas B2 and B1a cells were elevated in the Irgm1-/- lung, B1a cells were reduced in the peritoneal cavity, suggesting abnormal trafficking of B cell subsets. Irgm1-/- mice had increased influx of neutrophils, chemokines, cytokines, and IgM into the airspace after exposure to inhaled LPS and/or S. pneumoniae. Pathogen clearance from the lung and survival were increased in Irgm1-/- mice upon challenge with S. pneumoniae. Future studies are planned to better define the mechanism of B cell dys-homeostasis, determine whether Irgm1-/- mice have autoimmunity, and define whether the enhanced host defense response and survival of Irgm1-/- mice against S. pneumoniae derives from increased number and function of innate B cells and their products.
Increasing intestinal reactive oxygen species reverses diabetes-induced inflammation (MUC2P.928)
Authors: Chen Lee-Wei , Wu Ying-Ying , Chen Pei-Hsuan , Hsu Ching-Mei
Published: January 2023

Systemic inflammation appears to persist in diabetic patient. To examine the role of reactive oxygen species (ROS) of intestine in DM-induced inflammation, streptozotocin (STZ)-induced diabetes and Ins2Akita (Ins2Akita mutation mutant) mice were used. STZ mice demonstrated a significant decrease of Lacobacilli as well as ROS production and a marked increase of p-JNK as well as iNOS expression in the intestinal mucosa. Oral supplementation of fructooligosaccharides (FOS) or dead Lacobacilli increased the production of ROS of intestine and decreased IL-1β expression of Kupffer cells in STZ and Ins2Akita mice. Feeding of L-NAME, an iNOS inhibitor, but not D-NAME increased the production of ROS of intestinal mucosa and decreased endotoxin in portal vein and IL-1β expression of Kupffer cells in STZ mice. Oral feeding of N-Acetylcysteine, a ROS scavenger, decreased ROS production and inhibited the effects of FOS as well as L-NAME on reversing DM-induced ICAM expression of liver. Using STZ JNK1-/- and Ins2Akita JNK1-/- mice to reverse DM-induced pJNK expression of intestinal mucosa induced a significant decrease of IL-1β expression of Kupffer cells and ICAM expression of liver. Collectively, diabetes induces systemic inflammation through decreasing ROS production and increasing pJNK expression of intestine. Increasing intestinal ROS production could reverse diabetes-induced systemic inflammation.
Impact of a high energy diet on obesity-associated immune biomarkers in Sprague Dawley rats (MUC2P.929)
Authors: Clarke Sandra , Kalmokoff Martin , Brooks Stephen , Green Judy , Caldwell Don , Inglis G. , Green-Johnson Julia
Published: January 2023

A trial was designed to examine effects of a high energy diet on obesity-associated biomarkers in Sprague-Dawley (SD) rats. Male rats (n = 176) were fed an AIN93G high-energy diet containing 16% dietary fat for 2 weeks, and segregated into obese-prone and obese-resistant phenotypes. Necropsies of animals displaying the highest (n = 10) and those displaying the lowest weight gain (n = 10) were conducted to examine potential differences in the concentration of immune biomarkers. Adipose, mucosal (ileum, ileal Peyer’s patches, cecum, proximal and distal colon), mesenteric lymph nodes (mln) and systemic (serum, liver, spleen) tissues were comparatively analyzed for cytokine or adipokine profiles. Obese rats had higher levels of leptin (P = 0.0001) and IL-22 (P = 0.059) in the distal colon and higher levels of IL-10 (P = 0.032) and TNF-α (P = 0.016) in the proximal colon compared to their obese-resistant counterparts. In obese-resistant rats, higher levels of CINC-2αβ (P = 0.048) occurred in mesenteric adipose tissue along with higher levels of soluble ICAM-1 (P = 0.018) in the mln. These findings suggest a high energy diet imparted differential effects on mucosal cytokine and adipokine production in colonic and mesenteric tissue between obese-prone and obese-resistant SD rats. Elevated levels of pro-inflammatory and regulatory cytokines are evident as little as 2 weeks after the initiation of a high-energy diet.