bluuebunny/crossref_metadata_2025_split huggingface dataset:
IL-22 producing NK cells aid in the repair of damaged tissues (INC5P.326)
Authors: Aguilar Ethan , Sungur Can , Zamora Anthony , Murphy William 
Published: January 2023

Abstract
Natural killer (NK) cells are lymphocytes of the innate immune system that are associated with killing of virally infected and neoplastic cells. They have been shown to produce large amounts of pro-inflammatory cytokines, such as IFN-γ, and cytotoxic molecules, such as perforin and granzyme. Recent studies have identified unique subsets of natural killer cells, with distinct effector functions not limited to their classic cytotoxic function. These functions include producing a new array of cytokines, including IL-22, which has been shown to aid in tissue regeneration. Here, we provide evidence for a NK cell subset with the capacity to aid in the repair of tissue damage following viral infection as well as radiation damage. Using an in vitro co-culture system where we culture unstimulated NK cells with virally infected fibroblasts or irradiated epithelial cells, we were able to identify triggers that induce the expression of IL-22. In order to test the biological significance of IL-22 production by NK cells, we next used an in vivo murine cytomegalovirus (MCMV) model where we are able to deplete or adoptively transfer the IL-22 producing subset. With this model we are able show differences in liver pathology following MCMV infection, dependent on an IL-22 producing NK cell subset. A better understanding of IL-22 production by natural killer cells, and delineating a specific IL-22 producing subset could offer therapeutic benefits to patients who receive radiation therapy.
Thymic stromal lymphopoietin-mediated extramedullary hematopoiesis promotes allergic inflammation (INC5P.328)
Authors: Siracusa Mark , Saenz Steven , Tait Wojno Elia , Kim Brain , Osborne Lisa , Ruymann Kathryn , Hakonarson Hakon , Spergel Jonathan , Comeau Michael , Artis David 
Published: January 2023

Abstract
Extramedullary hematopoiesis (EMH) refers to the differentiation of hematopoietic stem cells (HSCs) into effector cells that occurs in compartments outside of the bone marrow. Previous studies linked pattern recognition receptor (PRR)-expressing HSCs, EMH and immune responses to microbial stimuli. However, whether EMH operates in broader immune contexts remains unknown. Here, we demonstrate a previously unrecognized role for thymic stromal lymphopoietin (TSLP) in promoting the population expansion of progenitor cells in the periphery and identify that TSLP-elicited progenitors differentiated into effector cells including macrophages and granulocytes; and that these cells selectively contributed to type 2 cytokine responses. The frequency of circulating progenitor cells was also increased in allergic patients with a gain-of-function polymorphism in TSLP, suggesting the TSLP-EMH pathway may operate in human disease. These data demonstrate that TSLP-induced EMH contributes to the development of Th2 cytokine-mediated inflammation and indicate that EMH is a conserved mechanism of innate immunity.
hBD-3 facilitates mDC-NK crosstalk (INC6P.348)
Authors: Judge Chelsey , Reyes Elane , Conry Sara , Sieg Scott , Feng Zhimin , Weinberg Aaron , Anthony Donald 
Published: January 2023

Abstract
mDC and NK cells are involved in the innate immune response to pathogen, and bidirectional mDC-NK interactions are well described. hBD-3, an anti-microbial peptide, is known to activate mDC via TLR1/2. Here we investigated the effects of hBD-3 on mDC-NK interactions. We observed hBD-3 to activate PBMC to secrete IFN-γ and kill K562 and HUH hepatoma target cells in an mDC and NK dependent fashion, and both TLR1/2 and CCR2 were involved. In purified mDC-NK coculture experiments we observed hBD-3 to facilitate mDC and NK activation, IFNγ secretion, and killer activity. Activity was variable among healthy subject samples, in some cases dependent on TLR1/2, and in other cases dependent on CCR2. We observed TLR1/2 and CCR2 expression on both mDC and NK cells, with variability in expression among individuals. These results implicate hBD-3 in ability to directly activate NK cells, mDC, and facilitate mDC-NK interactions. HBD3 may therefore act as a mediator of innate cell interactions that result in bridging of innate and adaptive immunity.
NK1R- and NK2R-dependent neuropeptide signaling regulates Type-1 immunity through activation of dendritic cells (INC6P.336)
Authors: Kaneumi Shun , Ohtake Junya , Masuko Kazutaka , Sumida Kentaro , Terada Satoshi , Kishikawa Takuto , Ohno Yousuke , Kita Toshiyuki , Kobayashi Hiroya , Kitamura Hidemitsu 
Published: January 2023

Abstract
Neurotransmitters such as Substance P (SP) and Neurokinin A (NKA) are widely distributed in both central and peripheral nervous system. Recently, we demonstrated that IFN-γ remarkably induced neurokinin-2 receptor (NK2R) expression in human dendritic cells (DCs). These findings strongly suggested that neuropeptide-signaling might regulate function of human DCs DC-mediated immune responses. In this study, we examined the effect of neuropeptide signaling on DC function. Human DCs generated from peripheral blood mononuclear cells (PBMCs) were treated with various type I cytokines or toll-like receptor ligands. Both neurokinin-1 receptor (NK1R) and NK2R mRNA expressions in DCs were significantly enhanced by poly I:C stimulation. The upregulation of NK1R and NK2R mRNA expressions was blocked in the presence of STAT-1 inhibitor. Surface expression levels of HLA-DR and costimulatory molecules on DCs, augmented by poly I:C, were modulated by NK1R or NK2R inhibitor. In addition, we found that blockade of NK1R- and NK2R-mediated signaling significantly suppressed various cytokine productions by antigen-specific CD4+ T cells stimulated with DCs. These findings indicate that NK1R- and NK2R-dependent neuropeptide signaling regulate Type-1 immunity through the activation of DCs, suggesting that such neuro-immune cross-talk through NK1R and NK2R signaling might be involved in various diseases with chronic inflammation caused by excessive Type-1-dominant immunity.
Multiplex analysis of secreted biomarkers from stimulated natural killer cells (INC6P.337)
Authors: Fuerstenberg Richard , Anderson Shaya , Brumbaugh Kathy , Schmidt Jane 
Published: January 2023

Abstract
NK (natural killer) cells are a component of the innate immune system that destroy compromised host cells, such as tumor cells or virus-infected cells, by recognizing such cells through a "missing self" mechanism. Over the past decade, NK cells have come under greater scrutiny as major influencers of the host’s immune response via secretion of cytokines, chemokines and other factors. In addition, studies of the mechanisms of NK cells as antitumor effector cells have advanced our understanding of potential cancer therapies. The traditional phenotypic definition of NK cells has been through cell surface markers using flow cytometric methods. The known ability of NK cells to secrete cytokines and other factors provides an opportunity to identify the secretory profile of NK cells under specific conditions as an alternative to flow cytometry. In this study, human NK cells were enriched from peripheral blood using a negative selection method, grown overnight in tissue culture flasks either unstimulated or stimulated with IL-12. The cell culture supernates from both conditions were assayed for 160 secreted biomarkers in large multiplex immunoassays using Luminex Screening Assays from R&D Systems, Inc. The markers that showed a positive response were identified for further analysis, thereby establishing a secretory profile for the study of the actions of NK cells.
Inflammasome responses to Legionella pneumophila in human macrophages (INM6P.414)
Authors: Casson Cierra , Yadav Anjana , Shin Sunny 
Published: January 2023

Abstract
Inflammasome activation is important for defense against intracellular pathogens because it induces cell death and regulates the secretion of IL-1 family cytokines. The canonical inflammasome activates caspase-1 to mediate cell death and cytokine release. In murine cells, caspase-11 also contributes to inflammasome activation. For Legionella pneumophila and other Gram-negative bacteria, caspase-11 controls IL-1α release and cell death and contributes to IL-1β and IL-18 secretion. However, humans do not encode caspase-11. Instead, humans encode caspase-4 and caspase-5, two closely related homologs of caspase-11. Therefore, we aim to determine the inflammasome components in human cells that respond to intracellular pathogens, using L. pneumophila, the causative agent of the severe pneumonia known as Legionnaires’ disease in humans, as a model. We find that both immortalized human monocytes and primary human monocyte-derived macrophages activate the inflammasome in a manner dependent on the type IV secretion system, a virulence factor the bacterium uses to establish its intracellular niche. While IL-1β secretion requires caspase-1 catalytic activity, IL-1α release and cell death are independent of caspase-1 activity, implicating caspase-4 or -5 in the response to pathogenic L. pneumophila. Our findings demonstrate that human caspase-4 and/or -5 may function similarly to mouse caspase-11 to facilitate inflammasome activation in human macrophages.
Single-cell stochastic propagation of IFNβ responses to viral infection (INM6P.420)
Authors: Fribourg Miguel , Patil Sonali , Ge Yongchao , Batish Mona , Tyagi Sanjay , Hayot Fernand , Sealfon Stuart 
Published: January 2023

Abstract
The immune responses to viral infection are highly stochastic processes which initiate in a limited number of cells that then propagate the immune signal. A main component of the response to virus infection entails the synthesis and secretion of type I interferons, among them IFNβ. The mechanism of propagation of the critical IFNβ response at the single cell level is an important unresolved question. Prior single cell studies of the IFNβ response propagation have often been limited by the absence of accurate, detailed single cell data on IFNβ mRNA production at early time points from the two copies of the intact IFNb genes present in each cell due to lack of assay sensitivity, late time measurements, and altered stoichiometry introduced by reporter assays. Here we used a recently developed single-cell single-molecule assay to study early induction and propagation of the IFNβ response in human primary monocyte-derived dendritic cells. We then applied model-based computational methods to extract the stochastic features that explain the observed cell-to-cell gene expression variability. Our results show that extracellular signaling plays a critical role in shaping the single-cell IFNβ response to viral infection. Furthermore, we also find that extracellular signaling contributes to cell-to-cell gene expression variability by introducing temporal dispersion. This mechanism may be of importance in shaping the immune response to viral infection.
The DNA sensor, cyclic GMP-AMP synthase (cGAS) is essential for induction of IFN beta during Chlamydia trachoma tis infection (INM6P.407)
Authors: Zhang Yugen , Yeruva Venkat , Marinov Anthony , Wyrick Priscilla , Lupashin Vladimir , Nagarajan Uma 
Published: January 2023

Abstract
IFNβ has been implicated as an effector of oviduct pathology resulting from genital Chlamydia infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFNβ expression during Chlamydia infection. Using siRNA technique and gene knock out fibroblasts, we determined that TREX-1, a host 3’exonuclease reduced IFNβ expression significantly during Chlamydia infection, implicating cytosolic DNA as a ligand for this response. STING is a signaling adaptor required for IFNβ expression and the DNA sensor, cGAS binds cytosolic DNA to generate the STING ligand cGAMP. We establish that cGAS is required for IFNβ expression during Chlamydia infection in multiple cell types. Infected cells knocked down for STING or cGAS alone failed to induce IFNβ. Interestingly, IFNβ expression was rescued by co-culture of cells depleted for either STING or cGAS suggesting that cGAMP produced in cGAS+STING- cells migrated to adjacent cells via gap junctions to bind to STING in cGAS-STING+ cells. Further, cGAS was localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane, indicating that chlamydial DNA is likely sensed outside the inclusion as infection progresses. These novel data provides evidence that cGAS-mediated-DNA sensing directs IFNβ expression during C.trachomatis infection and suggest that uninfected cells adjacent to infected cells can also induce IFNβ during in vivo infection, leading to oviduct pathology.
Apoptotic cells suppress TNF production by tissue resident macrophages through a CD73-dependent mechanism (INM7P.433)
Authors: Murphy Patrick , Elliott Michael 
Published: January 2023

Abstract
Apoptotic cells (AC) trigger an anti-inflammatory, pro-resolution phenotype in macrophages, but the mechanisms of this suppression and the consequences for resolution of tissue inflammation remain poorly understood. ACs also facilitate phagocyte recruitment through the release of the ‘find-me’ signal ATP. Interestingly, extracellular ATP can be hydrolyzed to adenosine (Ado), a potent inhibitor of macrophage inflammatory responses, via the ecto-enzymes CD39 (ATP→AMP) and CD73 (AMP→Ado). We therefore hypothesized that ATP released by AC could act to suppress macrophage inflammation via CD73-dependent generation of Ado. CD73 function in macrophages remains largely unexplored, and in fact we found that the commonly used macrophage models BMDM, RAW264.7, J774 and BV-2 express CD39 but not CD73. By contrast, we observed high levels of both CD39 and CD73 on murine CD11b+/F4/80hi resident peritoneal macrophages (RPM). In co-culture experiments, AC potently suppressed TNF production by LPS-treated WT but not Cd73-/-RPM. Inhibition of CD73 enzymatic activity, depletion of extracellular Ado and inhibition of adenosine receptors also abrogated the suppressive effects of AC on WT RPM. Together these studies reveal a novel role for CD73 in controlling the immunoregulatory actions of AC on tissue resident macrophages and further our understanding of apoptotic cell clearance as an important component of immune resolution.
Suppressed pulmonary innate immune responses after burn injury are regulated by mTOR (INM7P.429)
Authors: Malik Julia , Thurlow Lance , Gast Karli , Kartchner Laurel , Linz Brandon , Richardson Anthony , Maile Robert , Cairns Bruce 
Published: January 2023

Abstract
Severe trauma, such as burn injury, leads to metabolic and immune dysregulation often resulting in overwhelming bacterial pulmonary infection. Neutrophils accumulate in the lung and exhibit decreased oxidative burst late after injury, which may lead to susceptibility to infection. Substrates of the mammalian target of Rapamycin (mTOR), a key kinase in metabolic signaling, have been implicated in neutrophil trafficking and oxidative burst. We hypothesize that mTOR regulates the innate immune response after burn injury. Mice received a 20% total body surface area burn, and half of the burned mice were treated 5 times per week with Rapamycin to inhibit mTOR. After 14 days we assayed the bactericidal response of pulmonary neutrophils by quantifying dihydrorhodamine (DHR) oxidation. While mTOR inhibition did not impact the number of pulmonary neutrophils, oxidative burst was increased compared to untreated burn mice (DHR mean fluorescence intensity, untreated=56.42; Rapamycin=92.29; p&lt;0.05, n=5). Following pulmonary infection with Pseudomonas aeruginosa, neutrophil oxidative burst was decreased by Rapamycin treatment, corresponding to a 10-fold increase in bacterial load compared to untreated mice. These data suggest that mTOR plays a central role in regulating bactericidal oxidative burst by neutrophils after trauma. We propose that metabolic regulators may be relevant targets to improve innate immune function and consequently infection clearance in burn patients.
AMPK regulates innate immune signaling and viral control through ULK-1 dependent decrease of STING expression (INM7P.430)
Authors: Prantner Daniel , Perkins Darren , Vogel Stefanie 
Published: January 2023

Abstract
The host protein STING has been shown to be essential for recognition of both viral and intracellular bacterial pathogens, but its regulation remains unclear. Previously, we have described how mitochondrial membrane potential regulates STING-dependent IFN-β induction. The kinase activity of AMPK is controlled by cellular metabolism and intracellular calcium concentration. The goal of our study was to examine whether STING-dependent signaling is regulated by AMPK. Addition of an intracellular calcium chelator or an AMPK inhibitor suppressed IFN-β induction in mouse macrophages stimulated with the STING-dependent ligand DMXAA. Similarly, mouse embryonic fibroblasts (MEFS) lacking AMPK alpha failed to up-regulate IFN-β after DMXAA treatment. Additionally, these AMPK-/- MEFS failed to activate the IRF3-kinase TBK-1 and phosphorylate the transcription factor IRF3 after DMXAA treatment, confirming that STING-dependent signaling is AMPK-dependent. Loss of AMPK led to constitutive activation of the known STING negative regulator ULK-1 and a decrease in the basal level of STING protein. As a result, AMPK-/- MEFS exhibit impaired control of Vesicular Stomatitis Virus, a virus sensed by STING. ULK-1 has been shown to phosphorylate and degrade STING in the presence of dsDNA through the sensor protein cGAS. But in WT MEFS, STING was degraded in response to DMXAA in a cGAS- and ULK-1- independent manner, illustrating a novel regulatory pathway for this crucial signaling protein.
On the multiple role of NLRP3 on the pathogenesis of the viral infection (INM8P.444)
Authors: Gimenez Fernanda , Nuñez Gabriel , Rouse Bary 
Published: January 2023

Abstract
Herpes simplex 1 (HSV-1) infection of the eye can be a cause of blindness with lesions attributable to inflammatory events that include components of both adaptive and innate immunity. Several innate immune receptors are triggered by HSV-1 but it is unclear how such innate immune sensing relates to the subsequent control of virus and the ensuing inflammatory process. The present study focuses on the role of the inflammasome NLRP3, which is known to influence the outcome of inflammatory disease. Comparison of ocular and intradermal infection with HSV-1 in control and NLRP3 knockout mice revealed that mice lacking NLRP3 had significantly increased viral replication and delayed clearance together with increased neutrophil recruitment and proinflammatory chemokines and cytokines production compared to WT animals. Dramatic differences were also evident in the extent of skin zosteriform lesions in WT and NLRP3 KO mice. Since immunoinflammatory diseases are minimized in NLRP3 KO mice, we anticipated that SK lesions would diminish in KO mice, but instead they occurred earlier and were more severe. We demonstrate that the cellular and molecular events in SK in KO vs. WT animals were different especially in the balance of T cells type involvement and neutrophil participation. Our results indicate that NLRP3 can influence the outcome of a virus infection by affecting multiple steps in the pathogenesis.
Liver myeloid-derived suppressor cells inhibit genetically modified T cells in an IDO-dependent manner. (IRC5P.464)
Authors: Burga Rachel , Thorn Mitchell , Point Gary , Espat N. Joseph , Junghans Richard , Katz Steven 
Published: January 2023

Abstract
Introduction: The immunosuppressive environment in the liver limits the potency of immunotherapy for liver metastases (LM). Myeloid-derived suppressor cells (MDSC) expand in response to LM and mediate T cell suppression via indoleamine 2,3-dioxygenase (IDO). We hypothesized that IDO inhibition rescues genetically modified T cell (GMT) performance through blockade of MDSC-mediated suppression. Methods: Anti-CEA GMT were produced from activated murine splenocytes. C57BL/6 mice were injected via spleen with CEA+ MC38 tumor cells, and MDSC isolated by CD11b+ immunomagnetic beads. MDSC were evaluated by flow cytometry and ex vivo co-culture with CFSE-labeled GMT stimulated by CEA+ tumor, with and without IDO-blockade. Results: IDO expression was demonstrated in MDSC isolated from tumor-bearing (15.7-46.2%) and control livers (15.1%-37.7%). Increased IDO expression was dependent on STAT3 activity in the setting of LM, as blockade with cucurbitacin I (JSI-124) led to downregulated IDO expression on MDSC (48% vs. 21.5%, p=0.005). Liver MDSC suppressed GMT proliferation two-fold (p=0.004). IDO blockade with competitive inhibitor 1-methyltryptophan (1-MT) reversed GMT suppression, resulting in a five-fold increase in GMT division (p=0.016). Conclusions: Liver MDSC express IDO, and IDO represents a mechanism through which MDSC suppress anti-CEA GMT. Our results indicate that blockade of IDO activity may be a viable strategy for enhancing GMT targeted immunotherapy for liver metastases.
STAT3 is necessary for liver myeloid-derived suppressor cell PDL-1-dependent inhibition of genetically modified T cell function. (IRC5P.466)
Authors: Burga Rachel , Thorn Mitchell , Point Gary , Espat N. Joseph , Junghans Richard , Katz Steven 
Published: January 2023

Abstract
Introduction: Liver myeloid-derived suppressor cells (L-MDSC) can suppress genetically-modified T cells (GMT). STAT3 is a regulator of myeloid progenitors and promotes MDSC expansion. We hypothesized an increase in L-MDSC STAT3 activity in the setting of liver metastases (LM), contributing to suppression of GMT. Methods: LM were established in C57BL/6 mice by splenic injection of CEA+ MC38 tumor cells. L-MDSC were isolated with anti-CD11b immunomagnetic beads and flow cytometry used for phenotypic analysis. Ex vivo blockade of STAT3 was performed with cucurbitacin I (JSI-124) and Celastrol. Serum STAT3 and phospohorylated STAT3 (pSTAT3) levels were quantified by ELISA. Results: Both STAT3 and pSTAT3 levels were elevated (1.9-fold, p=0.005 and 1.6-fold, p=0.019) in serum of mice with established LM. L-MDSC isolated from LM-mice contained more pSTAT3 than normal mice (4.9% vs. 1.1%, p=0.007). Having previously demonstrated PDL-1-dependent suppression of anti-CEA GMT by L-MDSC, we asked if increased STAT3 activity contributed to immunosuppression. Blockade of STAT3 led to decreased PDL-1 expression on L-MDSC [52.7% vs. 9.9% (JSI-124 p=0.002) vs. 15.4% (Celastrol p=0.002)], likely hampering PDL-1 dependent immunosuppression. Conclusion: We demonstrated increased STAT3 and pSTAT3 in L-MDSC of mice with LM, and STAT3-dependent PDL-1 expression. Blockade of STAT3 is a potential clinical strategy for preventing L-MDSC PDL-1 expression and suppression of GMT via the PD-1/PDL-1 axis.
Perivascular leukocyte cluster: an essential structure for efficient effector T cell activation in the skin (IRC5P.467)
Authors: Kabashima Kenji , Natsuaki Yohei , Egawa Gyohei 
Published: January 2023

Abstract
It remains largely unclear how antigen-presenting cells encounter effector/memory T cells efficiently in the periphery. Here we used a murine contact hypersensitivity model to analyze antigen presentation in the skin. By antigen challenge, dendritic cells (DCs) formed clusters with effector T cells in dermal perivascular areas to promote in situ proliferation and activation of effector T cells in antigen- and integrin LFA-1-dependent manners. Intriguingly, DCs were attracted toward perivascular macrophages and DC clustering was abrogated by macrophage-depletion. IL-1α treatment induced CXCL2 production from dermal macrophages, and DC clustering was suppressed with blockade of either IL-1R or CXCR2. These findings suggest that dermal leukocyte cluster is an essential structure for elicitation of the acquired cutaneous immunity.