bluuebunny/crossref_metadata_2025 huggingface dataset:
Crosstalk between lungs and bone marrow during allergic inflammation (HYP7P.278)
Authors: Anderson Erik , Kobayashi Takao , Iijima Koji , Kephart Gail , Bartemes Kathleen , Chen Chien-Chang , Kita Hirohito
Published: January 2023

We used an established model of asthma that involves intranasal administration of the fungal extract of Alternaria. Upon lung exposure to Alternaria, IL-33 was released by epithelial cells within one hour, followed by increased production of IL-5 in the lungs. We also found elevated serum levels of IL-5 in response to Alternaria exposure, which reached a peak at 6 hours post Alternaria administration. After prolonged exposure to Alternaria over 6 days, a significant increase in mature eosinophils was observed in bone marrow. In addition, the serum from mice challenged with Alternaria was able to stimulate bone marrow cells to undergo eosinophil maturation in vitro. Intraperitoneal administration of IL-5-blocking antibody in vivo decreased eosinophil maturation in bone marrow in response to intranasal Alternaria below intranasal PBS baseline levels. We have shown the source of IL-5 is by type 2 innate lymphoid cells directly through IL-5 reporter mice, and indirectly through IL-5 serum levels in CD127 knockout mice. Taken together, this study illustrates that the type 2 innate immune responses in the lungs leads to enhanced production of eosinophils in the bone marrow through the increase in lung and blood levels of IL-5. Thus, distant organs communicate during allergic inflammation by using cytokines as mediators. This concept will aid in the identification of novel therapeutic strategies to alleviate symptoms of asthma and allergic diseases.
A role for beta-2 adrenergic receptors in the moderate aerobic exercise-modulated T regulatory cell function within a murine OVA-driven allergic asthmatic lung (HYP7P.287)
Authors: Dugger Kari , Watson Kacie , Sayner Sarah , Chastain Parker
Published: January 2023

We have shown moderate aerobic exercise can increase both the number and function of T regulatory (TREG) cells that suppress Th2 responses of an ovalbumin (OVA)-driven murine asthma model by an unknown mechanism. Exercise can communicate with TREG cells with the systemic release of catecholamines that bind to Beta-2-adrenergic receptors (β2-AR) and generate cAMP signals that can affect cell function. In these studies, we investigated a role for β2-AR in the exercise-mediated modulation of TREG cell function. For these studies, TREG cells (CD4+CD25+) were depleted from female BALB/cJ mice by i.v. injection of anti-CD25. Either 500,000 wt or β2-AR-/- TREG cells were adoptively transferred into the TREG-depleted BALB/cJ mice. Mice were sensitized/challenged with OVA to mimic allergic asthma pathogenesis. Mice were moderately exercised 45 min post-OVA challenge on a treadmill for 1hr 3x/wk for 4wks. Upon protocol completion, mice were examined for changes in TREG cell suppressive function and their resulting TREG cAMP levels. Our results show that exercise increases TREG function in vitro as compared with sedentary controls. However, the increase in TREG function of exercised TREG cells was abrogated in the absence of β2-ARs. We conclude that moderate aerobic exercise attenuates asthma-related airway inflammation via a mechanism that involves increased TREG cell function modulated by their expression of β2-ARs and subsequent production of cAMP.
Induction of regulatory alveolar macrophages in a biphasic model of house dust mite-induced asthma (HYP7P.279)
Authors: Bracken Sonali , Adami Alexander , Szczepanek Steven , Natarajan Prabitha , Guernsey Linda , Thrall Roger
Published: January 2023

Alveolar macrophages (AMs) are among the first cells to encounter inhaled allergens. While pro-inflammatory AMs are known to enhance the severity of asthma, the role of regulatory (IL-10+TGF-β+) AMs in suppression of allergic inflammation is not well defined. To determine whether suppression of asthma is associated with an increase in regulatory AMs, Vert-X IL-10-eGFP mice (n=5-6 per group) were challenged daily with 2 or 11 wks intranasal (i.n.) house dust mite (HDM). We have established that acute (2 wk) HDM exposure induces hallmarks of allergic inflammation whereas chronic (11 wk) exposure promotes tolerance to HDM. Tolerant (11 wk) mice exhibited a 2-fold increase in frequency and number of AMs when compared to diseased (2 wk) mice (p<0.001). AMs isolated from 11 wk mice produced higher amounts of IL-10 than 2 wk AMs, as demonstrated by a significant increase in frequency of GFP+ AMs and in mean fluorescence intensity of GFP (p<0.001). AMs from 11 wk mice also exhibited a 3-fold increase in TGF-β production (p<0.06) and a 2-fold decrease in MHC-II expression (p<0.05) when compared to AMs from 2 wk mice, indicating conversion to a regulatory phenotype. Moreover, the appearance of regulatory AMs in 11 wk mice was associated with a decrease in pro-inflammatory AM populations (p<0.01). These findings suggest that skewing of AMs towards a regulatory phenotype may be of critical importance to the generation of immunological tolerance against chronically inhaled allergens.
Increased severity of murine anaphylaxis in the absence of signaling through the C3a and C5a receptors (HYP7P.307)
Authors: Strait Richard , Koehl Joerg , Finkelman Fred
Published: January 2023

Because the complement activation products C3a and C5a can induce anaphylaxis by activating mast cells, basophils, macrophages, neutrophils and other cell types, we hypothesized that murine anaphylaxis mediated by a complement-activating antibody isotype would be less severe in mice deficient in the receptors for these anaphylatoxins or in the central complement component, C3, than in wild-type mice. Surprisingly, anaphylaxis mediated by the complement-activating isotype, IgG2a, was considerably more severe in mice deficient in C3 or in both C3aR and C5aR; with approximately twice the drop in body temperature in the mutant as in the wild-type mice on the same genetic background and ~75% mortality in the mutant mice vs. no deaths in the wild-type mice. Anaphylaxis mediated by the non-complement-activating isotypes, IgG1 and IgE, was also considerably more severe in C3aR/C5aR double-deficient mice than in wild-type mice. Furthermore, injecting histamine or platelet activating factor induced more severe shock in the receptor double-deficient than in wild-type mice. In contrast, mice solely deficient in C3aR or C5aR resembled wild-type mice, rather than the double-deficient or C3-deficient mice. These observations suggest that signaling through either the C3aR or C5aR protects against anaphylaxis by decreasing sensitivity to vasoactive mediators and raises the concern that chronic suppression of signaling through both receptors might increase the severity of allergic disorders.
Curcumin inhibits the development of food allergy by suppressing mast cell function in an NF-κB-dependent manner (HYP7P.308)
Authors: Mathias Clinton , Xing Wei , Kinney Shannon , Mazzamurro Jeffrey , Carlson Logan , Schneider Sallie
Published: January 2023

Mast cells play critical roles in the establishment of allergic responses to food antigens. We have previously shown that frequent ingestion of the curry spice curcumin, inhibits mast cell expansion and suppresses intestinal anaphylaxis in a murine model of ovalbumin (OVA)-induced food allergy. Here, we demonstrate that the protective effects of curcumin are conferred during the mast cell-dependent, OVA-challenge phase of the model and are mediated by inhibition of NF-κB activation. Exposure to curcumin during OVA-challenge, but not OVA-sensitization, results in inhibition of allergic diarrhea, intestinal mastocytosis and Th2 cytokine production. This suggests that while curcumin has limited inhibitory effects on Th2 sensitization, it has direct suppressive effects on mast cell homeostasis and function. Suppression of mast cell activation was also associated with inhibition of NF-κB in OVA-gavaged, curcumin-treated animals suggesting that the inhibitory effects of curcumin may be mediated in an NF-κB-dependent manner. In summary, our data demonstrate that curcumin inhibits allergic responses to food antigens by blocking NF-κB activation and suppression of mast cell-dependent responses.
Kinetics of inflammation and leukocyte recruitment in a murine model of asthma: eosinophils versus neutrophils (HYP7P.284)
Authors: Berdnikovs Sergejs , Abdala-Valencia Hiam
Published: January 2023

Murine model of ovalbumin-induced allergic inflammation remains to be a popular choice for approximating human acute asthma condition. Despite its widespread usage, exact timing and sequence of inflammatory events in this model remain obscure. We used a BALB/cJ mouse model with two intraperitoneal sensitizations and six intranasal challenges, and monitored inflammatory parameters at 6 and 24 hours after each ovalbumin challenge. Multicolor flow cytometry was used to assess recruitment dynamics of leukocytes in the digested lung tissue and bronchoalveolar lavage (BAL). Although neutrophil recruitment dominated the initial response to allergen, eosinophils started to appear almost simultaneously, gradually increasing their presence with each challenge. Interestingly, while still present in the lung tissue, neutrophils ceased mucosal surveillance (measured as BAL infiltrate) after three challenges, at which time point eosinophilia increased dramatically. This increase in BAL eosinophilia correlated with upregulation of integrin CD11c by CD11b+Siglec-F+Ly6Glow eosinophils in the tissue. Mediators such as IL-4, IL-6, IL-9 and IL-33 exhibited peak expression during the first three challenges. Other mediators, such as CCL11, IL-5, IL-13 and Slit2-roundabout signals were more prevalent later in the model. Collectively, our results demonstrate progression of events that govern presence of eosinophils versus neutrophils in the lung tissue and airways during allergic inflammation.
Kainate receptors are critical for Th2 immune responses in allergic disease (HYP7P.286)
Authors: Sturgill Jamie , Martin Rebecca , Elkovich Andrea , Conrad Daniel
Published: January 2023

Asthma is a complex disease in which many cell types play a pathogenic role. The asthmatic response requires T cells to make appropriate cytokines, B cells to make antigen specific IgE, mast cells to bind IgE and react upon subsequent antigen encounter, and lung cells to respond to MC derived mediators. To date there have been no pharmacologic interventions that can address all of these aspects of the disease, thus polytherapy is the norm. A serendipitous discovery made in our laboratory led to the pivotal finding that kainate receptors (KARs) are present in the immune system. In our hands, an antagonist of this receptor was shown to strongly inhibit polyclonal IgE production in in vitro human cultures. We have extended these studies to demonstrate that in the murine in vitro system, the KAR is also critical for the in vitro production of IgE and that signaling through this receptor also leads to increased ADAM10 activity, production of soluble CD23 release, and subsequent IgE production. Furthermore, we determined in vivo relevance for this receptor. Mice deficient in the KAR post antigen challenge produce significantly less antigen specific IgE and mucus production in a house dust mite (HDM) model of allergic asthma. Interestingly, these mice have normal Th1 immune response upon challenge with Ova/CFA. Thus, KARs may serve as an innovative specific pharmacological target for the treatment of asthma and other IgE mediated disease.
Complement mediated phagocytosis induces the activation of the NALP3 Inflammasome (INC5P.331)
Authors: Suresh Rahul , Sutterwala Fayyaz , Mosser David
Published: January 2023

The Complement System is an important component of innate immunity, whose activation has been associated with acute inflammation. Activation of complement leads to the generation of a Membrane Attack Complex (MAC) composed of C5b-C9. The function of the MAC has been primarily associated with target cell death though the polymerization and insertion of C9 on the surface of the activating particle. In this study we provide evidence that MAC assembled on the surface of complement activating particles, can be transferred to host macrophages during the process of phagocytosis. This “bystander activation” onto macrophages causes potassium efflux and ROS production, which can induce the activation of the NALP3 inflammasome. The activation of caspase-1 then induces the secretion of IL-1β and IL-18 to regulate both innate and adaptive immunity. We show that macrophages activated by MAC, can induce T cell differentiation into Th17 cells when used as APCs. This study demonstrates that the phagocytosis of complement-opsonized particles can induce inflammasome activation by a novel mechanism involving MAC mediated “bystander” activation of host macrophages. This work provides another mechanism whereby complement activation can lead to acute inflammation and it emphasizes the importance of a previously undescribed function of the MAC to activate inflammasomes on phagocytic macrophages.
Microglial CX3CR1 required for M2 polarization and recovery after intracerebral hemorrhage (INC5P.322)
Authors: Taylor Roslyn , Hammond Matthew , Ai Youxi , Sansing Lauren
Published: January 2023

Intracerebral hemorrhage (ICH) occurs when a blood vessel in the brain ruptures, resulting in the activation of microglia. Upon activation, microglia polarize into a pro-inflammatory, M1 phenotype or an M2 phenotype associated with repair. Microglia and monocyte subsets express the chemokine receptor CX3CR1. CX3CR1-null microglia have been shown to have dysregulated inflammation. We hypothesize that microglial CX3CR1-deficiency promotes dysregulated, M1 microglia that do not aid in repair. C57BL/6 (WT) and CX3CR1GFP/GFP (CX3CR1-null) mice were irradiated and reconstituted with bone marrow from WT mice (CD45.1) to generate bone marrow chimeras (CD45.1→WT or CD45.1→CX3CR1-null). ICH was modeled by injecting 20μl of blood into the right striatum. Behavioral outcomes were measured using gait analysis, cylinder test, and beam walking. Mice were sacrificed 14 days after ICH; perihematomal regions were harvested for intracellular staining. The CD45.1→CX3CR1-null mice display greater neurological deficit 14 days after ICH by cylinder test (p=0.002), beam walking (p=<0.001) and gait analysis (p=0.02). By intracellular staining, WT microglia have produce more for IL-10 (p=0.002) and IL-4 (p=0.0003) than CX3CR1-null microglia. CX3CR1-null microglia have less SIRPα (p=0.02) and higher CD36 expression (p=0.0004) compared to WT microglia. Our results suggest CX3CR1 signaling is necessary for microglia to transition from M1 to M2 and contributes to recovery after ICH.
Oxidative stress-induced pulmonary neutrophil recruitment is independent of conventional dendritic cells (INC5P.325)
Authors: Brand Jeffrey , Mathews Joel , Kasahara David , Neto Felippe , Shore Stephanie
Published: January 2023

Oxidative stress and airway neutrophilia are key features of some asthma phenotypes. Ozone (O3), a potent oxidant, induces pulmonary neutrophil recruitment which requires IL-17A+ γδ T cells. IL-23 is required for IL-17A production by T cells and conventional dendritic cells (cDC) are a potent source of IL-23. We therefore hypothesized that oxidative stress-induced IL-17A+ γδ T cells require IL-23 production from cDC. Wild type (WT), Tcrd-/- (lacking γδ T cells), and flt3l-/- (deficient in cDC) mice were exposed to air or 0.3 ppm O3 for 72 h. Bronchoalveolar lavage (BAL) was collected for cell differentials and lungs were processed for qPCR analysis. Lungs from mice which were not lavaged were used for flow cytometric analysis. In WT mice, O3 caused a 4 fold increase in the percentage of lung CD11c+/MHCII+/Siglec-F+ dendritic cells. IL-23 mRNA was also increased after O3 exposure. Flt3l-/- and WT mice had similar amounts of IL-23 and IL-17A mRNA and BAL neutrophils, thus indicating that cDC are not the source of IL-23 after such oxidative stress. However, IL-23 mRNA was reduced in Tcrd-/- mice which had fewer CD11c-/ CD11b+/F4/80+ myeloid cells. These cells are thus the potential source of IL-23. Altogether these data suggest oxidative stress induces IL-23 production by non-dendritic myeloid cells leading to IL-17A+ γδ T cell driven neutrophil recruitment.
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

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

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

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

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

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

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

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

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

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

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<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

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

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

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

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

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.
Helminth-conditioned dendritic cells prime CD4+ T cells to IL4 production in vivo (IRC5P.469)
Authors: Ronchese Franca , Connor Lisa , Tang Shiau Choot , Camberis Mali , Le Gros Graham
Published: January 2023

Dendritic cells are critical for the initiation of immune responses, however, their role in priming IL4-producing Th2 cells is controversial and remains an area of intense investigation. We found that after skin injection of the nematode parasite Nippostrongylus brasiliensis (Nb), a strong inducer of Th2 responses, Nb material is taken up by a population of dermal dendritic cells in lymph node, and that this population is necessary and sufficient for IL4 priming. Blocking the migration of dermal dendritic cells to the lymph node also prevented the priming of IL4-producing cells, and chicken ovalbumin (OVA)-loaded dDC from Nb-treated mice were sufficient to sensitize OVA-specific CD4+ T cells to IL4 production in vivo. Nb-loaded dermal dendritic cells were CD11b+, CD326neg, CD103neg, PDL2hi and IRF4+, and modestly upregulated expression of OX40L. However, OX40L and TSLP were not required for Th2 priming. Thus, our data suggest that the appropriate stimuli can induce DC to express the unique signals that direct CD4+ T cells to Th2 differentiation.
Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation (IRC5P.453)
Authors: Wu Yung-Hsuan , Kuo Wen-Chi , Lai Ming-Zong
Published: January 2023

Cellular FLICE-inhibitory protein (c-FLIP) is an inhibitor of caspase-8 and is required for macrophage survival. Recent studies have revealed a selective role of caspase-8 in noncanonical IL-1b production that is independent of caspase-1 or inflammasome. Here we demonstrated that c-FLIPL is an unexpected contributor to canonical inflammasome activation for the generation of caspase-1 and active IL-1b. Hemizygotic deletion of c-FLIP impaired ATP- and monosodium uric acid (MSU)-induced IL-1b production in macrophages primed through Toll-like receptors (TLRs). Decreased IL-1b expression was attributed to a reduced activation of caspase-1 in c-FLIP hemizygotic cells. In contrast, the production of TNF-a was not affected by downregulation in c-FLIP. c-FLIPL interacted with NLRP3 or procaspase-1. c-FLIP is required for the full NLRP3 inflammasome assembly, and c-FLIP is associated with NLRP3 inflammasome. c-FLIP downregulation also reduced AIM2 inflammasome activation. In contrast, c-FLIP inhibited SMAC mimetic-, FasL-, or Dectin-1-induced IL-1b generation that is caspase-8-mediated. Our results demonstrate a prominent role of c-FLIPL in the optimal activation of the NLRP3 and AIM2 inflammasomes, and suggest that c-FLIP could be a valid target for treatment of inflammatory diseases caused by overactivation of inflammasomes.
AMP-activated protein kinase suppresses autoimmune CNS disease by regulating M1 type macrophage-Th17 axis (IRC5P.474)
Authors: Giri Shailendra , Mangalam Ashutosh , Rattan Ramandeep , Denic Aleksandar , Viollet Benoit , Rodriguez Moses
Published: January 2023

AMPK is an energy-sensing metabolic switch implicating in various metabolic disorders. Here we report its new role in controlling the immunopathology of experimental autoimmune encephalitis by polarizing pro-inflammatory phenotype of Macrophage. AMPK alpha1 knockout (alpha1KO) developed severe EAE due to higher expression of IL17 with reduced IFNy and IL4. . AMPK alpha1KO also exhibited increased number of infiltrated mononuclear cells in CNS due to impaired blood brain barrier integrity. Macrophage from alpha1KO mice exhibited M1 type phenotype (IL10lowIL12/23high) with stronger capacity to induce allogenic and antigen-specific T-cell response and also heightened the encephalitogenic property of MOG35-55-primed CD4 T cells in B6 mice. Collectively, our data shows AMPK controls the clinical outcome of disease by regulating M1 phenotype-Th17 axis in animal model of MS.
TRIM22 enhances staurosporine-induced apoptosis in monocytes (IRM7P.492)
Authors: Shu Qiang , Chen Chi , Zhao Dongyan , Liu Xiwang , Chen QiXing , Fang XiangMing
Published: January 2023

Background TRIM22 was first identified as an interferon-induced gene, and constitutively expressed in peripheral blood leukocytes and lymphoid tissues, especially expressed at high levels in monocytes. Researches suggest that TRIM22 may have an impact on monocyte fate in immune response. However, the direct evidence of TRIM22 on monocyte apoptosis remains absent. Methods TRIM22 was constructed and transfected into a monocytic cell line THP-1 cell. Apoptosis of transfected cells was induced by STS and tested by flow cytometry. The caspase pathways involved in TRIM22-related apoptosis were illustrated by inhibitory experiments. Proapoptotic/antiapoptotic protein were measured by immunoblot. Results Overexpression of TRIM22 in THP-1 cells caused extensive apoptosis induced by STS; The activation of caspase-9 and caspase-3, and the release of cytochrome c were increased in TRIM22-overexpressing cells; Inhibition of caspase activation protected the cells from apoptosis augmented by TRIM22; Bak was upregulated in TRIM22 overexpressing monocytes. Conclusions Overexpression of TRIM22 in human acute monocytic leukemia cell THP-1 caused extensive apoptosis induced by STS both in absence and presence of lipopolysaccharide, which was associated with elevated cleavage of caspase-9 and caspase-3, and release of cytochrome c. Z-VAD-fmk, a pan caspase inhibitor, prevented TRIM22-induced apoptosis. Western blot found that Bak was upregulated in TRIM22 overexpressing monocytes.
Staphylococcal enterotoxin B (SEB)-induced miR-155 targets SOCS-1 to promote acute inflammatory lung injury. (IRM7P.481)
Authors: Rao Roshni , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Staphylococcal enterotoxin B (SEB) initiates an exaggerated inflammatory response characterized by immune cell infiltration, the release of inflammatory cytokines and tissue damage. However, the role of microRNAs (miR) associated with the damaging effects of SEB have not yet been delineated. In this study, we show that inhalation exposure to SEB results in acute inflammatory lung injury accompanied by altered miR expression profile in lung infiltrating cells. Amongst the miRNA that were significantly elevated, miR-155 is the most over-expressed. In contrast, miR-155-/- mice were protected from SEB-mediated inflammation. Interestingly, there was a positive correlation between miR-155 levels and IFN-γ secretion, suggesting a functional link between SEB-induced miR-155 and pro-inflammatory cytokine IFN-γ. Through the use of bioinformatics tools, suppressor of cytokine signaling -1 (SOCS1), a negative regulator of IFN-γ, was identified as a potential target of miR-155. While miR-155-/- mice displayed increased SOCS1, the overexpression of miR-155 led to its suppression, thereby enhancing IFN-γ levels. Additionally, the inhibition of miR-155 and IFN-γ resulted in restored SOCS1 expression. Our data, therefore, confirm an important role of miR-155 in facilitating SEB-mediated inflammation and proposes that miR-155 may be an important target in the treatment of acute lung inflammation.