bluuebunny/crossref_metadata_2025 huggingface dataset:
Developmental changes in Tcrb architecture are independent of thymocyte proliferation (HEM2P.248)
Authors: Majumder Kinjal , Koues Olivia , Rupp Levi , Kyle Katherine , Collins Patrick , Bassing Craig , Oltz Eugene
Published: January 2023

Assembly of the genetic components of the adaptive immune system by V(D)J recombination must be tightly controlled to prevent chromosomal translocations that cause genomic instability. This control is exerted at the level of tissue, stage and allele specificity by genetic and epigenetic mechanisms. Changes in chromosome conformation is one of the mechanisms of epigenetic control. In DN thymocytes, the Tcrb locus adopts a conformation which brings together distally located V, D and J gene segments by looping out intervening DNA to facilitate rearrangement. As the thymocyte proliferates and proceeds to the DP stage of development, the distal ends of Tcrb dissociate from one another. We applied high-throughput 4C-sequencing strategy to study the developmental changes in germline Tcrb looping at greater resolution. Characterization of the interactomes of multiple viewpoints revealed that: (i) the D-J region interacts with central Vβs in DP thymocytes despite losing distal Vβ contacts and (ii) the distal Vβs maintain a unique architecture in DP thymocytes. This conformation of Tcrb is maintained in DP thymocytes independent of proliferation, suggesting that lineage-specific factors can stabilize chromosome loops. Current studies are focused on deciphering the molecular mechanisms that regulate these developmental changes in Tcrb looping including structural protein binding and perturbation of chromatin barrier elements.
Cardiac myosin and the Th17 immunophenotype in human autoimmune myocarditis (HUM1P.252)
Authors: Cunningham Madeleine , Myers Jennifer , Cooper Leslie , Kem David , Stavrakis Stavros , Shevach Ethan , Fairweather DeLisa , Stoner Julie , Cox Carol
Published: January 2023

The immunopathogenesis of autoimmune myocarditis and dilated cardiomyopathy (DCM) in humans is unpredictable and pathogenic mechanisms undefined. The aim was to investigate potential autoimmune mechanisms in human autoimmune myocarditis and heart failure. Our evidence for a Th17 immunophenotype in human myocarditis/DCM(n=40) included reduced CD4+CD25+FoxP3+ Tregulatory cells(0.0006), elevated Th17 cells(0.0008) and increased Th17-promoting cytokines(IL-6 and TGF-beta). Th1(IFNγ+) cells were not significantly elevated(p=0.7184). IL-6(<0.0001), TGF-beta(0.0001), IL-23(0.0003), and GM-CSF+ T cells(0.0006) were significantly elevated. IL-17A+ cells were prevalent in biopsies from human myocarditis, non-recovery associated with persistence of IL-17A cytokines and heart failure associated with elevated Th17 cells in peripheral blood. To identify a potential mechanism to promote a Th17 pro-inflammatory pathogenesis in the heart, novel TLR ligands of human cardiac myosin (HCM) (peptides S2-16 and S2-28) promoted myocarditic PBMCs/CD14+ monocytes to produce Th17 cytokines(IL-23 p=0.0289; IL-6,TGF-beta p<0.0001). TLR ligand stimulation of PBMCs/ monocytes was abrogated by antibodies blocking TLR2, IL-6, IL-17A/23, and GM-CSF. Our translational study suggested human myocarditis/DCM may be controlled by a Th17 phenotype including reduced Treg, elevated Th17 cells, and pro-inflammatory and pro-fibrotic Th17 cytokines triggered by cardiac myosin TLR ligands potentially released from heart.
PARP inhibitor, olaparib ameliorates acute lung injury by modulating oxidative stress and NF-κB mediated inflammatory response in mice (HUM1P.265)
Authors: Naura Amarjit , Kapoor Kunal , Singla Esha , Sahu Bijayani
Published: January 2023

We have previously shown that poly (ADP-ribose) polymerase (PARP)-1 gene deletion in mice provide robust protection against lung inflammation in the context of asthma and acute lung injury. Olaparib is a potent new generation PARP inhibitor that has been approved for human testing in cancer patients. The present work was designed to evaluate its beneficial potential against LPS-induced acute lung injury upon intra-tracheal administration of the endotoxin in mice. Administration of olaparib at different doses, 30 min after LPS treatment showed that single injection of the drug effectively reduced the total number of inflammatory cells particularly neutrophils in the lungs. This was associated with reduced pulmonary edema as the total protein content in bronchoalveolar fluid was found to be decreased substantially. The drug restored the LPS mediated oxidative stress toward normal in lungs as assessed by measuring malondialdehyde and GSH levels. Finally, RT-PCR data revealed that olaparib downregulates the LPS induced expression of NF-κB dependent genes namely TNF-α, IL-1β and VCAM-1 in the lungs without altering the expression of total p65NF-κB. Overall, the data suggests that olaparib possesses a strong therapeutic potential against LPS induced lung injury in mice. Given the fact that olaparib is approved by FDA for human testing, our findings can pave the way for testing of the drug on humans inflicted with acute lung injury.
Th1, Th2, Th17 and Treg levels in umbilical cord blood of preeclamptic women. (HUM1P.300)
Authors: Soto-Vega Elena , Solleiro-Villavicencio Helena , Vargas-Rojas Maria
Published: January 2023

Preeclampsia is one of the major causes of maternal and neonatal mortality. The immune system during pregnancy has to maintain the tolerance to the fetus, thus changes in the cytokine balance may result in a disturbed pregnancy. T helper cells play an important role in the modulation of the immune system. Many studies have been performed to study the T cell composition in different compartments during pregnancy, although this is the first study in which the levels of Th1, Th2, Th17 and Treg cells are evaluated in umbilical cord blood from normal and preeclamptic women. We did not find any difference in Th1 and Th17 levels between groups, but Th2 and Treg levels were significantly diminished in preeclamptic women. Even if we found no differences between pro-inflammatory profiles, their counterparts Th2 and Treg are diminished, suggesting that the balance is broken encouraging the development of an exacerbated inflammatory response. Our results show that there is a shift, not only in the Th1/Th2, but also in the Th17/Treg balance, favoring skewness towards a proinflammatory status in preeclampsia, so the innate and adaptative immune system are involved in the development of an exaggerated maternal systemic inflammation.
Immune signatures of Alzheimer’s disease: profiles of neutrophils. (HUM1P.301)
Authors: LE PAGE Aurélie , Lamoureux Julie , Bourgade Karine , Frost Eric , Dupuis Gilles , Fulop Tamas
Published: January 2023

Alzheimer's disease (AD) is the most common form of dementia. Amyloïd plaques accumulation and microbial infections have been proposed as an important components of AD aetiology. Defects in amyloïd-β clearance by monocyte/macrophage/microglia, imply that the immune system may participate in development and progression of AD. Phenotype and functional analysis of polymorphonuclear neutrophils (PMN) in peripheral blood were performed by flow cytometry. Our cohort of patients comprised amnestic Mild Cognitive Impairment (aMCI) (n = 10), mild stage AD (mAD) (n = 10) and healthy elderly controls (n = 10). aMCI patients had memory impairment but no functional disorders. However, these patients have a 50% chance of developing AD within 3 years following the diagnostic. PMN number was unchanged between the three groups. In contrast, the CD177+ population was significantly increased in mAD patients (p < 0.05). Phagocytosis of opsonized bacteria was less in aMCI and AD patients than controls. This observation was associated in aMCI with a decrease in CD33 (siglec 3) and complement receptor for C5A expression. Decreased expression of CD14 (a pattern recognition receptor), CD15 (carbohydrate adhesion molecule) and CD16 (FcγRIII) expression was found in mAD patients. Our results describe for the first time alterations of neutrophils in aMCI patients. PMN analysis may provide a useful tool in early identification of AD patients.
Peripheral inflammation reflects cortical Aβ load in patients with amnestic mild cognitive impairment (HUM1P.302)
Authors: Stowe Ann , Ireland Sara , Ortega Sterling , Chen Ding , Huebringer Ryan , Cullum C , Greenberg Benjamin , Monson Nancy , Zhang Rong
Published: January 2023

The National Plan to address Alzheimer’s disease (AD) underscores the urgent need for the prevention and treatment of AD, especially with a new patient diagnosed in the U.S. every 67 seconds. We recently published data showing increased levels of central nervous system inflammation in patients with amnestic mild cognitive impairment (aMCI), a precursor disease to Alzheimer’s disease (AD). We found that lymphocytes and pro-inflammatory cytokines in the cerebrospinal fluid (CSF) of aMCI patients were elevated similar to or exceeding those found in patients with multiple sclerosis. To determine any potential contribution of central and peripheral inflammation to AD pathology, we analyzed within-patient leukocyte populations and correlated these measures with brain amyloid (Aβ) burden, quantified by PET imaging at time of CSF and blood collection. Fifteen patients (55-76 yr old) and/or their study partners signed written informed consent. Age significantly increased plasmablast representation and decreased NK T cell populations in the CSF, but this did not reflect brain Aβ levels. Independent of patient age, higher cortical Aβ levels were significantly correlated to increased CD8 T cell and decreased CD4 T cell populations in the peripheral blood. Additionally, peripheral memory B cells increased with Aβ burden but not with age. These data suggest a potential role of the adaptive immune system in the development of cortical Aβ burden at a preclincial stage of AD.
Low ambient oxygen prevents atherosclerosis (HUM1P.254)
Authors: Kang Ju-Gyeong , Sung Ho Joong , Amar Marcelo , Remaley Alan , Springer Danielle , Kwon Jaeyul , Chen Jichun , Wang Pingyuan , Hwang Paul
Published: January 2023

Large epidemiolgic studies show that living at high altitude, which decreases oxygen exposure, is associated with reduced cardiovascular and cancer mortality. However, some experimental data show that acute hypoxia exposure can increase atherosclerosis by worsening inflammation and blood lipid profiles. We sought to address these disparate observations by chronically controlling the oxygen exposure of ApoE-/- mice and examining its effect on atherosclerosis. Herein, we report that reducing oxygen exposure by 50% caused a marked decrease in aortic atherosclerosis, associated with the inhibition of pro-inflammatory cytokines in circulation and increased anti-inflammatory IL10 levels in aortic tissue. We further show that the expression of IL10, well established to prevent atherosclerosis, was induced by low oxygen in a hypoxia inducible factor-1α (HIF1 α) dependent manner. We propose that this anti-inflammatory mechanism may contribute to the lower incidence of ischemic heart disease in populations living at high altitudes and suggest a potential therapeutic pathway for disease prevention.
Increased sensitivity of peripheral CD27+ memory B cells to Fas-induced apoptosis contributes to their disappearance in cirrhosis (HUM1P.309)
Authors: Kaplan David , Chang Li-Yuan , Tanoue Shiroh , Li Yonghai
Published: January 2023

Liver cirrhosis is associated with a 70% decrease of peripheral CD27+ memory B cells. The mechanism for CD27+ B-cell loss in cirrhosis remains unknown. We hypothesize that increased sensitivity to fas-mediated apoptosis leads to reduced peripheral CD27+ B-cells survival. Methods: Ex vivo B-cell fas-receptor (CD95) expression and in vitro measurement of spontaneous and fas-induced apoptosis (annexin V/PI) in 11 cirrhotic (CIR) and 11 healthy donors (HD). We found that expression of CD95 was significantly higher in CD27+ B cells than in CD27- B-cells in both CIR and HD (CIR, 50.2% versus 3.8%, p< 0.0001). The ex vivo percentage and gMFI of CD95 on CD27+ B-cells was significantly higher in CIR than HD (CIR 50.2% versus HD 34.6%; CIR 624 versus HD 337, p<0.02). Surprisingly, ex vivo CD27+ B cells were resistant to fas-mediated apoptosis in both CIR and HD. Anti-CD40 activation of sort-purified CD27+ and CD27- B cells resulted in upregulation of CD95 (1.05-1.17-log-fold) to similar degrees in CIR and HD. Addition of agonist anti-fas resulted in significantly greater apoptosis in cirrhotic CD27+ B cells relative to HD (CIR 33.3% versus HD 18.5%, p=0.05). Conclusion: These data suggest that peripheral CD27+ memory B-cell in cirrhosis exhibit increased sensitivity to fas-induced apoptosis in an activation-dependent manner. This sensitivity may be associated with their reduced frequency in cirrhosis and possibly with resultant derangements of humoral immunity seen in cirrhosis.
Endoscopy and FACS analysis in classic and humanized murine models of GVHD (HUM1P.316)
Authors: Beal Dominic , Graham Sean , Van Dam Brett , Lyng Gregory , Sonis Stephen
Published: January 2023

It is perplexing that standard murine GVHD model endpoints of weight loss (WL) and composite clinical changes (CCC) often fail to respond to standard forms of therapy (tacrolimus and anti-p40 monoclonal antibody). To improve on the translatability of GVHD models we evaluated serial endoscopy as a means to directly assess injury and serial blood counts to predict onset of severe disease. Two models were used. CD3+ cells were magnetically depleted from bone marrow harvested from male Balb/c mice. Varying numbers of (depleted) bone marrow and splenocytes were then injected IV into lethally irradiated (8Gy) C57Bl/6 hosts. Serial endoscopy performed after GVHD onset (~2 weeks post transfer) demonstrated changes which strongly correlated with WL and CCC (R2=-0.84 and 0.79). Next we induced GVHD in a humanized model by the adoptive transfer of human PBMCs into severely immunocompromised mice (NSG). Serial blood draws demonstrated a strong correlation between peripheral blood human T cell (PBTC) numbers and WL and CCC (R2=-0.85 and 0.67 on day 42) suggesting that assessment of PBTC numbers may be applicable to the evaluation of therapies directly targeting these cell types. Day 7 human PBTC counts predicted final WL and CCC scores with high correlation (R2=-0.86 and 0.90) indicating that this metric can be used to stratify animals into groups prior to the onset of observable disease and treatment.
A transgenic mouse model correlates anomalous immune dynamics in human sickle cell disease (HUM1P.256)
Authors: Pryor Julianna , Collins Brittany , Joshi Sunil
Published: January 2023

Sickle cell disease, SCD, is an inherited blood disorder characterized by hardened, sticky C-shaped RBCs is a major cause of morbidity and mortality. It is the most prevalent genetic disease affecting 1/500 African Americans and 1/36000 Hispanics Americans, most residing in southeastern Virginia. Severe, frequent infections occur as a result of immune dysregulation associated with disease progression, impairing both innate and adaptive immunity. APCs such as DCs, B cells and macrophages express MHC II bind antigen that is recognized by TCR expressed on CD4+ Th cells. Thus, the interaction between receptor-ligand clusters on Th and APCs led to the activation of naïve CD4+ Th cells, stimulating the release of cytokines that influenced the activity of many cell types, including the NKT, CD8+ CTLs and the APCs that activated them. A “humanized” sickle mouse model of SCD was created by generating bone marrow chimeras using sickling mice donors and Pepboy recipients. We recently found that the transition from SPF to ‘dirty’, conventional, housing significantly dysregulated the immune system. These changes were characterized by a hyper-inflammatory state with the activation of innate-immune response, upregulation of TLR4, and loss of antigen-presentation machinery, loss of MHC II expression, with disease progression. Results indicate the loss of MHC II expression on APCs from various tissue of sickle mouse with the disease progression.
The ETS family transcription factors ETV5 and PU.1 co-regulate Th9 cell differentiation (HYP2P.334)
Authors: Hufford Matthew , Koh Byunghee , Pham Duy , Kaplan Mark
Published: January 2023

T helper 9 (Th9) cells, a CD4+ T cell subset characterized by their robust production of interleukin (IL)-9, can promote allergic inflammation in mice and are associated with atopic disease in humans. We have previously demonstrated that the E26 transformation-specific (ETS)-family transcription factor PU.1 is integral in Th9 mediated IL-9 production and is the only transcription factor, to date, that induces IL-9 production in another Th subset. Because conditional PU.1 deletion in T cells does not completely eliminate Th9 mediated IL-9 production, we investigated whether other ETS proteins were important transcription factors in Th9 cell development. Herein, we demonstrate that ectopic expression of ETS Variant 5 (ETV5) can also enhance Th9 cell differentiation and promote IL-9 production in Th2 differentiated cells. Conversely, conditional deletion of ETV5 impaired IL-9 production in Th9 cultures while promoting Th2 cytokine production. Unlike PU.1 that binds the Il9 promoter, ETV5 preferentially binds potential enhancer regions of Il9, and recruits distinct histone acetyltransferases from those recruited by PU.1. Finally, conditional deficiencies of both ETV5 and PU.1 in T cells effectively eliminated IL-9 production in Th9 cultured cells. The effects of deficiency in both factors are being tested in a model of allergic pulmonary inflammation. Collectively, these data indicate ETV5 and PU.1 co-regulate Th9 cell differentiation in partially non-redundant mechanisms.
miR-23~27~24 clusters restrict Th2 immunity and associated immunopathology during airway allergic reaction (HYP2P.335)
Authors: Wu Cheng-Jang , Cho Sunglim , Yasuda Tomoharu , Cruz Leilani , Lin Ling-Li , Miller Marina , Lee Hyang-Mi , Broide David , Rajewsky Klaus , Lu Li-Fan
Published: January 2023

MiRNAs (miRNAs) are important regulators in T cell differentiation and function. Here we show miR-23~27~24 clusters play a pivotal role in controlling type II immunity. Specifically, under Th2 polarizing condition, T cells with overexpression of miR-23~27~24 clusters exhibited reduced IL-4 secretion whereas T cells devoid of miR-23~27~24 clusters produced elevated amounts of IL-4. Further mechanistic studies revealed miR-24 and miR-27 could repress Gata3, a key regulator of Th2 cell differentiation through an indirect and a direct manner, respectively. Finally, by using an OVA-induced asthma model, we have shown that mice with T cell-specific ablation of miR-23~27~24 clusters developed a more severe airway inflammation characterized by increased IL-4 secretion, lung eosinophil infiltration, mucin production and serum IgE levels. Taken together, our studies identify a miRNA family with important biological function particularly in controlling Th2 immunity and suggest that a tight regulation of this miRNA family is required to maintain optimal effector T cell function and to prevent aberrant immune responses.
Deciphering ther ole of CD8+ T cells in allergic inflammation (HYP2P.339)
Authors: Anderson Erik , Kobayashi Takao , Iijima Koji , Kephart Gail , Chen Chien-Chang , Kita Hirohito
Published: January 2023

CD8+ T cells represent a relatively under studied cell type in allergic lung inflammation, despite their presence in the mucosal tissues. This is because CD8+ T cells are normally associated with a Th1 response as opposed to Th2 or Th17 responses, as are the most cases with allergic inflammation. However, recent papers have shown the production of Th2 and Th17 cytokines in these cells, yet knowledge of their physiological role in allergic inflammation is incomplete. The presence of IL-17 in asthma patients contributes to a severe, steroid resistant phenotype. Therefore, determining contributors of IL-17 and their physiological role is paramount to understanding the disease. Our preliminary data which suggests memory Tc17 cells are generated in CD4-/- mice when sensitized intranasally to ovalbumin with IL-33. We believe Tc17 cells are responsible for the inflammation because the inflamed lung tissue contained elevated IL-17, and stimulated T cells from inflamed tissue produced elevated levels of IL-17 as well. Additionally, we were unable to induce inflammation in Rag-/- mice or TCRβ-/- mice, which pinpoints the cell responsible to CD8+ T cells in CD4-/- mice. Using these mice to study an exaggerated phenotype will allow us to more easily detect the contributions that happen to a lesser degree in wild type animals. This work create a more complete picture of the disease environment and lead to therapies which can account for the contribution of this cell type.
The role of Notch signaling in T follicular helper cells during allergic immunity (HYP2P.326)
Authors: Dell Aringa Mark , Reinhardt Richard
Published: January 2023

The generation of Immunglobulin (Ig) E and high-affinity IgG1 by B cells is a hallmark of allergic inflammation. These hallmarks are heavily reliant on interactions with T follicular helper (Tfh) cells. A critical cytokine provided by Tfh cells to the germinal center B cells is Interleukin-4 (IL-4). The mechanisms controlling IL-4 production by Tfh cells remain unknown. Given that Notch signaling is required for the differentiation of Tfh cells and it is known to influence cytokine production in T cells, we hypothesized that Notch also plays an important role in regulating the function of Tfh cells. Here, we use intestinal helminth infection to characterize whether persistent Notch signaling is required to maintain Tfh cell fate and function during allergic immunity. Genetic deletion of Notch receptors in infected mice results in reduced IL-4 production by Tfh and Th2 cells. Conversely, over-expression of Notch signaling leads to increased IL-4 production by Tfh cells. However, increased Notch signaling also appears to alter Tfh cell fate and phenotype. In support, chemical inhibition of Notch signaling after Tfh differentiation results in decreased expression of the Tfh chemokine receptor, CXCR5 among IL-4 producing T cells in the lymph node. These findings suggest that Notch signaling is not only important for Tfh differentiation, but that strength of Notch signaling is important for regulating Tfh cell fate, function, and maintenance.
The effects of thermal Injury and sepsis on the production of leukocyte microvesicles (INC1P.360)
Authors: Babcock George , Domenico Karen , Robinson Chad
Published: January 2023

Microvesicles (MV) are membranous extra cellular vesicles derived from various cells following cell activation or apoptosis. MVs are typically defined as being from 0.1 to 1 µ in size. There have been several reports of biologically active MVs in the blood in certain clinical conditions. The goal of this study was to examine the effects of thermal injury plus sepsis on the number and type of MVs released from leukocytes. C57BL/J mice received 15% total body surface area burn followed by infection with Pseudomonas aeruginosa. At times post injury the blood and bone marrow were examined for MV expressing CD11b, CD19, CD34, CD41, and CD117 using flow cytometry. Generally,the absolute number of MV in the blood dropped following, however, the percentage of the various cell types changed. The percentage of CD11b, 34, and 42 increase slightly following burns but significantly following burn/infection.The absolute numbers of all MV dropped with the exception of CD 19 which increased both in percentage and absolute number in the burn group but not the burned/infected group. The absolute number of CD117+ MV increased significantly in the burn/infected group.The bone marrow remained relative stable with a slight increase in CD117+ MV. These data indicate that the pattern of MVs production changes following burn injury and burn infection. Because the number of CD34+ MV did not increase it would appear that the large increase in CD117+ MV was due to increase in common myeloid progenitors.
Antigen archiving and management by lymphatic endothelial cells. (INC1P.363)
Authors: Tamburini Beth , Burchill Matthew , Kedl Ross
Published: January 2023

Antigen derived from virus infections such as influenza and Vesicular Stomatitis Virus (VSV) can persist well beyond the natural rise and fall of the adaptive immune response against the infection. Antigen can similarly persist following both viral Vaccinia challenge and subunit vaccination. Surprisingly, persisting antigen from either Vaccinia or subunit vaccination is captured and maintained by a stromal cell subset: Lymphatic Endothelial Cells (LECs). The duration of antigen persistence is directly correlated with antigen dose and pattern recognition receptor (PRR) activation. PRR induced inflammation leads to LEC proliferation followed by antigen persistence in the lymph node. The coupling of LEC proliferation and antigen capture identifies a novel mechanism by which the secondary lymphoid stroma stores, or “archives”, antigens for extended periods of time after antigen challenge. Consistent with the idea that viral antigen persistence impacts the function of circulating memory T cells, we find that vaccine elicited antigen archiving on LECs has a significant impact on the fate of circulating memory T cells. As such, antigen archiving on LECs leads to increased production of interferon gamma and interleukin 2 by antigen specific CD8 T cells and protection against secondary infection. These findings may broadly impact our understanding of how lymph node stroma, specifically LECs, are involved in i) managing antigen and ii) protecting against re-infection.
Co-inhibitory factor induced NK cell exhaustion and immune escape meachanism in HBV-related hepatocellular carcinoma (INC1P.400)
Authors: sun cheng , Sun Rui , Tian Zhigang
Published: January 2023

The effectiveness of cancer immunotherapies targeting co-inhibitory molecules CTLA-4 and PD-1 have fully validated that reversal of tumor immune tolerance is an important new pathway for the treatment of tumor. Recent evidence shows that NK cell exhaustion in HBV related HCC patients. However, the underlying mechanism remains unclear. In our study, we found that the expression of NKG2A on NK cells from peripheral blood and different tissue regions of HCC was significantly increased, which then impaired NK cell-mediated cytotoxic capacity and interferon-γ production. We further found the same inhibition and exhaustion of NK cell in the different disease-progressing groups of HCC patients. Furthermore, we confirmed that IL-10 amounts in the immune microenvironment of hepatitis and HCC were highly related to the development of HCC. This study will help to understand the mechanism of immune tolerance of NK cells induced by HCC and verify the potential value of NKG2A as a novel intervention target for cancer immunotherapy.
Allergen-experienced ILC2s display memory-like functions and enhance type 2 lung inflammation (INC2P.410)
Authors: Martinez-Gonzalez Itziar , Mathä Laura , Takei Fumio
Published: January 2023

The ability of lymphocytes to remember their previous encounter with antigens and rapidly and vigorously respond to re-exposure to the same antigen is a key feature of the immune system. Whereas this immunological memory is thought to be antigen specific, here we show that group 2 innate lymphoid cells (ILC2s), which play a key role in type 2 immunity, display memory-like functions. Allergen-stimulated lung ILC2s rapidly proliferate and secrete cytokines that induce allergic lung inflammation, followed by a contraction phase, in which the inflammation is resolved. Some of the allergen-experienced ILC2s persist and vigorously react to an unrelated allergen, mediating a stronger allergic lung inflammation than naive ILC2s. Gene expression profiles of memory ILC2s are different from those of naive and effector ILC2s and display a gene signature of memory T cells. Antigen non-specific memory ILC2s may make individuals sensitive to a broad range of allergens.
Proteomic analysis of macrophages activated with salmonella lipopolysaccharide (INC2P.413)
Authors: makkar sarbjeet , Liyanage rohana , Lay Jackson , Narayan Rath
Published: January 2023

Macrophages play pivotal role in immunity. They are activated by pathogen derived molecules such as lipopolysaccharides (LPS) which triggers the production of various proteins that drive and resolve inflammation. There are many studies on the effect of LPS at the genome level but the knowledge of its proteomic effects is limited. To determine the proteomic effects of LPS, we used Stable Isotope Labelling of Amino acids in Cell culture (SILAC) with a chicken macrophage cell line HTC, stimulated with Salmonella typhimurium LPS. The heavy (H) (13C-lysine) labelled cells were treated with LPS and the regular or light lysine (L) grown cells treated as controls. After 16h of stimulation, the proteins were extracted, quantified, and an equal amount from both H and L cells mixed. The protein mixtures were subjected to reduction/alkylation, and trypsin digestion followed by high throughput liquid chromatography/ tandem mass spectrometry. We quantified 133 proteins using Skyline software, out of which 13 were upregulated and 11 down regulated. Some of the upregulated proteins included HSP 70 and 90, DNA helicases, prostaglandin D synthase and CCL5 ligand. The down regulated proteins were ubiquitin, proteasome, and proteins related to protease activity. In conclusion this study suggests that activation of macrophages with LPS results in differential expressions of proteins involved in cytoskeleton remodeling, cell migration and cell signaling pathways involved in immune response.
Ethyl pyruvate, a modulator of dendritic cell activation and survival (INM1P.437)
Authors: Chakhtoura Marita , Chain Robert , Varghese Linda , Gallucci Stefania
Published: January 2023

Danger Signals induce dendritic cells (DCs) to mature and present antigen in a pro-inflammatory context. When remaining immature however, DCs confer tolerance. Hence the need for new protocols inducing tolerogenic DCs. We assessed the effects of ethyl pyruvate (EP), ester derivative of pyruvate with anti-inflammatory properties, on DC responses. We found that EP (10mM) abolished pro-inflammatory cytokine induction (IL-12, TNFa, IL-6) by the TLR ligand and strong DC activator LPS in bone marrow-derived dendritic cells (BMDCs) at 8, 24, 48 and 72h post-stimulation. Furthermore, EP inhibited the IFN-I response and the anti-inflammatory cytokine IL-10 induced by LPS stimulation. Determining the effects of EP on BMDC viability with a dose titration response showed that EP (10mM) strongly inhibited in vitro DC activation without inducing death. Moreover, EP could prevent LPS-induced killing that normally occurs 72h post-stimulation. EP also decreased the up-regulation of BMDC surface MHC-II and co-stimulatory molecules, suggesting a role in inhibiting antigen presentation. Investigating the mechanism of action, EP decreased Erk and JNK phosphorylation but not p38 phosphorylation. Our results indicate that EP inhibits most of the biological responses of DCs to LPS. We are currently examining further the molecular mechanisms of EP’s action in BMDCs to highlight novel targets to inhibit DC activation.
Estrogen receptor alpha is required for macrophage iron sequestration and homeostasis in female mice (INM1P.438)
Authors: Fluitt Amy , Hevener Andrea
Published: January 2023

Sexual dimorphisms in immunity are known to confer protection against infections at the cost of heightened risk for autoimmunity in women. The female sex hormone, estradiol, can modulate both pro/anti-inflammatory macrophage signals and the mechanisms of action remain unclear. Iron sequestration by macrophages is fundamentally important to limit bacterial infection, and since estradiol is thought to influence iron metabolism in females, we asked whether ERα impacts the innate immune response by regulating macrophage iron metabolism. To address this question, we crossed floxed Esr1 (fl/fl control) and LysM-Cre transgenic animals to generate myeloid-specific ERα knockout (MACER) mice. We studied the effect of ERα deletion on iron homeostasis upon endotoxin treatment in female MACER and fl/fl conrols. MACER mice had increased serum iron (92%; p=0.01) compared to fl/fl under basal conditions. As expected, endotoxin treatment reduced serum iron levels in fl/fl controls (41%, p=.008), however there was no change in serum iron in MACER mice. Consistent with impaired macrophage iron homeostasis, expression of genes associated with iron absorption (TfR, Slc11a2, Cybrd1), efflux (Fpn), and storage (Ftl) were significantly altered in female MACER mice compared to fl/fl controls. Together, these data suggest that ERα regulates macrophage iron metabolism in females, is required for systemic iron homeostasis, and may play a previously unrecognized role in female innate immunity.
The NF-κB p65 and p50 homodimer cooperate with pSTAT1 to synergistically activate iNOS transcription in cancer cells and myeloid cells (INM1P.440)
Authors: Simon Priscilla , Sharman Sarah , Lu Chunwan , Yang Dafeng , Paschall Amy , Liu Kebin
Published: January 2023

Inducible nitric oxide synthase (iNOS) metabolizes L-arginine to produce NO, which was originally identified in myeloid cells as a host defense mechanism against pathogens. However, recent studies have revealed that iNOS is often induced in both tumor and myeloid cells in the tumor microenvironment. Compelling experimental data have shown that iNOS can promote or suppress tumor development in certain cellular conditions. The molecular mechanisms underlying this contrasting function of iNOS is unknown. Because iNOS is often induced by inflammatory signals, it is likely that the opposite functions of iNOS might be controlled by inflammatory signaling pathways. We show here that proinflammatory IFN-γ and NF-κB signals synergistically induce iNOS expression in human colon cancer cells. We further demonstrated that NF-κB directly binds to the NOS2 promoter to regulate iNOS expression. Although the IFN-γ and NF-κB signaling pathways alone are insufficient to induce iNOS expression in myeloid cells, IFN-γ and NF-κB can synergistically induce iNOS expression. We determined that IFN-γ up-regulates IRF8 expression to augment NF-κB induction of iNOS expression. We observed that the p65/p65 and p50/p50 homodimers, not the common canonical p65/p50 heterodimer, directly binds to the nos2 promoter to regulate iNOS expression in myeloid cells. Our results thus determine that the IFN-γ-induced IRF8 acts in concert with the NF-κB p65/p65 and p50/p50 homodimers to regulate iNOS expression.
Calcium binding protein S100a9 protects male lupus-prone BWF1 mice from disease development (IRC4P.448)
Authors: Davison Laura , Alberto Andrés , Liegl Lauren , Vogl Thomas , Jorgensen Trine
Published: January 2023

Testosterone protects from SLE pathogenesis, yet the mechanism of protection remains unknown. Using the (NZB x NZW)F1 (BWF1) mouse model of SLE, we have previously shown that immunosuppressive Gr1+CD11b+ cells are elevated in protected male BWF1 mice compared with females. Female and male Gr1+ cells use different mechanisms of suppression: reactive oxygen and nitrogen species (ROS/NOS) (female), or a secreted component independent of ROS/NOS production (male). Furthermore, female Gr1+ cells lose their capacity to suppress as the mice age. In support hereof, in vivo depletion of Gr1+ cells accelerated disease development in male mice only. Calcium-binding protein S100a9 is induced by inflammation and has been shown to exert either proinflammatory or immunosuppressive functions. Finding that male-derived Gr1+ cells express 3-4 fold higher levels of S100a9 mRNA than females, we hypothesized that S100a9 is the immunosuppressive mechanism used by male Gr1+ cells. Using S100a9-/- BWF1 mice we show that male-derived Gr1+ cells indeed use S100a9 to suppress plasma cell differentiation in vitro. Moreover, BWF1.S100a9-/- males developed increased serum ANA levels and IgG-IC deposition in the kidney glomeruli, and accelerated renal failure, while only the kidney phenotype was somewhat affected in BWF1.S100a9-/- female mice. In conclusion, differential regulation of S100a9 by male and female Gr1+ cells may contribute to the differential development of lupus in male and female BWF1 mice.
Mast cell 2B4 induces antigen-specific CD8+ T cells function (IRC4P.457)
Authors: Kim Jiyoung , Lee Junho , Kim Kwanghee , Lim Seon Ah
Published: January 2023

The signaling lymphocyte activation molecule (SLAM) family receptor, 2B4 (CD244), is expressed on NK cells, a subpopulation of T cells (including CD8+ T cells and γ/δ T cells), monocytes/macrophages, basophils, eosinophils, and mast cells (MCs). Although 2B4 is known to play an important role in the biological function of NK cells, the role of 2B4 on MCs remains unclear. Here, we identified a critical role for 2B4 on MCs in antigen-specific CD8+ T cell activation. Using a co-culture system, we demonstrated that OT-I CD8+ T cells acquired higher expression of CD69 and CD25 activation markers in the presence of OVA-pulsed wild type (WT) bone marrow-derived MCs (BMMCs) but not 2B4 KO BMMCs. In addition, CD8+ T cell activation was significantly reduced by incubation with 2B4-blocked MCs. This effect was potentiated by incubation with CD48-blocked MCs, indicating that while 2B4/CD48 interactions influenced CD8+ T cell activation, CD2/CD48 interactions were also involved in MC regulation of CD8+ T cells. Moreover, CD8+ T cell proliferation was enhanced by 2B4-expressing MCs. Thus, this study provides insight on how MCs can influence CD8+ T cells via 2B4/CD48 interactions.
C1q-CD33 inhibitory immunoreceptor interactions occur via C1q globular heads and CD33 C2-like domains (IRC4P.601)
Authors: Santiago-Schwarz Frances , Son Myoungsun , Wright Janell
Published: January 2023

Complement independent functions of C1q include C1q’s collagen-like region (CLR) engaging the immunoreceptor LAIR-1 to restrict monocyte-derived dendritic cell (mono-DC) differentiation/activation. If C1q’s globular region (gC1q) directly engages distinct inhibitory immunoreceptors is unknown. CD33, a C-type lectin, is highly expressed on monocytes. Like LAIR-1, triggering through CD33 inhibitory motifs (ITIM) restricts monocyte/DC activity. CD33 has C2-like domains potentially recognized by gC1q; thus we hypothesized that C1q would engage CD33. We tested whole (w) C1q/gC1q/C1qCLR for binding to CD33 isoforms (CD33M: one V & one C2 domain and CD33m: C2 domain only). Analyses with purified proteins revealed that wC1q binds to both isoforms and that gC1q, but not C1qCLR, binds to CD33 C2 domains. CD33M and CD33m transfected HEK293T cells bound C1q. On THP-1 cells, masking of CD33 C2 domains by sialic acid impeded binding of C1q. Domain oriented inhibitory partnering was substantiated by coincident triggering of CD33/LAIR-1 ITIM phosphorylation in monocytes by C1q and phosphorylation of LAIR-1 but not CD33 ITIM by C1qCLR. C1q also prompted CD33-LAIR-1 crosslinking on monocytes in proximity ligation assays. Thus, CD33 is a receptor for C1q; CD33 C2 domains are biologically relevant; C1q/CD33/LAIR-1 partnering occurs in monocytes. Masking of CD33 C2 domains by sialic acid suggests a mechanism for controlling C1q-CD33 inhibitory activity.
Natural indoles, indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM), attenuate staphylococcal enterotoxin B-mediated liver injury by downregulating miR-31 expression and promoting caspase-2-mediated apoptosis (IRC4P.605)
Authors: Busbee Philip , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. In the current study, we investigated the effect of natural indoles, I3C and DIM on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme AST levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. I3C and DIM also caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells.
STAT6-dependent regulation of airway inflammatory responses via OX40L expression on dendritic cells (IRC4P.610)
Authors: Lee Soojin , Shan Jichuan , Jin Manli , Srinivasa Bharat , Fixman Elizabeth
Published: January 2023

Epithelial and other structural cells drive Th2 responses in vivo indirectly upon TLR4 or NOD receptor stimulation and production TSLP and other innate cytokines, which upregulate OX40L on dendritic cells (DCs). These DCs are potent inducers of Th2 cell differentiation. We have investigated the ability of a cell penetrating peptide targeting STAT6, STAT6-IP, to modulate OX40L expression and inhibit Th2 responses. Bone marrow derived CD11c+ DCs (BMDCs) were incubated with OVA (containing LPS) ± STAT6-IP (or control peptides) and MFI of OX40L surface expression was quantified by FACS. OVA-induced increases in OX40L MFI were reduced by STAT6-IP. Our data suggest that STAT6-IP treated, OVA-loaded BMDCs also have reduced Th2 cytokine production from target CD4+ T cells. In vivo, lung OX40L mRNA levels increased in OVA+MDP (a NOD2 agonist) treated mice were inhibited by STAT6-IP treatment. Airway inflammatory responses were assessed in OVA+MDP (± STAT6-IP) treated mice, upon OVA challenge 28 days later. Lung resistance, bronchoalveolar lavage levels of eosinophils and neutrophils, Th2 cytokine production & TARC mRNA levels were all reduced by STAT6-IP treatment at the time of OVA+MDP sensitization. Our data are consistent with a model in which STAT6-IP inhibits antigen-induced OX40L expression on DCs, reducing Th2 cytokine production from target T cells and the subsequent Th2-biased airway inflammatory responses that contribute to pathogenesis in allergic airways disease.
The roles of HMGB1 in mitophagy, as a targeted defense against cellular stress (IRC4P.616)
Authors: Jeong Jang , Yoo Jong , Shin Jeon-Soo
Published: January 2023

High mobility group box 1(HMGB1), an abundant non-histone nuclear protein is translocated to cytosol under cellular stress like oxidative stimuli. Translocated HMGB1 has been reported as regulator of autophagy, which is a process where eukaryotes degrade various intracellular materials such as damaged mitochondria. Autophagy consists of three types: macro, micro, and chaperone-mediated autophagy. In this study, we show that HMGB1 could translocate to mitochondria; migration of HMGB1 was dramatically increased in stress condition. And, we also found that HMGB1 interact with mitophagy related protein. To determine whether HMGB1 is a positive regulator of mitophagy, we confirmed the difference in degree of mitophagy induction with HMGB1 deficient cell line under stress condition. We demonstrated that HMGB1 deficient cells showed higher rate of mitophagy induction than wild type cells did. In summary, HMGB1 could migrate to mitochondria and bind with mitophagy related protein, which may diminish mitophagy induction. Furthermore, because many neurodegenerative diseases are known to be related to autophagy, these findings suggest that HMGB1 might trigger onset of neurodegenerative disease, which can be a novel therapeutic target.
Natural killer cells suppress B cell immunity through perforin-dependent mechanisms (IRC4P.617)
Authors: Rydyznski Carolyn , Karmele Erik , Mahl Sarah , Waggoner Stephen
Published: January 2023

Accumulating evidence suggests that natural killer (NK) cells are critical regulators of antiviral T cells during chronic infection. We find that NK cells are also potent inhibitors of germinal center (GC) responses and the generation of long-lived B cell immunity after acute infection. A similar suppressive function was noted for NK cells after immunization with the non-replicating T cell-dependent antigen NP-KLH, suggesting that strategies to subvert NK cell-mediated inhibition may be able to improve vaccine responses. To determine whether cytotoxic function is important for this regulatory activity, we examined the contributions of perforin-containing granules and the death receptor Fas. NK cell inhibition of GCs was intact in Fas-deficient lpr mice but absent in perforin-deficient mice, thereby implicating granule-mediated cytotoxicity in this process. Moreover, we found that NK cells acted in a perforin-dependent manner during the first three days post-immunization to suppress generation of follicular helper CD4 T cell (Tfh) responses. The suppression of Tfh by NK cells was independent of the presence or absence of B cells, while CD4 T cells were required for development of virus-specific B cells. Thus, NK cells determine the quality and magnitude of B cell responses following immunization predominately via perforin-dependent repression of Tfh responses. This immunoregulatory process represents an innovative target for interventions aimed at enhancing vaccine efficacy.
Scavenger receptor SREC-I mediated entry of TLR4 into lipid microdomains and triggered inflammatory cytokine release in macrophages upon LPS activation (IRC4P.454)
Authors: Murshid Ayesha , Gong Jianlin , Borges Thiago , Calderwood Stuart
Published: January 2023

Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its immune functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS). The receptors then became internalized together in intracellular endosomes. SREC-I then promoted TLR4-induced signal transduction through the NFĸB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I / TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contain the small GTPase Cdc42 and the non-receptor tyrosine kinase c-Src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NFkB and MAP kinase pathways. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NFkB pathway. Our experiments indicated that SREC-I could bind LPS and might be involved in innate/inflammatory immune responses to extracellular danger signals.