bluuebunny/crossref_metadata_2025 huggingface dataset:
Inadequate CD18 shedding and increased depletion of soluble CD18 contributes to inflammatory activity in spondyloarthritis (P5090)
Authors: Kragstrup Tue , Jalilian Babak , Hvid Malene , Kjærgaard Anders , Østgård René , Schiøttz-Christensen Berit , Jurik Anne , Vorup-Jensen Thomas , Deleuran Bent
Published: January 2023

Spondyloarthritis (SpA) is characterized by inflammation in the axial skeleton and entheses. Integrin adhesion molecules containing the CD18 chain are pivotal regulators of leukocyte extravasation from the blood into zones of inflammation. Recently, we showed that CD18 is shed from leukocytes forming a soluble form of CD18 (sCD18) in the blood. Here we show that plasma sCD18 is a marker of inflammatory activity in SpA. The plasma levels of sCD18 were decreased in SpA patients compared with healthy controls and showed inverse correlations with disease activity scores including the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score. Remarkably, this situation could be simulated in vitro. Thus, the degree of CD18 shedding from SpA patient-derived PBMC correlated inversely with the BASDAI score. According to flow cytometric analyses, CD18 was primarily shed from intermediate (CD14++ CD16+) monocytes. In agreement with earlier reports identifying CD18 as a substrate for MMP-9, the concentration of this enzyme in the culture supernatants correlated with the sCD18 concentration. Also, sCD18 complexes adhered to TNFα-stimulated endothelial cells expressing high levels of ICAM-1 but not to unstimulated cells. Taken together, our data links alterations in plasma sCD18 and inflammatory activity in SpA. The low plasma level of sCD18 in SpA seems to be a combination of inadequate CD18 shedding and increased depletion of sCD18 potentially affecting leukocyte migration.
Ephedrine hydrochloride inhibits PGN-induced inflammatory responses by promoting IL-10 production and decreasing proinflammatory cytokine secretion via PI3K/Akt/GSK3β pathway (P5048)
Authors: Yu Yizhi , Zheng Yuejuan , Cao Xuetao
Published: January 2023

The approaches to control the inflammatory responses and reduce the mortality rate of sepsis are still not very effective in clinics and new potential drugs are needed to be confirmed to be useful in the induction of anti-inflammatory response. Ephedrine hydrochloride (EH) is a compound widely used for cardiovascular diseases, especially to treat hypotension caused by anesthesia or overdose of antihypertensive drugs. In this study, we reported that EH also plays an important role in the control of inflammatory response. It increases interleukin 10 (IL-10) and decreases proinflammatory cytokine (IL-6, TNF-α, IL-12, and IL-1β) expression in response to gram-positive cell wall component peptidoglycan (PGN) in primary peritoneal macrophages and Raw264.7 cells. The anti-inflammatory role of EH was also observed in experimental peritonitis mouse model induced by intraperitoneally PGN injection. Phosphatidylinositol 3-kinase (PI3K)/Akt pathway was found to be responsible for EH-enhanced IL-10 production and EH-decreased IL-6 expression. Therefore, our results illustrated that EH can help to maintain immune equilibrium and diminish host damage by balancing the production of proinflammatory cytokines and anti-inflammatory cytokine after PGN challenge. EH may be a new potential anti-inflammatory drug in treatment of severe invasive gram-positive bacterial infection.
Identification of a G-Protein coupled receptor for esterified 15(S)hydroxyeicosatetraenoic acid on human primary bronchial epithelial cells (P6255)
Authors: Vonakis Becky , Hise Kathleen , Liu Yun , Plunkett Beverly , Fang Xi , Haddad El-Bdaoui , Cox Paul
Published: January 2023

In asthmatic epithelium, the TH2 cytokine milieu results in production of phospholipid-esterified 15-HETE and elevated mucus secretion. A high-throughput 15-HETE binding assay was developed using 15-HETE esterified to biotin and Streptavidin-AlexaFluor488 to form the ligand (15HETE-Bt:SA-AlF). 15HETE-Bt:SA-AlF binding to Beas-2B cells and PBEC grown under air-liquid interface were assessed from 2- 60 min. Specificity of binding was monitored with unlabeled fatty acids [0.05-10 μM], biotin-hydrazide and SA-ALF. CyclicAMP (cAMP) was quantified using a competitive immunoassay kit. Intracellular calcium [Ca2+]i was measured (Fluo-3 / Fura-red) by flow cytometry. Beas-2B (n=8) and PBEC (n=3 donors) demonstrated time-dependent binding of 15HETE-Bt:SA-AlF, reaching equilibrium at 30 min., 4 deg.C. Binding of 15HETE-Bt:SA-AlF was dose-dependently displaced on Beas2B with 15-HETE>12-HETE, 5-HETE > 5,15diHETE,>15-KETE >> LTB4 (p< .05, 15-HETE vs. other fatty acids) whereas biotin-hydrazide:SA-ALF or SA-ALF were ineffective. In Beas2B cells, 15-HETE-Bt [0.1 - 10 μM] induced pM [cAMP] (n =4) whereas cholera toxin pretreatment (1 μg/ml, 5 hr.) inhibited 55-100% of cAMP production (n=2). A transient increase in [Ca2+]i was detected with 3-10 μM 15-HETE-Bt or 15-HETE in Beas2B (n=2). Bronchial epithelial cells exhibit specific, reversible binding of esterified 15-HETE suggesting a unique Gs- GPCR for esterified 15-HETE that may regulate airway inflammation in asthma.
Programmed cell death ligand 2 regulates Th9 differentiation and induction of chronic airway hyperreactivity (P6261)
Authors: Akbari Omid , Maazi Hadi , Kerzerho Jerome , Speak Anneliese , Szely Natacha , Lombardi Vincent , Lam Jonathan , Van Snick Jacques
Published: January 2023

Asthma is defined as a chronic inflammatory disease of the airways; however, the underlying physiologic and immunologic processes are not fully understood. The aim of this study was to determine whether Th9 cells develop in vivo in a model of chronic airway hyperreactivity (AHR) and what factors control this development. We have developed a novel chronic allergen exposure model using the clinically relevant antigen Aspergillus fumigatus to determine the time kinetics of Th9 development in vivo. Th9 cells were detectable in the lungs after chronic allergen exposure. The number of Th9 cells directly correlated with the severity of AHR, and anti-IL-9 treatment decreased airway inflammation. Moreover, we have identified programmed cell death ligand (PD-L) 2 as a negative regulator of Th9 cell differentiation. Lack of PD-L2 was associated with significantly increased TGF-β and IL-1α levels in the lungs, enhanced pulmonary Th9 differentiation, and higher morbidity in the sensitized mice. Our findings suggest that PD-L2 plays a pivotal role in the regulation of Th9 cell development in chronic AHR, providing novel strategies for modulating adaptive immunity during chronic allergic responses.
Dendritic cell expression of interferon regulatory factor 4 drives Th2 responses and regulates IL-10 and IL-33 production (P6239)
Authors: Williams Jesse , Tjota Melissa , Clay Bryan , Fixsen Bethany , Singh Harinder , Sciammas Roger , Sperling Anne
Published: January 2023

Dendritic cells (DCs) are known for promoting adaptive immune responses; however the molecular mechanisms that drive DCs to promote Th2-responses are poorly understood. We now demonstrate that house dust mite (HDM) allergen stimulation induces Interferon Regulatory Factor 4 (IRF4) expression in bone-marrow derived DCs as well as respiratory DCs. Mice deficient for IRF4 specifically in CD11c+ cells (CD11c-IRF4-/-) had dramatic defects in eliciting Th2-mediated HDM allergic inflammation, as measured by lung eosinophilia and Th2 cytokine production. Yet, the ability of CD11c-IRF4-/- mice to mount protective pulmonary Th1 viral responses remained intact. To determine whether defects in Th2 responses in vivo were due to direct effects of DCs on T cells, in vitro co-culture experiments were performed. CD11c-IRF4-/- DCs failed to induce Th2 differentiation compared to T cells stimulated by WT DCs. Further, genetically increasing IRF4 specifically in DCs augmented Th2 differentiation. Interestingly, we found that stimulation with HDM of WT DCs, but not CD11c-IRF4-/- DCs, increased expression of IL-10 and IL-33; both of which have been reported to play central roles in the development of Th2 responses. Together, these findings identify IRF4 expression in DCs and its ability to modulate cytokine production as a novel mechanism by which DCs regulate Th2 inflammatory responses.
Peanut allergic humans display increased Ag-dependent IL-33/IL-33R responses that regulate recall responses (P6241)
Authors: HayGlass Kent , Xie Jungang , Chooniedass Rishma , Becker Allan , Simons F Estelle
Published: January 2023

Peanut allergy is severe and persistent. IL-33 plays key roles in modulating murine Th2-like responses, shaping serum Ig production, and anaphylactic shock. The potential involvement of IL-33 and receptor components ST2 or IL-1RAcP in food allergy is unexplored as is the impact blocking endogenous IL-33 function. We hypothesized that (i) peanut allergic humans exhibit elevated systemic production and responsiveness to IL-33 vs non-allergic controls and (ii) that neutralizing endogenous IL-33 inhibits undesirable Ag-specific recall responses characteristic of food allergy. Acute peanut-driven restimulation revealed elevated IL-33, ST2 and IL-1RAcP production by PBMC of both peanut allergic and clinically tolerant groups (p<0.001). The intensity of peanut-dependent IL-33 mRNA production is linked to clinical phenotype(p<0.0006). Exogenous IL-33 increased Ag-driven CD4 T cell dependent IL-5, IL-9, IL-13 in both groups, with allergic volunteers most responsive (p<0.005). Soluble IL-33 receptors sST2-Fc and sIL-1RAcP-Fc inhibit elevated IL-5,9,13 recall responses in peanut allergic humans by median 80% (p<0.005). Thus, the data reveal a hitherto unrecognized and prominent role for excessive IL-33/IL-33R production and responsiveness in food allergic humans. Unexpectedly potent inhibition of peanut Ag-driven Type 2 recall responses in peanut-allergic individuals by sIL-33R components suggests potential therapeutic utility in food allergy and other human allergic disorders.
IL-1 released by skn dendritic cells activated through Syk and CARD9 pathway is essential for the sensitization of allergic contact dermatitis. (P6245)
Authors: Hara Hiromitsu , Yasukawa Shinsuke , Furue Masutaka , Yoshida Hiroki
Published: January 2023

Allergic contact dermatitis (ACD) is caused by T cells reactive to environmental and industrial allergens. Optimal T cell priming requires co-stimulatory signals from DCs that are activated through innate immune sensors. However, innate immune pathways through which contact allergens activate skin DCs are not clear. Here we show that stimulation of contact sensitizers (haptens) induces activation of the tyrosine kinase Syk in DCs. This not only induces the synthesis of pro-IL-1α/β and but also engages the NLRP3 inflammasome, resulting in DC release of IL-1α/β, which is essential for the sensitization of effector T cells. Both CARD9 and MyD88-deficient mice failed to induce contact hypersensitivity (CHS) to haptens. DAP12, Syk and CARD9 were required for hapten-induced IL-1 secretion from DCs, whereas IL-1R1-MyD88 signaling was required in T cells being primed by hapten-activated DCs. The CHS-sensitizing ability of DCs required inflammasome activation that is dependent on ROS generation. The DAP12-Syk signaling was essential for the ROS-regulated inflammasome activation induced by haptens, whereas CARD9/BCL10 signaling selectively controlled pro-IL-1 synthesis by regulating NF-κB activation. Finally, DC-specific deletion of CARD9, DAP12, Syk or NLRP3, but not MyD88 in mice by our newly-developed MixC-Treck system resulted in impaired CHS sensitization. Thus, our results identified an essential molecular axis of innate immunity required for the sensitization of ACD.
Using the lymphocytic choriomeningitis virus mouse model of infection to link specific immune responses with epidemiological outcomes (P6318)
Authors: Holechek Susan , Nieves Lizbeth , McAfee Megan , Guzman Vanessa , Lussier Danielle , Bruce Courtney , Blattman Joseph
Published: January 2023

The adaptive immune system can act to prevent infection, reduce disease, and/or prevent shedding of viral pathogens. However, little is known about which specific adaptive immune responses affect each of these epidemiologic outcomes. Lymphocytic choriomeningitis virus (LCMV) is a natural pathogen of both mice and humans. We have shown that transmission from congenitally infected carrier mice occurs at high frequency while transmission from adult persistently infected mice is relatively inefficient. The major difference between these two groups is the presence (in adult infected) or absence (in congenitally infected) of an anti-viral adaptive immune response. We have investigated how CD4 T cell and B cell/antibody responses affect virus shedding from adult LCMV persistently infected mice. CD4-/- or immunocompetent C57BL/6 mice were infected with the clone-13 strain of LCMV and shedding of virus from body fluids sampled. Although we detected high titers of virus in the saliva and blood of all infected animals, we observed higher levels of transmission to susceptible mice from CD4-/- hosts compared to wild-type mice. Because we observed no detectable LCMV-specific antibody responses in the CD4-/- infected mice, these results suggest that CD4 T cells and antibodies may play a role in limiting virus transmission. We have used these data to parameterize mathematical models linking immunological responses with epidemiological outcomes of infection to generate new avenues of inquiry.
Lactobacillus rhamnosus GG (LGG) enhances virus-specific effector T cell responses induced by human rotavirus vaccine in neonatal human gut microbiota associated gnotobiotic pigs (P6320)
Authors: Yuan Lijuan , Wen Ke , Wang Haifeng , Shepherd Megan , Li Guohua , Yang Xingdong , Kocher Jacob , Giri-Rachman Ernawati , Karyala Saikumar , Settlage Robert
Published: January 2023

This study aims to enhance rotavirus vaccine efficacy through modulation of gut microbiota by probiotics and identify the underlying mechanism. We generated a human gut microbiota (HGM) associated neonatal pig model using stool samples from a newborn infant. Germ-free pigs were fed the fecal suspension daily starting at 12 hrs after birth for 3 days to establish human microflora in the pigs. The HGM pigs were divided into three groups: AttHRV only and AttHRV plus 9 or 14 doses of LGG. Daily LGG feeding started at 3 days of age. All the HGM pigs received the oral attenuated human rotavirus (AttHRV) vaccine at 5 days of age and then an oral booster dose 10 days later. A subset of the pigs in each group was challenged with a homotypic virulent HRV at post-AttHRV inoculation day 28. Frequencies of virus-specific IFN-γ producing T cells and TGF-β+/IL-10+FoxP3+ Treg cells in ileum, spleen and blood were determined. The 14-dose LGG regimen significantly increased LGG counts in large intestinal contents at 28 days of age and IFN-γ+CD8+ T cells at challenge and postchallenge and IFN-γ+CD4+ T cells postchallenge in ileum. LGG did not significantly alter frequencies of Tregs. The influence of LGG on the HGM is being characterized by 16sRNA gene V4 region Illumina MiSeq® sequencing. Studies using this novel pig model with defined human microbiota will provide clear evidence of LGG’s adjuvant effect and will facilitate establishment of effective dosing regimens of probiotics for humans.
Epstein-Barr virus infection induces aberrant TLR/MyD88 activation pathway and fibroblast-myofibroblast conversion in systemic sclerosis. (P6322)
Authors: Farina Giuseppina Alessandra , Farina Antonella , Cirone Mara , York Michael , Lenna Stefania , Padilla Cristina , Mclaughlin Sarah , Faggioni Alberto , Trojanowska Maria , Lafyatis Robert
Published: January 2023

Activated fibroblasts, mainly myofibroblasts are considered the principal mediators of fibrogenesis in systemic sclerosis (SSc), although the origin of persistent activation of fibroblasts and myofibroblasts is unclear. Activation of Interferon by Toll-like receptors in immune cells may play a role in the pathogenesis of inflammation in many autoimmune diseases, including SSc. We investigated whether fibroblasts persistently activated by innate immune and infectious stimuli might induce IFNs and TGFβ, two major markers of inflammation and fibrosis implicated in SSc pathogenesis. Since Epstein-Barr virus has been a leading candidate in triggering several autoimmune diseases, we developed methodology for infecting SSc fibroblasts with this virus. We found that EBV signals through the TLR/MyD88-pathway in infected-fibroblasts, activating a distinct innate immune response characterized by the expression of selected IFN- and TGFβ-inducible genes. Intriguingly, activation of the TLR/MyD88-pathway by CpGODN2006 or R837 ligand, significantly induced IFN-regulated genes, but no expression of TGFβ-regulated genes was detected in TLR/MyD88-stimulated fibroblasts. EBV-TLR/MyD88 aberrant activation induces the expression of selected IRFs, ISGs, TGFβ1 and several markers of fibroblast activation, such as smooth-muscle-actin and Endothelin-1, and all of these genes play a key role in determining the pro-fibrotic phenotype in SSc-fibroblasts.
Deconvoluting protective immune responses elicited by the live-attenuated 17D yellow fever virus vaccine (P6324)
Authors: Watson Alan , Lam LK Metthew , Klimstra William , Ryman Kate
Published: January 2023

The safe and efficacious live-attenuated 17D yellow fever virus (YFV) vaccines provide a benchmark for immunization. The contribution of 17D elicited humoral and cell-mediated responses to protection from challenge with wtYFV remains unknown due to a lack of tractable animal models of immunization and disease. By artificially reproducing the primate-specific phenotype of wtYFV to circumvent type I IFN control, we developed a murine model in which challenge with an otherwise lethal wtYFV is limited to low-level replication in the draining lymph nodes following 17D immunization. Moreover, the fatal viscerotropic disease and cytokine storm afflicting naive animals is completely abrogated. Similar to humans, 17D immunization in our model elicits neutralizing antibodies as well as robust YFV-specific CD4+ and CD8+ T cell accumulation with polyfunctional cytokine profiles and markers of early memory differentiation. Transfer of immune sera, CD4+/CD8+ T cells and/or B cell populations from 17D-immunized mice into naïve mice prior to challenge with wtYFV indicates that antibodies are not solely responsible for protection. However, YFV-specific T cells elicited in naive mice infected with wtYFV accumulate to higher numbers than in 17D vaccinated mice, yet fail to protect. We are currently comparing 17D and wtYFV infections to deconvolute mechanisms of 17D-induced immunogenicity and wild-type YFV immune evasion.
The significance of tyrosine kinase 2 gene in encephalomyocarditis-D virus-induced diabetes as studied in Tyk2 gene knockout mice (P6334)
Authors: Nagafuchi Seiho , Teshima Miho , Kai Yuji , Kai Katsuya , Izumi Kenichi , Kurisaki Hironori , Katsuta Hitoshi , Nagafuchi Seiho
Published: January 2023

Type 1 diabetes mellitus results form progressive loss of pancreatic β cells. The virus, as one of environmental factors, has been long considered to play a role in the development of type 1 diabetes. The D variant of encephalomyocarditis (EMC-D) virus induced susceptible strain of mice to become type 1 diabetes. We have already reported that innate immunity may play an important role in protection against EMC-D virus-induced diabetes (Arch Virol, 2008). In the present study, we analyzed the role of Tyk2 gene in the pathogenic mechanism of EMC-D virus-induced diabetes by using Tyk2 gene knockout (KO) mice. Tyk2 gene KO C57BL/6J mice, mouse strain resistant to EMC-D virus-induced diabetes, developed diabetes. In order to examine whether Tyk2 gene expression was important in parenchymal or immune cells to protect against EMC-D virus-induced diabetes, we developed splenic chimera mice. Tyk2 gene expression was important in parenchymal cells, but not in spleen cells, to resist against EMC-D virus-induced diabetes. Consistently, mice specifically expressed Tyk2 gene only in pancreatic β cells prevented EMC-D virus-induced diabetes. These observations suggest that Tyk2 gene expressed in pancreatic β cells plays a significant role in preventing EMC-D virus-induced diabetes in mice.
Dengue virus nonstructural protein 1 vaccine protects against lethal challenge in interferon α/β receptor-deficient mice (P6348)
Authors: Beatty P Robert , Orozco Susana , Killingbeck Sarah , Zompi Simona , Harris Eva
Published: January 2023

Dengue virus (DENV) is a flavivirus that infects 100 million people and causes severe disease in ~500,000 people annually. DENV nonstructural protein 1 (NS1) is secreted by infected cells and found at high levels in patient sera during the acute illness. To investigate the potential for NS1 as a vaccine candidate, we examined the protective efficacy of immunization with recombinant NS1 protein against lethal DENV infection in a mouse model. Interferon α/β receptor-deficient mice were injected intraperitoneally 3 times over an 8-week period with 20 ug recombinant NS1 with different adjuvants, including alum, Sigma adjuvant system (SAS), CpG DNA, MF59 and/or monophosphoryl lipid A (MPLA). Two weeks after the third immunization, vaccinated mice were infected with a lethal dose of DENV2. Vaccination with NS1 combined with SAS and CpG DNA provided complete protection against mortality along with reduced morbidity whereas mice immunized with NS1 combined with alum and/or CpG DNA displayed little or no protection against DENV2 challenge. All vaccination groups demonstrated comparable levels of total anti-NS1 IgG; however, mice immunized with SAS+CpG had higher levels of anti-NS1 IgG2b as compared to alum (p=0.057). Data comparing mice immunized with NS1 together with MPLA and MF59 will also be presented. Thus, immune responses to a DENV nonstructural protein can provide protection against severe disease and NS1 may provide an alternative vaccine against dengue.
T cell mediated mortality in neonatal mice upon LCMV infection (P6302)
Authors: Kenney Laurie , Carter Erik , Selin Liisa
Published: January 2023

Newborns are more susceptible to infections due to their lack of immunological memory and under-developed immune systems. Passive maternal immunity protects neonates until their immune systems have matured. We questioned if a noncytolytic virus that produces strong T cell responses in adults would also induce a response in neonates. Neonates were infected with LCMV at 5e4, 500, 50, 5 and 0.5 PFU. Surprisingly, at all doses the majority of pups succumbed to infection during the peak of the T cell response. Death was caused by T cell-dependent pathology and not viral load as 100% of T cell deficient pups survived with minimal lung and liver pathology. However, passive immunity from LCMV-immune mothers protected 100% of pups from death by helping control viral load early in infection. In adult mice high antigenic stimulation can cause over-activation of T cells leading to clonal exhaustion, where cells undergo a stepwise loss of function. Clonal exhaustion allows for survival from some high dose viral infections and results in persistent infection. We found that neonates without passive immunity that survived to day 14 did not show signs of T cell clonal exhaustion. Furthermore, surviving pups were not persistently infected, clearing virus by week 3. Neonates are commonly thought to have less functional immature immune systems, but these results show that neonates are capable of producing strong T cell responses that contribute to increased mortality.
IL-22 driven neutrophils afford critical protection from acute murine cytomegalovirus infection (P6310)
Authors: Stacey Maria , Marsden Morgan , Dolton Garry , Stack Gabrielle , Jones Emma , Taylor Philip , Jones Simon , Snelgrove Robert , Wilkinson Gavin , Humphreys Ian
Published: January 2023

Pathogenic herpesviruses including cytomegalovirus target numerous host organs during infection, but the immunological mechanisms that afford antiviral protection in different tissue microenvironments require a better understanding. Utilizing the murine cytomegalovirus (MCMV) model, we demonstrate that neutrophils are critical antiviral effector cells that counter acute herpesvirus infection. Depletion of Ly6G+ neutrophils exacerbated virus-induced weight loss and elevated virus load in a tissue-restricted manner. Neutrophils profoundly and directly inhibited virus replication in vitro. MCMV-elicited neutrophil responses were orchestrated by interleukin-22 (IL-22), which was expressed by conventional NK cells and induced neutrophil-attractant chemokines in MCMV-infected peripheral organs. These data demonstrate that IL-22 affords anti-herpesvirus infection in peripheral tissue and highlights a previously unappreciated and critical role for neutrophils in countering cytomegalovirus infection.
Memory CD8 T cells that are bystander-activated during an infection control early pathogen replication in an innate-like manner (P4371)
Authors: Prlic Martin , Chu Talyn
Published: January 2023

Recent studies illustrate that during an infection the antigen non-specific memory CD8 T cell compartment is not simply an inert pool of cells, but can participate in the immune response. This bystander activation of memory CD8 T cells occurs in the absence of cognate antigen recognition, but the overall role of T cell receptor (TCR) mediated signals has not been defined. Furthermore, it is unknown if and how these cells contribute to the clearance of an infection. We measured the strength of TCR signals that bystander activated CD8 T cells (BA-CTLs) receive in vivo and found evidence of TCR signaling, albeit limited. Given this marginal contribution of the TCR, we sought to determine how BA-CTLs efficiently kill target cells. We demonstrate that BA-CTLs recognize and directly eliminate target cells in an innate-like, granzyme-dependent manner. We next selectively inhibited BA-CTL mediated killing in vivo during the course of an infection, which led to a significant defect in pathogen clearance. Our data suggest that BA-CTLs play a previously unappreciated, essential role in the early immune response to pathogens before the onset of a de-novo generated, primary CD8 T cell response.
Switching on memory CD8+ T cells for enlarged pathogen-specific protection (P4375)
Authors: M'Homa Soudja Saidi , Chandrabos Ceena , Veenstra Mike , Ruiz Anne L. , Julien Marie , Lauvau Gregoire
Published: January 2023

Memory CD8+ T cells induced upon immunization exhibit improved functional features that contribute to protection of immunized hosts. Although both cognate antigen recognition and inflammation are important for memory CD8+ T cell reactivation, the relative contribution of these factors and the cell types providing these signals in vivo are poorly defined. In recent work, we have shown that Ly6C+CCR2+ inflammatory monocytes, a subset of monocytes, largely orchestrate memory CD8+ T and NK lymphocytes to become potent effector cells by sensing IL-18 and IL-15-derived inflammatory cytokines from monocytes independently of their cognate antigen. Like NK cells, they underwent rapid mobilization, upregulated intense and sustained effector functions during bacterial, viral and parasitic infections, and contributed to innate responses and protection in vivo. In our most recent experiments, we investigate further the molecular mechanisms of cross-talk between activated memory T cells and innate immune effector cells that allow for pathogen-specific protective immunity. Our results unravel a novel and unexpected level of antigen-dependent spatio-temporal tissue organization that is essential to achieve quick and efficient pathogen confinement and killing. This mechanism depends on memory T cell-derived IFN-γ and chemotactic cues, and on effector monocytes. We will present and discuss these data.
Differential regulation of human T cell and NK cell function by tobacco product preparations (P4339)
Authors: Arimilli Subhashini , Damratoski Brad , Gaddamanugu Prasad
Published: January 2023

NK cells and T cells play essential roles in innate and adaptive immune responses by protecting against microbial infections and in tumor surveillance. While smoking is known to cause immunosuppression, it is unclear how the use of smokeless tobacco (ST) products affects immune responses. We investigated immunological responses to exposure of reference tobacco product preparations (TPPs) using poly I:C and LPS-stimulated PBMCs. The TPPs included whole smoke-conditioned media (WS-CM) and total particulate matter (TPM) prepared from 3R4F reference cigarettes, 2S3 moist snuff smokeless tobacco extracted in complete artificial saliva (ST/CAS), and nicotine. WS-CM and TPM treatment resulted in dose-dependent reductions in the expression of intracellular IFN-γand TNF-α in NK cells and T cells, secretion of several cytokines by PBMCs and the inhibition of cytolytic activity of effector cells in PBMCs. Although interference from the vehicle effects confounded the interpretation of data from cells exposed to ST/CAS, some immunosuppressive effects were evident only at high doses. Treatment with nicotine caused minimal immunosuppression in this system. These data suggest that the function of human NK cells and T cells is impacted by exposure to TPPs, on an equi-nicotine unit basis, as follows: WS-CM > TPM >ST/CAS> nicotine. These findings support the hypothesis that immune-suppression from smoking is linked to a higher incidence of microbial infections and cancer among smokers.
NKp65 hallmarks a rare subset of human innate lymphocytes (P4347)
Authors: Steinle Alexander , Döring Claudia , Hansmann Martin-Leo , Vogler Isabel
Published: January 2023

We previously provided the first characterization of the human activating C-type lectin-like receptor (CTLR) NKp65. Like its close relative NKp80, NKp65 is a homodimeric CTLR with a cytoplasmic hemITAM-like signaling motif and encoded in the Natural Killer Gene Complex (NKC) juxtaposed to its ligand. While NKp80 ligates AICL expressed by myeloid cells, NKp65 was found to be a high-affinity receptor for KACL, like AICL a member of the human CLEC2 family of CTLR. Strikingly, KACL expression is almost exclusively confined to human skin, i.e. keratinocytes. NKp65/KACL interaction has been shown to activate cytotoxicity and IFNg secretion of an NKp65- expressing NK cell line. However, while NKp80 is expressed by virtually all human NK cells, physiological expression of NKp65 remained enigmatic. In order to identify NKp65 expressing cells we generated NKp65-specific mAb that specifically stain a small subset of human innate lymphocytes isolated from peripheral blood. Transcriptional profiling of NKp65-expressing cells suggested that NKp65 expression is associated with a distinct subset of innate lymphocytes. Phenotypic analyses confirmed exclusive expression of NKp65 on this innate lymphocytes subset, while no NKp65 expression on NK cells was observed. Hence NKp65 hallmarks this subset of innate lymphocytes and may endow this subset with the capacity for dedicated immunosurveillance of human skin.
Vitamin D insufficiency modulates the pro- and anti-inflammatory cytokine balance without altering regulators of circulating 25-hydroxyvitamin D (P6301)
Authors: Barker Tyler , Dixon Brian , Schneider Erik , Weaver Lindell
Published: January 2023

Although evident in pathophysiological conditions, it is unknown if regulators of vitamin D are disrupted, if pro-inflammatory cytokines are increased, or if anti-inflammatory cytokines are decreased with vitamin D insufficiency in young adults. The purpose of this study was, therefore, to evaluate circulating regulators of vitamin D, and circulating pro- and anti-inflammatory cytokines in young adults with low serum 25-hydroxyvitamin D (25(OH)D) concentrations. Circulating 25(OH)D, pro- and anti-inflammatory cytokines, parathyroid hormone, calcium, and albumin were measured in young (25 to 42 y), reportedly healthy adults during the winter. Subjects were separated into one of two groups: vitamin D insufficient (25(OH)D < 32 ng/mL, n = 14) or vitamin D sufficient (25(OH)D > 32 ng/mL, n = 14). Age, height, body mass, and body mass index, and circulating parathyroid hormone, calcium, and albumin concentrations were not significantly (all p > 0.05) different between groups. However, pro-inflammatory cytokines (interleukin (IL)-2, IL-1β, tumor necrosis factor-α, and interferon-γ) were significantly (all p < 0.05) greater in the vitamin D insufficient group. The anti-inflammatory cytokines, IL-5 and IL-10, were not significantly (both p > 0.05) different between groups. We conclude that regulators of vitamin D are not disrupted with vitamin D insufficiency, but insufficiency could result in pro-inflammatory stress without modulating anti-inflammatory cytokines in young adults.
Regulation of IL-4-induced STAT6 activation by SUMOylation (P6307)
Authors: Lee Choong-Eun , Tran Tam , Kim Seol-Hee , Sung Ki-Sa , Choi Cheol-Yong
Published: January 2023

As a key signaling molecule in the cytokine-mediated transcriptional activation STATs are shown to undergo PTMs including phosphorylation, acetylation, methylation, ubiquitination and SUMOylation. The SUMO-modification of STAT1 has been implicated in the regulation of protein stability, nuclear translocation, and transcriptional activation, probably through the reduction of the formation and life-span of the active dimer. As a part of regulation of IL-4 signaling mechanism, we have investigated the role of SUMOylation on STAT6 activation. We have identified potential SUMOylation sites on STAT6 by SUMOplot prediction program analysis and observed the IL-4-induced SUMOlyation of STAT6 in HeLa cells. The introduction of mutation at a potential SUMO site K636 and transfection of shRNA of SUMO-conjugating enzyme UBC9 resulted in the inhibition of tyrosine phosphorylation at Y641 induced by IL-4. The results indicate that endogenous SUMOylation reaction of STAT6 is critical for IL-4-induced STAT6 phosphorylation. Conversely, the forced expression of SUMO-1 and STAT6 caused cytosolic relocalization of both STAT6 and phospho-STAT6 induced by IL-4 as demonstrated by Western blot and confocal analysis. Our data suggest that SUMO-modification of STAT6 may play dual roles in the regulation of phosphorylation and nuclear translocation of STAT6 during IL-4 signal transduction.
A critical role of Toll-like receptor 2 in chronic stress-induced immune suppression (P6309)
Authors: Yin Deling , Tan Yang , Feng Yimin , Caudle Yi , Xiang Yanxiang , Hu Dan
Published: January 2023

Stress can enhance or suppress immune functions depending on the duration and severity of stressful situation. Our previous studies observed that Toll-like receptor (TLR) participates in chronic restraint stress-induced immune dysfunction. However, the mechanism by which TLR2 contributes to immune suppression induced by chronic restraint stress remains elusive. Our investigation found that stimulation of TLR2 by peptidoglycan (PGN), a TLR2 ligand, significantly attenuates cell apoptosis in splenocytes induced by chronic restraint stress. Furthermore, we found that administration of PGN markedly blocks chronic restraint stress-induced alterations of anti-apoptotic and apoptotic genes, including Bcl/2 and glycogen synthase kinase-3β (GSK-3β). We also found that activation of TLR2 by PGN inhibits chronic stress-reduced level of c-Jun N-terminal kinase (JNK) phosphorylation. This effect of PGN is abolished in TLR2 deficient mice, suggesting PGN-induced protection of JNK phosphorylation through a TLR2 dependent manner. Moreover, our data have shown that chronic stress caused a dramatic decrease in cytokine IL-2 level but an increase in the cytokines IL-4 and IL-17 in CD4+ T cells. Interestingly, stimulation of TLR2 by PGN could block these alterations of cytokine levels induced by chronic stress. Collectively, our studies thus demonstrate that stimulation of TLR2 attenuates chronic stress-induced immune suppression by modulating apoptosis-related proteins and cytokines.
Comparison of IFN-γ and IFN-γ in antiviral, immune regulatory and apoptotic roles in fish (P6311)
Authors: Nie Pin , Fu Jian , Chang Ming , Huang Bei , Chen Shan
Published: January 2023

The cDNA and genomic sequences of grass carp IFN-γ and IFN-γ related (IFN-γrel) genes were cloned, with the absence of an NLS motif in IFN-γrel. IFN-γ and IFN-γrel differed in many aspects. Quantitative analysis revealed that IFN-γ was mainly expressed in spleen and intestine, while IFN-γrel was mainly expressed in gills and intestine, moderately in thymus and muscle. In peripheral blood leucocytes, the expression of IFN-γ and IFN-γrel were both up-regulated after PolyI:C and LPS treatment. Luciferase activities showed that IFN-γ could enhance interferon promoter activity and NF-κB promoter activity. The function of antivirus and antitumor roles was compared between IFN-γ and IFN-γrel in CIK cells. IFN-γ could induce expression of Mx, PKR, IRF1, SOCS1, TNF-α, while IFN-γrel did not have such roles. Nevertheless, IFN-γ and IFN-γrel could not induce the expression of Casp-3, Casp-9 and cathD. Furthermore, IFN-γ but not IFN-γrel inhibited the replication of GCRV, grass carp reovirus in CIK cells and prompted the apoptosis in CIK cells. Surprisingly, whatever IFN-γ was mutated with the deletion of 14 aa in C terminal or using GGGG substitution of RRRR had the similar effect as the intact IFN-γ in inducing the gene expression and the apoptosis. These results thus indicate IFN-γ is a functional homologous gene similar to IFN-γ in other higher vertebrates, while the role of IFN-γrel may need further investigation.
Jun activation domain-binding protein 1 (JAB1)/CSN5 is required for the optimal response to interferon-α. (P6313)
Authors: Muromoto Ryuta , Matsuda Tadashi
Published: January 2023

Degradation of IFNAR1 protein is one of the mechanisms limiting the extent of cellular responses to type I interferon. Tyrosine kinase 2 (TYK2), a JAK family kinase, has been reported to bind and stabilize the basal IFNAR1 protein level, indicating that TYK2 is a fundamental component of IFN receptor complex. In this study, we identified Jun activation domain-binding protein 1 (JAB1; also known as fifth subunit of COP9-signalosome, CSN5) as a new TYK2 binding partner and investigated the role of JAB1 in the regulation of IFN responses. Knock-down of JAB1 by siRNA resulted in the suppression of IFN-α-induced phosphorylation of STAT proteins and their transcriptional activation. Importantly, JAB1 knock-down induced an activation of SCF ubiquitin ligase complex containing Cullin 1 (CUL1), as judged by the enhancement of covalent modification of CUL1 with the ubiquitin-like protein NEDD8, and markedly reduced the basal protein level of IFNAR1. In contrast, NEDD8 knock-down or inhibition of NEDD8-modification by NEDD8-activating enzyme (NAE) inhibitor resulted in an increase of IFNAR1 protein with concomitant reduction of NEDD8-modified CUL1. Furthermore, NAE inhibitor treatment enhanced HeLa cell susceptibility to growth suppression effect by IFN-α. These data suggested that NEDD8-modification pathway is involved in the proteolysis of IFNAR1 and that JAB1 acts as a positive regulator of IFN responses by stabilizing IFNAR1 protein through antagonizing NEDD8 pathway.
Interleukin 22 receptor alpha1 is expressed by both the luminal and glandular epithelia of the day 34 pregnant mare uterus (P6319)
Authors: Brosnahan Margaret , Miller Donald , Adams Mackenzie , Antczak Douglas
Published: January 2023

Invasive trophoblast cells of the equine chorionic girdle (CG) migrate through maternal endometrium to form endometrial cups, dense accumulations of cells that produce equine chorionic gonadotropin from days 40 to 120 of pregnancy. At day 37 CG cells attach to the luminal epithelium, passing through and phagocytosing the epithelial cells. The CG cells then migrate into the stroma to form the mature endometrial cups and the luminal surface of the endometrium re-epithelializes. Penetration of the stroma occurs in association with endometrial glands, but the glandular epithelium is undisturbed. Interleukin 22 (IL22) is involved in the preservation of epithelia. Previously we identified high expression of IL22 in CG cells using a gene expression array on placental tissues from day 34 of gestation. qRT-PCR analysis showed a striking increase in IL22 mRNA in CG cells from days 32 to 35, and absence of IL22 in other conceptus tissues. Our discovery of IL22 in the CG was novel, as this cytokine had been reported only in immune cells. qRT-PCR also detected IL22 receptor mRNA in pregnant endometrium at levels similar to other equine epithelia. Using in situ hybridization we have now visualized IL22RA1 mRNA in both the luminal and glandular epithelia of the pregnant mare uterus. We hypothesize that IL22 produced by the CG binds IL22RA1 on luminal endometrium and glands, serving as a mechanism of fetal-maternal communication and ensuring the integrity of epithelial surfaces.
In vitro cytokine release assays for commercially available biologics for predicting cytokine storm in human patients (P6321)
Authors: Scesney Susanne , Ibraghimov Alexander
Published: January 2023

Therapeutic antibodies undergo numerous safety tests to ensure in vivo safety. One required safety assay looks at the cytokine release from either human whole blood or human peripheral blood mononuclear cells (PBMC) when incubated with the therapeutic antibody. Recently TGN1412, a CD28 super-agonist antibody, while not causing cytokine release from purified PBMC, when administered in vivo, resulted in cytokine storm in all six volunteers. Since that incident, many laboratories have been working to design a cytokine release assay (CRA) that will show cytokine release in response to TGN1412. In this study we compare two CRA formats. In one assay, test antibody is coated to a polypropylene plate, then incubated with freshly purified PBMC for two days. In the second assay, human PBMC are pre-incubated at high density to make the cells more “lymph node like” (LNL) before incubation with soluble antibody. The response of PBMC from multiple donors to TGN1412 and multiple commercially available antibodies were compared. The plastic fixed antibody tends to generate exaggerated cytokine responses probably due to excessive crosslinking. The LNL format has the advantage of using soluble antibody which may better reflect how PBMC in vivo encounter antibody.
The differential regulation of CCR6 biological functions by site-specific phosphorylation of CCR6 C-terminal tail (P6323)
Authors: Lu Mei-Yi , Liao Fang
Published: January 2023

Chemokine receptors are seven-transmembrane G-protein-coupled receptors (GPCRs). For most GPCRs, agonist-induced receptor phosphorylation at C-terminus leads to the recruitment of β-arrestins, resulting in receptor desensitization and internalization. CCR6 is a CC chemokine receptor whose ligand is CCL20. The role of site-specific phosphorylation of CCR6 at C-terminus in CCR6 biological function has not been investigated yet. In this study, we have intended to identify the specific serine/threonine residues at C-terminus of CCR6, and studied their role in the regulation of CCR6 biological function. We have generated a series of site-specific mutants in which specific serine/threonine residues at C-terminal tail of CCR6 were mutated into alanine as well as deletion mutants in which progressive truncation of C-terminus of CCR6 were generated. We have used these CCR6 mutants to characterize the role of site-specific serine/threonine residues in the regulation of CCR6 biology including the association of β-arrestins with CCR6, the internalization and desensitization of CCR6, the intracellular signaling of CCR6, the intracellular trafficking of CCR6, and the CCR6-mediated cell migration. We found that site-specific phosphorylation of threonine/serine residues at C-terminus of CCR6 differentially regulates CCR6 biological functions. The results will be presented and discussed.
A new chemoattractant protein found by PBMC screening platform (P6347)
Authors: Pei Xiaolei , Zhang Yan , Sun Qianying , Xu Enquan , Zheng Yi , Guo Changyuan , Wang Pingzhang , Ma Jing , Peng Xinjian , Chen Dixin , Zhang Yang , Zhang Yingmei , Guo Shuai , Shi Taiping , Ma Dalong , Wang Ying
Published: January 2023

To find new human gene encoding protein which has potential to be chemokine like or chemokine, our laboratory has established the peripheral blood mononuclear cell (PBMC) chemoattractant platform, which includes four steps. First, we screened out 212 genes potential to be cytokines from the genes library in which the genes’ cytokine-related function was unknown or unreported. And then, we constructed the eukaryotic vectors expressing the candidate genes. And third, the proteins encoded by the candidate genes, which could be secreted, were chosen. At last, the secretive proteins were applied on the PBMC chemoattractant platform to find the protein which had the chemoattractant ability. The PBMC chemoattractant platform was based on the Boyden Chamber Assay. Among the proteins which were potential to be chemokine or chemokine like, one protein was found having the ability to chemoattract peripheral blood monocyte and lymphocyte, which was low expressed in normal tissue and detected in human breast cancer and prostate cancer. What’s more, we have found that CCR2B was the protein’s chemoattractant receptor in vitro. In consideration of CCR2B is normally expressed in lots of cancer sites, the protein which we have found may play some kind role in the procession of cancer development.
FAM19A4: a novel cytokine promotes phagocytosis of zymosan but negatively regulates TNF-α secretion associated with NF-κB pathway in macrophages. (P6349)
Authors: Wang Wenyan , Wang Pingzhang , Li Ting , Wang Xiaolin , Zhang Heyu , Yuan Wanqiong , Zhang Yingmei , Ma Dalong , Han Wenling
Published: January 2023

Cytokines play a key role in the immune system. When pathogen infection occurs, macrophages undergo phagocytosis first, accompanied with inflammatory cytokines secretion. FAM19A4 is a novel cytokine identified by the strategy of immunogenomics. It is a classical secreted protein, low expressed in most normal tissues, but is up-regulated in inflammatory stimulated monocytes-macrophages. FAM19A4 can enhance phagocytosis of murine macrophages against zymosan both in vitro and in vivo by noticeably increasing the phosphorylation of Akt, but reduce TNF-α secretion in zymosan-challenged macrophages by impacting activation of p38 and ERK1/2 in NF-κB pathway, due to the degradation of p65 both in nucleus and cytoplasm. These findings provide a novel cytokine FAM19A4 expressed by stimulated macrophages, which can promote phagocytosis of macrophages by activating Akt, but impact TNF-α increasing with challenge by inhibiting NF-κB pathway.
Suppressor of cytokine signaling proteins are induced by IL-7 and regulate CD127 expression in human CD8 T cells (P6291)
Authors: Al-Ghazawi Feras , Parmar Parmvir , Faller Elliott , El Salfiti Abdulkareem , MacPherson Paul
Published: January 2023

Interleukin (IL)-7 is an essential non-redundant cytokine influencing cell survival and proliferation throughout the life-span of a T cell. IL-7 is currently being investigated as a potential therapy in HIV+ individuals with poor immunological response to antiretroviral therapy. In order to optimize the use of IL-7 in therapeutic settings, it is crucial to understand how expression of the IL-7 receptor-α (CD127) is regulated. We elucidate in this study the mechanism by which IL-7 targets surface CD127 protein for degradation and describe the role of SOCS proteins, a family of eight proteins (CIS and SOCS1-7), in this process. We show here that in primary human CD8 T cells, upon binding IL-7, surface CD127 is rapidly internalized and phosphorylated at Y449 within 20 minutes of IL-7 stimulation, while activation of the JAK/STAT5 pathway stimulates expression of CIS transcripts and proteins. Following IL-7 stimulation, CIS binds directly to CD127 as demonstrated by Co-IP and co-localizes with both CD127 and the early endosomal marker EEA1, then with the proteasomal marker MCP20, as demonstrated by confocal microscopy. Subsequent proteasomal degradation of CD127 and CIS is dependent on an E3 ligase and can be blocked with the E3 ligase inhibitor SMER3. Through siRNA-mediated knockdowns we confirm roles of CIS in the IL-7 mediated degradation of the CD127 protein.