bluuebunny/crossref_metadata_2025 huggingface dataset:
MAPK/ERK-dependent TCR signaling induces ADAM17-mediated processing of TNFR2 by CD8+ T cells (CCR5P.249)
Authors: DeBerge Matthew , Ely Kenneth , Enelow Richard
Published: January 2023

Elevated levels of soluble TNF receptor 2 (sTNFR2) are observed in a variety of human pathophysiological conditions. Following influenza infection in mice, we found that sTNFR2 levels were elevated in both the serum and airways. As the levels of sTNFR2 in the airways correlated with the CD8+ T-cell response, we were interested in the cellular mechanisms by which CD8+ T cells regulate TNFR2 processing. We found that effector CD8+ T cells express TNFR2 on the cell surface and constitutively release sTNFR2. Activation of the cells by TCR stimulation resulted in a temporary increase in processing of TNFR2. This activation-induced increase required both actin remodeling and lipid raft formation and was dependent on MAPK/ERK signaling. Furthermore, we identified ADAM17 as the protease responsible for TNFR2 processing by CD8+ T cells. Inhibition of ADAM17 resulted in an interaction of both membrane-bound TNF-α and TNFR2 on the CD8+ T-cell surface. Moreover, we observed similar activation thresholds for both TNF-α and TNFR2 processing suggesting that sTNFR2 is important for regulation of sTNF-α levels. Furthermore, genetic deletion or in vivo antibody blockade of TNFR2 during influenza infection in mice enhanced the levels of sTNF-α supporting this hypothesis. Taken together, this study elucidates some of the cellular mechanisms regulating TNFR2 processing and demonstrates that TNFR2 plays an important role in regulating TNF-α levels during infection.
Identification of Fli-1 as a novel regulator of the inflammatory chemokine CCL5/RANTES (CCR5P.253)
Authors: Lennard Richard Mara , Sato Shuzo , Suzuki Eiji , Williams Sarah , Nowling Tamara , Zhang Xian
Published: January 2023

The Fli-1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE) in both human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival and significantly decreased infiltrating inflammatory cells into the kidney. The chemokine, CCL5, attracts inflammatory cells and expression is increased in patients with SLE. In this study, decreased expression of CCL5 mRNA was observed in kidneys of NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly impaired in endothelial cells treated with Fli-1 specific siRNA compared to controls. Fli-1 binds to Ets binding sites in the CCL5 promoter and transfection assays show that Fli-1 drives transcription in a dose-dependent manner. Mutation of the Fli-1 DNA binding domain demonstrates that activation of the CCL5 promoter is through direct binding by Fli-1. Ets1 also drives transcription from the CCL5 promoter, although Fli-1 driven transactivation is significantly stronger. The Ets1 transcription factor acts as a dominant negative to Fli-1, indicating that the genes may share a DNA binding site. Fli-1 is a novel regulator of CCL5 gene transcription. Combined with previous results, demonstrating that Fli-1 regulates MCP-1, we conclude that Fli-1 is a critical regulator of proinflammatory chemokines and affects disease pathogenesis by regulating factors involved inflammatory cell recruitment.
Essential role of IL-17 in renal damage associated with autoimmune glomerulonephritis (CCR6P.279)
Authors: Ramani Kritika , Pawaria Sudesh , Maers Kelly , Biswas Partha
Published: January 2023

Autoimmune Glomerulonephritis results in significant mortality and morbidity in numerous auto-inflammatory kidney diseases. The chronic inflammatory state is considered to be a driving force for AGN. In recent years the IL-23/Th17 axis appears to contribute to the pathogenesis of human and experimental AGN. However, the role of IL-17 receptor signaling in renal pathology associated with AGN is poorly defined. Here, using a murine model of accelerated experimental autoimmune glomerulonephritis, we showed that IL-17RA signaling is critical for the development of renal pathology and inflammatory changes in the kidney. Consequently, mice deficient in IL-17RA signaling exhibited diminished renal inflammation and infiltration of the innate effector cells in the kidney, correlating with the reduced expression of IL-17 responsive genes. IL-17 enhanced the production of proinflammatory cytokines and chemokines from tubular epithelial cells, which are implicated in the recruitment of innate effector cells in the kidney. Finally, neutralization of IL-17A ameliorated renal pathology in wild type mice following aEAGN induction. These results clearly demonstrated that IL-17RA signaling significantly contributes to renal tissue injury in AGN and suggest that blocking IL-17RA may be a promising therapeutic strategy for the treatment of proliferative and crescentic glomerulonephritis.
Heat shock protein 90 involves IL-17-mediated skin inflammation under thermal stimuli (CCR6P.280)
Authors: Woo So-Youn , Kim Bo Kyung , Park Minhwa , Kim Ji-Yon , Cho Kyung-Ah , Lee Kyung Ho
Published: January 2023

The pathogenesis of inflammatory skin disease involves an interaction between immune cells and keratinocytes and Th17-mediated immune response is one of them. Several chemokines (CXCL1, CXCL5, and CXCL8) and antimicrobial peptides (β-defensin [BD1], LL37, S100A8 and S100A9) were transcriptionally upregulated in keratinocyte cell line (HaCaT) upon stimulation with IL-17. IL-17 binds to a heterodimeric receptor consisting of IL-17RA and IL-17RC, and adaptor protein Act1 binds to IL-17RA and mediates IL-17-induced signaling pathways. Because heat shock protein 90 (Hsp90) activity is required for IL-17 signalling via Act-1, we have evaluate the effect of Hsp90 inhibition on keratinocytes on IL-17-mediated cytokine and antimicrobial peptides. HaCaT cells were treated with 100 ng/ml of IL-17 or IL-22 for 24 hrs followed by 1 uM of 17-allylaminogeldanamycin (17-AAG) and collected for the analysis. We found that Hsp90 inhibition by 17-AAG under IL-17 stimulation led to decrease of CXCL1 but increase of CXCL5. In contrast to IL-17 and 17-AAG stimulation of HaCaT cells at 37°C, expression of Act1 and Act1 binding Hsp90 were increased after heat stimulation at 42°C for 30 min. These observation demonstrate the possible immune modulatory effect of the heat on keratinocytes during progression of inflammatory skin disease
Prostaglandin E2 inhibits IL-27 production by bone marrow-derived dendritic cells (CCR6P.281)
Authors: Hooper Kirsten , Yen Jui-Hung , Ganea Doina
Published: January 2023

Interleukin-27 (p28/EBI3) is expressed by activated antigen presenting cells and has been shown to have immunosuppressive functions including the inhibition of Th17 cell development and the induction of type 1 regulatory T cells (Tr1). In vivo studies demonstrated the anti-inflammatory effects of IL-27 in murine models of collagen-induced arthritis (CIA), inflammatory bowel disease (IBD), and experimental autoimmune encephalomyelitis. We showed previously that PGE2 has proinflammatory effects in models of CIA and IBD through the stimulation of IL-23 production and subsequent development of Th17 cells. Here we report on a novel proinflammatory function through the inhibition of IL-27 production by LPS-stimulated murine BMDC. A similar effect was observed in the dendritic, macrophage and microglia cell lines DC2.4, RAW264.7 and BV2. We determined that the effect of PGE2 was mediated through EP2/EP4 receptors and induction of cAMP. Studies targeting various downstream signaling pathways are in progress. These studies further elucidate the mechanisms involved in the inflammatory role of PGE2, and might identify additional therapeutic targets for the treatment of inflammatory autoimmune diseases.
Multiple TRIM proteins are targets of autoimmune response in lupus and Sjogren's syndrome. (HUM7P.308)
Authors: Rybakowska Paulina , Wolska Nina , Klopocki Arkadiusz , Sivils Kathy , James Judith , Bagavant Harini , Deshmukh Umesh
Published: January 2023

TRIM21 belongs to the large family of tripartite motif containing proteins, and is often targeted by autoantibodies in lupus and Sjogren’s syndrome. Considering the significant protein domain homology between different TRIM proteins, we hypothesized that additional TRIM proteins are targets of autoimmunity. Based on the literature, in this study we investigated autoantibody responses to TRIM38. While 9% of lupus patients (n=149) had anti-TRIM38 antibodies, the incidence in Sjogren’s syndrome patients (n=150) was 12%, and in controls (n=50) it was 4%. With respect to TRIM21, the incidence was 62%, 68%, and 4% respectively. In Sjogren’s syndrome patients, the presence of anti-TRIM38 antibodies was closely associated with the increased severity of dry eye parameters. Epitope mapping studies showed that anti-TRIM21 antibodies reacted with the RING, Coiled coil and PRY-SPRY domains of TRIM21, whereas anti-TRIM38 antibodies reacted only with the Coiled coil and PRY-SPRY domains of TRIM38. All anti-TRIM38 positive patients also had anti-TRIM21 antibodies. Affinity purified anti-TRIM21 antibodies from lupus patients did not immunoprecipitate TRIM38, indicating lack of cross-reactivity at B cell level. However, we observed T cell cross-reactivity between TRIM21298-312 and TRIM38302-316. Our study suggests that immune responses to TRIM proteins can evolve through epitope spreading and contribute towards exacerbating the pathogenesis in autoimmune disorders.
Soluble Fas ligand activates the CX3CL1-CX3CR1 axis by enhancing proteinase activity during arthritis in Fas-independent manner (HUM7P.312)
Authors: Kim Donghyun , Jeong Dongjin , Kim Hye Sung , Kim Hye Young , Lee Seung Hyo , Shin Eui-Cheol , Lee Eun Bong , Chung Doo Hyun
Published: January 2023

Fas/Fas ligand (FasL)-mediated apoptotic and non-apoptotic processes have long been known to regulate inflammatory responses. FasL exerts biological functions through two different forms in vivo: a membrane-bound FasL (mFasL) and soluble FasL (sFasL). It is unknown whether sFasL contributes to the pathogenesis of joint inflammation. Here, we found that sFasL triggered soluble CX3CL1 generation from fibroblast-like synovial cells by enhancing proteinase activity, which induced CX3CR1+ joint immune cell migration and chemokine production in an apoptosis-independent manner in mice and humans. Murine and human synovial cells deficient for Fas were minimally affected by sFasL-mediated induction of joint inflammation. Blockade of sFasL or CX3CR1 attenuated antibody-induced arthritis in mice. Therefore, we propose a novel function of sFasL in regulating joint inflammation via the CX3CL1-CX3CR1 axis, a process that occurs independently of Fas ligation. This novel axis may be a useful therapeutic target for arthritis.
Adults with indolent systemic mastocytosis have elevated peripheral blood myeloid-derived suppressor cells compared to chronic urticaria and healthy controls. (HUM7P.320)
Authors: Damle Sheela , Lofti-Emran Sahar , Ward Brant , Saleem Sheinei , Conrad Daniel , Schwartz Lawrence
Published: January 2023

MDSCs are a heterogeneous population of immature myeloid cells shown to be immunomodulatory in many diseases. In humans, they are identified by surface expression of CD33 and CD11b and lack of HLA DR. MDSCs are further divided into monocytic (M-MDSCs, CD14+) and granulocytic (G-MDSCs, CD15+) subsets. Our previous data showed that the prevalence and suppressive activity of murine MDSCs was augmented by the presence of mast cells (MCs). Indolent systemic mastocytosis (ISM) is a human disease caused by the clonal expansion of MCs. We propose that increased MCs and MC mediators frequently associated with mastocytosis produce an increase in peripheral blood MDSCs. Peripheral blood mononuclear cells (PBMCs) were isolated from blood obtained from adults with ISM and from chronic urticaria (CU, drug control), and healthy (H) age-matched controls. PBMCs, labeled with antibodies against HLA-DR, CD33, CD11b, CD15, and CD14, were analyzed by flow cytometry. MDSC concentrations in blood (x 106 cells/mL, mean ± standard error) were calculated. MDSC concentration was elevated (p<0.0001) in ISM (15±7) versus CU (1.4±0.4) and H (2.8±0.4). The G-MDSC subset (9.4±4, 0.6±0.1, 1.5±0.3 for ISM, CU, and H respectively, p<0.001) largely accounted for this elevation. A similar, non-significant trend (p=0.24) is seen in the M-MDSCs (1.6±0.5, 0.4±0.1, 0.7±0.3). Overall, these results demonstrate that MDSCs, particularly G-MDSCs, are elevated in the blood of patients with ISM, warranting further study.
Interleukin-17 Targets Ppp6c for epidermal hyperplasia via NF-kappaB-dependent MicroRNA miR-31 induction (HUM7P.300)
Authors: Wang Honglin , Yan Sha , Zhang Lingyun , Ke Fang , Bai Jing , Liu Zhaoyuan , Xu Zhenyao , Liu Jinliu
Published: January 2023

The efficacy of monoclonal antibodies against either interleukin (IL)-17 or the IL-17 receptor in psoriasis therapy provides strong evidence that IL-17 is the major inflammatory mediator in this disease. However, it is unclear how IL-17 induces epidermal basal keratinocyte hyperproliferation in psoriasis. To explore the mechanism of IL-17 in epidermal hyperplasia in psoriasis, we focused on microRNA(s) induced by IL-17 as these small non-coding RNAs play an important role in regulation of cellular proliferation. Among all the microRNAs we screened, microRNA-31 (miR-31) is the only one extremely sensitive towards IL-17 for its induction in keratinocytes. Its expression in both the epidermis of lesional skin of human psoriasis patients and mouse psoriasis models is IL-17 inducible and NF-kappaB dependent. A causal relationship between IL-17-dependent miR-31 induction and basal keratinocyte hyperproliferation has been then established by constructing and analyzing epidermal conditional knockout of miR-31 (miR-31fl/fl/Keratin 5-Cre) engineering animals. A careful genomic analysis led us to realize that miR-31 targets protein phosphatase 6 (ppp6c), a negative regulator restricting G1 to S phase progression. As expected, ppp6c is suppressed by IL-17 through the miR-31 induction and is diminished in human psoriatic epidermis. We thus have uncovered a novel mechanism that helps explain how T helper (Th)-17 cells signal to basal keratinocytes for their hyperproliferation in psoriasis.
Effect of CCL1 antisense ODN on the M2bMφ elimination and subsequent generation of M1Mφ in bacterial translocation sites of mice irradiated with whole body 137Cs γ-rays (WBI-mice) (INC7P.411)
Authors: Kobayashi Makiko , Ito Ichiaki , Nishiguchi Tomoki , Cannon Krystal , Suzuki Sumihiro , Loucas Bradford , Suzuki Fujio
Published: January 2023

An essential host defense against gut-associated sepsis is a local antibacterial innate immunity mainly expressed by M1Mφ (IL-10-IL-12+ Mφ with marked bactericidal activity). Although Mφ are radioresistant, M1Mφ are not generated in bacterial translocation sites (lamina propria and mesenteric lymph nodes) of WBI-mice. M2bMφ (IL-10+IL-12-CCL1+ Mφ) appearing in response to radiation injuries inhibit Mφ conversion from resident Mφ to M1Mφ. CCL1 has been characterized as an essential chemokine for the maintenance of M2bMφ properties, and M2bMφ are shown to be major CCL1 producer cells. In this study, we tried to eliminate M2bMφ and induce M1Mφ in the bacterial translocation sites of WBI-mice by treatment with CCL1 antisense ODN. The ODN was administered s.c. to WBI-mice twice a day for 1 week starting 3 days after γ-irradiation (8-11 Gy). One day after the final treatment, these mice were stimulated orally with 107 heat-killed E. faecalis. Then, F4/80+ Mφ were isolated from the bacterial translocation sties of these mice and tested for their M1Mφ and M2bMφ properties. In the results, M1Mφ were not detected in the translocation sites of WBI-mice stimulated with the antigen. However, M1Mφ were isolated from WBI-mice treated with the ODN and stimulated with the antigen. M2Mφ were not isolated significantly from these mice. These results indicate that M1Mφ are inducible by the bacterial antigen in 8-11 Gy WBI-mice after treatment with CCL1 antisense ODN.
Effect of CCL1 antisense ODN on spontaneous bacterial translocation and subsequent sepsis in mice irradiated with whole body 137Cs γ-rays (WBI-mice) (INC7P.412)
Authors: Suzuki Fujio , Ito Ichiaki , Nishiguchi Tomoki , Cannon Krystal , Suzuki Sumihiro , Loucas Bradford , Kobayashi Makiko
Published: January 2023

Radiation victims who suffer from gastrointestinal injuries are extremely susceptible to gut-associated sepsis. In our previous studies, M1Mφ located in the lamina propria and mesenteric lymph nodes were shown to be major effector cells against bacterial translocation. However, M1Mφ were not generated in these organs of WBI-mice who are carriers of M2bMφ (inhibitor cells for Mφ conversion from resident Mφ to M1Mφ). CCL1 produced by M2bMφ is essential for the maintenance of M2bMφ properties. In this study, we examined the effect of CCL1 antisense ODN on gut-associated sepsis stemming from endogenous intestinal microflora in 9 Gy WBI-mice. CCL1 antisense ODN was administered s.c. to WBI-mice twice a day for 1 week starting 3 days post-exposure to γ-rays. This group of mice was designated as treated mice. The mortality rates and bacterial growth in the organs and blood of treated mice were compared to those of control mice (WBI-mice treated with scrambled ODN). In the results, control mice died within 14 days of γ-irradiation, while 30% of treated mice survived more than 30 days after the irradiation. Bacteria progressively grew in the mesenteric lymph nodes, liver and blood of control mice, while the pathogens were not isolated significantly from these organs of treated mice. These results indicate that sepsis stemming from spontaneous bacterial translocation is noticeably mitigated in WBI-mice treated with CCL1 antisense ODN.
Respiratory epithelial cell innate immunity against Mycobacterium tuberculosis is deregulated by air pollution particulate matter (PM) (INC7P.417)
Authors: Schwander Stephan , Cantarella Pasquale , Sarkar Srijata , Rockafellow Megan , Rivas-Santiago César
Published: January 2023

Epidemiological studies demonstrate a link between exposure to indoor air pollution and cigarette smoke and risk of development of Tuberculosis (TB). The role of ambient air pollution PM in human antimycobacterial immunity has not been studied. We examined the effects of PM (<2.5 and <10 μm diameter, from Mexico City) on Mycobacterium tuberculosis (M.tb)-induced expression of mRNA and protein of human β-defensin 2 and 3 (HBD-2,3, antimicrobial peptides), IL-8 and MCP-1 by qRT-PCR and ELISA, respectively. In addition, cell-mediated growth control of M.tb was measured by colony forming units (CFU) assay. Respiratory alveolar type II epithelial (A549) cells were exposed for 18 hours to PM at 0.1, 1, 10 and 50 μg/mL followed by M.tb exposure at a multiplicity of infection of 10 (MOI 10) for an additional 18 hours. We observed that PM exposure decreased the expression of HBD-2 and HBD-3 (p<0.05), while IL-8 and MCP-1 expression remained unaltered in M.tb-infected A549 cells. Decreased antimicrobial peptide expression was not due to decreased cell viability upon PM exposure (shown by MTS and LDH assay). Interestingly, A549 cells exposed to PM exhibited a decreased capability to control M.tb growth (p<0.05, CFU assays). We conclude that PM inhibits the gene expression and protein production of antimicrobial peptides in A549 cells, thus enhancing intracellular growth of M.tb that could potentially increase the susceptibility to M.tb infection.
Neutrophils prime a long-lived effector macrophage phenotype that mediates accelerated helminth expulsion (INC7P.423)
Authors: Chen Fei , Wu Wenhui , Millman Ariel , Craft Joshua , Chen Eunice , Urban Joseph , Kim Charlie , Gause William
Published: January 2023

The innate immune cells mediating macroparasite clearance remain largely undefined. We examined their role in acquired resistance to the parasitic nematode, Nippostrongylus brasiliensis (Nb) hypothesizing that they may mediate the markedly accelerated CD4+ cell-independent worm clearance occurring after secondary inoculation. After secondary inoculation, parasitic larvae in the lung are surrounded by macrophages and showed reduced ATP, indicating impaired metabolism. As late as one month after Nb inoculation, lung macrophages, transferred to naïve recipients, accelerated parasite clearance. Primed macrophages adhered to larvae in vitro and triggered increased mortality through CD11b-dependent mechanisms. Neutrophil depletion impaired the recall response and depletion of neutrophils in primed mice abrogated the protective effects of transferred macrophages in recipient mice and inhibited their binding to L3. Global transcriptome analyses of sort-purified lung neutrophils from Nb inoculated mice revealed a markedly different expression pattern from lung neutrophils isolated from LPS inculated mice, indicating that neutrophils, like macrophages, develop a distinct alternatively activated phenotype (N2) after helminth infection. These data thus indicate that differentially activated neutrophils in the context of a type 2 immune response prime a long-lived effector macrophage phenotype that binds metazoan parasites and directly mediates rapid worm damage and clearance.
Complementary immune roles for infected and uninfected cells during bacterial infection (INC7P.425)
Authors: Copenhaver Alan , Casson Cierra , Nguyen Hieu , Fung Thomas , Duda Matthew , Roy Craig , Shin Sunny
Published: January 2023

The innate immune system responds to virulent pathogens, yet many pathogens manipulate host signaling pathways which should limit immune activation. To understand how the immune system overcomes pathogenic manipulation we study the intracellular bacterium Legionella pneumophila, the cause of the severe pneumonia Legionnaire’s disease. Legionella utilizes a type IV secretion system to inject bacterial proteins into the cytosol of infected cells. Several of these bacterial effector proteins inactivate host cell factors involved in protein translation. Despite Legionella's ability to block host protein translation, inflammatory cytokines are still made against Legionella. It is unclear how infected cells can mount a cytokine response when host protein synthesis is blocked. Our studies demonstrate that infected cells do not produce many cytokines, such as IL-6 and TNF, critical for controlling Legionella infection. Instead, uninfected, bystander cells produce these cytokines. Infected host cells do produce IL-1 de novo. These data suggest that infected cells have mechanisms to overcome protein synthesis inhibition to produce IL-1 and that uninfected, bystander cells are important contributors to the immune response during infection with Legionella. This mechanism of immune activation has broad significance as many other bacterial pathogens manipulate immune cell signaling.
TLR7 and CD28 regulate plasmacytoid dendritic cell IFN-I induction and silencing during chronic viral infection (INC8P.439)
Authors: Macal Monica , Tam Hurtado Miguel , Chang Aaron , Hesser Charles , Zuniga Elina
Published: January 2023

Type I Interferons (IFN-I) are potent cytokines essential for immune surveillance against many pathogens including persistent viruses such as human immunodeficiency virus (HIV) in humans and Lymphocytic Choriomeningitis Virus (LCMV) in mice. Plasmacytoid dendritic cells (pDCs) are specialized to produce IFN-I but the molecular mechanisms underlying their initial IFN-I production and the subsequent exhaustion that occurs during chronic viral infection remain to be fully elucidated. Using chronic LCMV infection in mice, we demonstrated that pDCs initially sensed and produced abundant IFN-I in a TLR7-dependent manner. Furthermore, gene expression profile comparisons of pDCs and conventional (c) DCs identified CD28, a prototypic T cell stimulatory receptor, as highly expressed in pDCs. Strikingly, CD28 acted in a cell-intrinsic manner to negatively regulate the magnitude of pDC IFN-I production upon TLR stimulation in vitro and early after in vivo LCMV infection, but was dispensable for the subsequent IFN-I silencing. In contrast, cell-intrinsic TLR7 signaling was essential for the later pDC IFN-I exhaustion observed in chronic LCMV infection and compromised innate responses against secondary opportunistic pathogens. These results highlight a novel role for CD28 in limiting the magnitude of pDC IFN-I production and a dual role for TLR7 in mediating both induction and subsequent exhaustion of innate responses during in vivo chronic viral infection.
Critical role of Neutrophils in Respiratory Syncytial Virus (RSV) induced disease pathogenesis (INC8P.440)
Authors: Kolli Deepthi , Yueqing Zhang , Palkowetz Kimberly , Garofalo Roberto , Casola Antonella
Published: January 2023

Neutrophils comprise the largest number of immune cells in the human body, and being the first immune cells to the site of injury and infection, neutrophils act as the first line of defense against harmful pathogens. RSV has been identified as a leading cause of respiratory tract illness in children worldwide. RSV induced disease is characterized by epithelial desquamation, neutrophilic bronchiolitis and pneumonia. The role of neutrophils in innate immune responses to RSV infection has not been fully characterized. We have employed anti-Ly6G clone monoclonal antibody 1A8 to specifically deplete neutrophils in Balb/c mice to study the role of neutrophils in RSV induced disease pathogenesis. Neutrophil depletion in mice infected with RSV A2 or Long strain caused a significant increase in body weight loss, airway hyper responsiveness and lung inflammation. Even though no effect on peak viral replication was observed; a significant reduction in early viral load and a delayed viral clearance in mice lacking neutrophils were observed. While depletion of neutrophils has no effect on the production of BAL type I IFNs, a significant increase in Th17 group of cytokines (IL-17A, IL-17F, IL-22, IL-23p19 and CD40L), G-CSF, KC and MIP-1β was observed. Moreover, we observed a significant reduction in lung DCs, pDCs and virus specific CD4 and CD8 cells in neutrophil depleted mice. Our study demonstrates that neutrophils play a critical role in disease progression of RSV infections.
Antiviral regulation in porcine monocytic cells at different activation states (INC8P.430)
Authors: Sang Yongming , Rowland Raymond , Blecha Frank
Published: January 2023

Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of macrophages and DCs and interferes in antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages at non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded to viral infection differently. Based on gene ontology analysis, two approaches were used: 1) pharmaceutical modulation of cellular lipid metabolism and 2) in situ PRRSV replication-competent expression of IFN-α; both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days post infection in macrophages. These findings suggest an intricate interaction of viral infection with activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity.
The role of interleukin-27 in influenza A and Staphylococcus aureus co-infection (INC8P.435)
Authors: Robinson Keven , Lee Benjamin , Mandalapu Sivanarayana , Scheller Erich , Kolls Jay , Alcorn John
Published: January 2023

Rationale: Influenza A represents a significant cause of worldwide morbidity and mortality and results in increased susceptibility to secondary bacterial infections in the lung. We have previously shown that Influenza A impairs bacterial host defense through inhibition of Type 17 immunity. IL-27 is known to inhibit Type 17 immunity, suggesting a potential critical role for IL-27 in viral and bacterial co-infection. Methods: Wild-type and IL-27 receptor α knock-out (IL-27Ra-/-) mice were infected with Influenza A H1N1 (PR/8/34) or vehicle for 6 days followed by challenge with Staphylococcus aureus (ATCC 49775) or vehicle for 24 hours. Lung inflammation, bacterial burden, gene expression, and cytokine production were determined. Results: IL-27Ra-/- mice challenged with influenza A had increased weight loss compared to controls. In addition, IL-27Ra-/- mice displayed significantly decreased IL-10 production. IL-27Ra-/- mice co-infected with influenza and S. aureus had improved bacterial clearance compared to wild-type controls despite worsened lung pathology. Importantly, there were increased IL-17 and IL-22 expression and significantly decreased IL-10 production in the IL-27Ra-/- mice. Conclusions: These data suggest that IL-27 contributes to enhanced susceptibility to S. aureus pneumonia following influenza infection through the induction of IL-10 and regulation of Type 17 immunity.
NKT cells act as helper T cells to cytotoxic T cells by direct recognition of CD1d on T cells (IRC8P.487)
Authors: Oh Sejin , Park Se-ho
Published: January 2023

Cytotoxic T lymphocytes (CTLs), known as CD8+ T cells, recognize their targets by binding to antigens associated with MHC class I. NKT cells recognize foreign glycolipid antigens and endogenous glycolipid antigens presented by CD1d which is an MHC class I- like molecule. NKT cells have been shown to be essential for the optimal activation of CTLs. In this study, we investigated the mechanism how NKT cells provide help for the CTL responses. We observed significantly higher production of IFN-γ by antigen-stimulated OT-1 CD8+ T cells in the presence of NKT cells. This enhanced CTL activation by NKT cells was observed even in the antigen presenting cell (APC)-free conditions. We also found that CD8+ T cells start to up-regulate surface expression of CD1d reaching highest level at around 18 hours upon antigen recognition. These results suggest that not only APC, but also activated CTLs receive direct help from NKT cells by increasing CD1d expression on their cell surface when they recognize cognate antigens.
Role of Itk in Th17 mediated inflammation model hypersensitivity pneumonitis (IRC8P.491)
Authors: Carter Chavez
Published: January 2023

Hypersensitivity Pneumonitis (HP) is a lung disease caused by repeated inhalation of environmental antigens leading to inflammation, tissue scarring, and loss of lung function. This pathology is believed to be due to the increased IL-17A, a cytokine secreted primarily by Th17 cells that induces recruitment of inflammatory cells such as neutrophils. The thermophile Sacharopolyspora rectivigula (SR) causes HP, using a murine model of SR exposure; we study the molecular mechanism of this disease development. Using novel IL-17A-GFP reporter mice, our data suggests that the high levels of IL-17A induced in response to SR are produced in part by CD4+ T cells and not neutrophils. The Tec family tyrosine kinase Itk regulates T cell activation and cytokine production in conventional Th17 cells but Itk-/- mice still develop HP. Histology shows an increase in inflammatory cells in lung airways as well as decline of lung tissue architecture. Itk-/- lungs also have high levels of IL-17A mRNA expression, and an increase in the number of CD4+ IL-17A producing cells. However, Itk-/- mice lack IL-17A producing γδ T cells in the early stage of HP, which recovers in the later stage. This directly coincides with the emergence of IL-17A producing CD4+γδ T cells in the lungs of Itk-/- mice exposed to SR. We conclude that Itk regulated signals are not critical for the production of IL-17A in response to SR, and Itk may differentially regulate the production of IL-17A in different types of T cells.
Anti-CD3 antibody treatment induces hypoglycemia and super tolerance to glucose challenge in mice through enhancing glucose consumption by activated lymphocytes (IRC8P.474)
Authors: Xia ChangQing , Chernatynskaya Anna , Looney Benjamin , Clare-Salzler Michael
Published: January 2023

Anti-CD3 antibody has been employed in several clinical settings. However, its effect on glucose metablolism has not been well investigated. In the current study, we investigated how anti-CD3 treatment affected blood glucose levels in mice. We found that anti-CD3 treatment induced immediate reduction of blood glucose as early as 3-4 hours post-treatment. Furthermore, we observed that a single dose of anti-CD3 treatment was able to correct hyperglycemia in all diabetic NOD mice. This glucose-lowering effect was not attributable to major T cell cytokines secreted by anti-CD3 activated T cells. Of interest, when tested in a normal strain of mice (C57BL/6), the C-peptide levels in anti-CD3-treated animals were significantly lower than those of control mice. Paradoxically, the anti-CD3-treated animals were highly tolerant to the exogenous glucose challenge. In addition, we found anti-CD3 treatment significantly induced activation of T and B cells in vitro and in vivo. Our further studies showed that anti-CD3 treatment lowered the glucose levels in T cell culture media, and increased the intracellular transportation of 2-NBDG particularly in activated T and B cells. In addition, injection of anti-CD3 antibodies upregulated Glut1 expression in spleen cells. The present study suggests that anti-CD3 therapy-induced hypoglycemia likely results from increased glucose transportation and consumption by the activated lymphocytes.
Late antigen recognition at the memory checkpoint is necessary for CD4+ T cell memory formation. (IRC9P.708)
Authors: Bautista Bianca , McKinstry Kai , Strutt Tara , Swain Susan
Published: January 2023

During the contraction phase of an influenza immune response, the majority of responding T cells undergo Bim-driven apoptosis leaving a small, heterogeneous population of T cells that will survive to form memory. The regulation of this process is poorly understood. Recently, our lab defined a “memory checkpoint” during which highly differentiated effector CD4+ T cells require autocrine IL-2 immediately preceding contraction to become memory. To determine whether antigen recognition is required for IL-2 production during the memory checkpoint, we developed an adoptive transfer model to modulate antigen. Donor CD4+ effectors transferred to hosts without antigen underwent immediate cell death and failed to form memory, suggesting that antigen recognition is indeed the key event at this checkpoint. The ex vivo evaluation of CD4+ effector cells at the memory checkpoint revealed that both co-stimulation and IL-2 signaling contribute to survival. TCR stimulation during the memory checkpoint resulted in higher sustained expression of memory markers CD127 and CXCR3. The survival to memory is mediated in part by a sustained reduction in Bim expression, as the requirement for late antigen interactions was eliminated in Bim heterozygous CD4+ T cells. These findings demonstrate that effectors generated by influenza must recognize antigen at the memory checkpoint to become memory.
Tapeworm antigens block LPS-induced inflammatory mediator production by macrophages via IL-10 and recruit myeloid derived suppressor cells (IRC10P.463)
Authors: Reyes Jose , Fernado Maria , Matisz Chelsea , McKay Derek
Published: January 2023

Parasites have been shown to suppress concomitant inflammatory disease in mouse models: mice infected with the rat tapeworm, Hymenolepis diminuta, are protected from colitis. Assessing the mechanism by which this worm may inhibit inflammation the objective was to determine if a PBS-soluble extract of H. diminuta antigens (HdAg) directly affected innate immune cell activity. Bone marrow macrophages (BMMs) exposed to LPS ± HdAg for 48 hr and IL-1β, IL-6, TNFα, NO and IL-10 measured in culture supernatants 1, 6, 24 and 48 hr later. Additional studies included the addition of an anti-IL-10 antibody to the cell culture. In other experiments, mice received HdAg (500 μg, ip) and 24 hr later peritoneal exudate cells were analyzed by flow cytometry. LPS-induced IL-1β and TNFα, but did IL-6 or NO, were significantly reduced by ~50% by HdAg co-treatment. BMM treated with HdAg showed a dose- and time-dependent increase in IL-10, paralleled by increased STAT3 phosphorylation suggesting IL-10 feedback on the BMM: anti-IL-10 prevented the HdAg inhibition of LPS-evoked TNFα by BMM. Mice given HdAg recruited a F480+Gr1+ cell population that boosted IL-10 production by anti-CD3/CD28-activated T cells. In conclusion, HdAg enhanced IL-10 production by LPS-stimulated BMMs which in turn down-regulated TNFα and IL-1β secretion. This, coupled to the recruitment/generation of MDSC, likely contributes to the suppression of pro-inflammatory events in H. diminuta infected mice.
Palmitoylation is required for Fas receptor clustering and cell death in CD4+ T cells (LYM7P.722)
Authors: Cruz Anthony , Ramaswamy Madhu , Sengupta Prabhudda , Lippincott-Schwartz Jennifer , Siegel Richard
Published: January 2023

Fas-Fas ligand (FasL) interactions are critical for apoptosis and T cell homeostasis, with deficiencies in either Fas or FasL leading to autoimmunity in both mice and humans. Fas can trigger apoptotic cell death directly through recruitment of a death-inducing signaling complex (DISC) consisting of FADD and Caspase-8. We have previously shown a correlation between lipid raft-associated Fas and sensitivity to apoptosis in CD4+ T cells, with the Fas-sensitive memory population having increased lipid raft receptor localization. Palmitoylation of human Fas at cysteine 199 (Cys-194 in mouse) is required for localization to lipid rafts. To investigate the role of palmitoylation in Fas signaling, we developed a transgenic mouse defective in palmitoylation of Fas receptor (Fas C194→V) backcrossed to the Fas-defective lpr/lpr mouse (FasC194Vlpr/lpr). These mice developed autoimmunity characteristic of lpr mice, but CD4-CD8- “Double-Negative” (DN) T cells did not accumulate. T cells expressing C194V mutant Fas were resistant to Fas- and TCR-induced cell death, indicating lipid raft localization is required for the apoptotic function of Fas, but not accumulation of DN T cells. High-resolution PALM imaging of cells expressing wild-type or C194V mutant Fas revealed receptor clustering into higher-order oligomers at the plasma membrane required palmitoylation. These findings identify palmitoylation as a key post-translational modification of Fas that controls the apoptotic signal.
High-throughput T cell receptor (TCR) sequence tracking reveals clonal intrinsic selection for skin resident T cells in human genital HSV-2 infection (MUC7P.771)
Authors: Zhu Jia , Kask Angela , Diem Kurt , Robins Harlan , Wald Anna
Published: January 2023

T cells resident in the barrier surface have been implicated to play an important role in rapid control of invading pathogen. Human genital herpes infection is characterized by frequent bursts of reactivating HSV-2 virus and rapid containments of host immune responses in genital skin and mucosa surface, thus a valuable human disease model for deciphering tissue resident immunity in vivo. Previously, we showed that both CD4 and CD8 T cells persisted locally for prolonged time period after complete resolution of HSV-2 infection. To understand the mechanism of T-cell resident immunity in the barrier tissue, we used high-throughput TCR sequencing to investigate the persistence, breadth and clonality of TCR vβ repertoire in sequential biopsy tissues obtained during HSV-2 lesion evolution from active lesion, healed, to 2, 4, and 8 weeks post healing. Each tissue TCR vβ repertoire is consisted of thousands unique productive sequences, a diverse yet oligoclonal T-cell profile. Interestingly, high frequency TCRVβ sequences dominated in post-healed tissues were poorly represented in circulating T cells from the blood, vice versa was also true. Although hundreds of TCRVB sequences were found commonly shared among sequential biopsies in the same subjects, only a few clones showed increased persistence, emerging from low frequency in lesion to dominance in post healed tissue. These data suggest a T-cell intrinsic mechanism in dictating T cell survival and/or self-renewal in human tissue.
KIR2DS1+ decidual natural killer cells show ex-vivo activation and cytotoxicity but not cytokine secretion (MUC7P.756)
Authors: Crespo Angela , Tilburgs Tamara , Strominger Jack
Published: January 2023

Decidual natural killer cells (dNK) are the most abundant lymphocytes at the fetal-maternal interface. dNK have been shown to have limited cytotoxic capacity but secrete high levels of cytokines to facilitate extravillous trophoblast (EVT) invasion. Previous studies have shown that mothers expressing the activating Killer Immunoglobulin-like Receptor-S1 (KIR2DS1) are protected from pregnancy complications when carrying a fetus expressing HLA-C2 (KIR2DS1 ligand) (Hiby et al, 2010). In this study, a significantly higher percentage of dNK expressed KIR2DS1 and KIR2DL1 than pNK. dNK and pNK expressed similar levels of perforin and granzyme B, but dNK contained markedly increased granulysin levels. Although pNK were more cytotoxic than dNK against HLA- targets, this cytotoxicity was inhibited by HLA-C1+ and HLA-C2+ targets. In contrast, the cytotoxicity of dNK from KIR2DS1+ mothers was not efficiently inhibited by HLA-C2+ targets. Moreover, higher phosphorylation of ZAP70/SYK in dNK from KIR2DS1+ mothers than KIR2DS1- mothers was showed. Thus, a higher activation state of KIR2DS1+ dNK cells interacting with HLA-C2+ targets may dominate over inhibition by HLA-C2 through KIR2DL1+ dNK. Contrary to stimulation of NK cell receptors with antibodies or cell lines, stimulation of dNK and pNK with EVT revealed no specific production of NK cytokines such as IFNγ or IL-8. These results suggest a dominant role for KIR2DS1+ dNK cells in cytotoxicity, but not cytokine secretion.
Expression and regulation of the TGF-β activating integrin αvβ8 on human mononuclear phagocytes (MUC8P.809)
Authors: Travis Mark , Fenton Thomas , Schulthess Julie , Arancibia Carolina , Lehtinen Markus , Powrie Fiona
Published: January 2023

Induction of Foxp3+ Tregs is important in suppressing immune responses to commensal bacteria and innocuous antigen in the gut. Previous work from our lab has shown that, in mice, expression of the integrin αvβ8 on intestinal dendritic cells (DC) is important for activation of the cytokine TGF-β and subsequent Treg induction, with lack of this pathway resulting in severe inflammatory bowel disease (IBD). Additionally, integrin αvβ8 is highly expressed on murine intestinal CD103+ DC compared to other intestinal mononuclear phagocytes, and is required for enhanced Treg induction by CD103+ intestinal DC and maintenance of immune homeostasis. However, pathways that control integrin αvβ8 expression are poorly understood and the role of integrin αvβ8 in human immunology is currently unknown. Here, we show that integrin αvβ8 is expressed preferentially on CD64+ macrophages and to a lesser extent on DC populations, in contrast to what is found in mouse models. Analysis of monocyte-derived macrophage populations indicated that M2-like macrophage populations preferentially expressed integrin αvβ8. Interestingly, treatment of monocyte-derived DCs with the LPS causes up-regulation of integrin αvβ8 expression, indicating that bacterial ligands may be an important cue in triggering integrin αvβ8 expression on DC. Thus, enhanced expression of integrin αvβ8 on certain human mononuclear phagocyte subsets may implicate these populations in the control of TGF-β dependent immune responses.
PD-1 and CTLA-4 signals are not important for the maintenance of tolerant self-reactive CD8 T cells in the small intestine (MUC8P.808)
Authors: Nelson Christine , Conemac Clare , McCurtain Jennifer , Fife Brian , Vezys Vaiva
Published: January 2023

Tolerance is an important mechanism by which the immune system distinguishes between self and non-self. Peripheral T cell tolerance is critical for the control of self-reactive cells that escape negative selection. Anergy is one mechanism that controls self-reactive cells in the periphery and prevents autoimmunity. Using a system that allows for the study self-specific CD8 T cell responses to a known intestinal auto-antigen, we determined that mucosal antigen signaling can lead to a long-term, stable population of cells in the intestine that have a classic anergic phenotype. These cells do not produce effector cytokines or induce intestinal damage. Inhibitory molecules, like PD-1 and CTLA-4, are believed to be important for both the induction and maintenance of T cell tolerance. We show that the induction phase of CD8 T cell tolerance to self was highly sensitive to control through both PD-1 and CTLA-4. However, once tolerance was established, the maintenance of tolerant CD8 T cells was independent of PD-1 or CTLA-4 signaling. Overall, this indicates that distinct mechanisms are responsible for the induction and maintenance of tolerant T cells. In addition, there may be many tolerant T cells resistant to immunotherapeutics targeting these pathways. Determining which subsets of self-reactive cells are susceptible to modulation through these pathways is important for understanding basic mechanisms tolerance and autoimmunity and for improving cancer therapeutics.
The structural basis for the anti-inflammatory activity of sialylated antibodies (THER5P.822)
Authors: Pincetic Andrew , Ahmed Alysia , Lomino Joseph , Wang Lai-Xi , Bjorkman Pamela , Ravetch Jeffrey
Published: January 2023

Antibodies (Ab) mediate pro-inflammatory effector functions through the engagement of Fc receptors (FcγRs) expressed on leukocytes. A complex, N-linked glycan attached to the Fc domain of IgG regulates the interaction to FcγRs by maintaining the Fc in a biologically active conformation. However, attaching sialic acid sugar residues to this Fc-associated glycan reduces affinity for FcγRs and confers binding to an alternate class of receptors, the C-type lectins (DC-SIGN). This switch in receptor specificity coincides with a switch in effector function in vivo as sialylated IgG (sFc) suppresses inflammation in mouse models of autoimmunity. The structural mechanism that regulates the sialic acid-dependent change in Ab effector function remains poorly understood. Biophysical analysis reveals that sialylation destabilizes the Fc fragment by increasing the hydrophobic surface area exposed to solvent. Upon solving a crystal structure of sFc, we observe that this loss of stability correlates with inter-domain flexibility such that the sFc in the crystal is found in both “open” and “closed” states. Mutagenic analysis of sFc further identifies key contact sites between the Fc-glycan and the Fc-backbone necessary to induce this conformational change and attain anti-inflammatory activity in vivo. These studies demonstrate that the conformational diversity of the Fc fragment serves as a general strategy to shift receptor specificity in order to effect different immunological outcomes.