bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute Epstein Barr virus-induced infectious mononucleosis leads to expansion of highly diverse CD8 T cell repertoires crossreactive with influenza A (HUM6P.249)
Authors: Gil Anna , Aslan Nuray , Mishra Rabi , Selin Liisa
Published: January 2023

The competence of T cell responses predominantly depends on how efficient T cell receptors (TCRs) are at recognizing antigenic epitopes. We show here that during acute infectious mononucleosis (IM) there was an expansion of IAV-M1-specific memory cells that are crossreactive with both EBV-BMLF1 and EBV-BRLF1 double-tetramer+ cells directly ex-vivo in HLA-A2+ patients. These crossreactive populations expanded dramatically in culture. For instance, there were multiple crossreactive populations in the IAV-M1 stimulated line. We systematically examined the characteristics of these crossreactive populations and compared them to the cognate IAV-M1, EBV-BMLF and -BRLF1 populations using TCR deep sequencing and gene array analysis of tetramer sorted cells. Surprisingly, both crossreactive and non-crossreactive populations were highly polyclonal with 1500-3500 different VA and VB clonotypes. The dominant clonotypes differed between the different populations. However, there were overlaps in either VB or VA usage consistent with their ability to bind both ligands. In one patient the M1-specific response used was an unusual dominant VB and was identical with the BMLF1-specific VB (VB2J2.7) but they differed in VA with M1 using VA13J50.1 and BMLF1 VA 12.1J12.1. Immune response signatures from gene array differed between the crossreactive and non-crossreactive populations consistent with the concept that crossreactive TCR selection may play a role in disease outcome. (NIHAI49320).
MicroRNA-21 drives severe, steroid-insensitive experimental asthma by amplifying PI3K-mediated suppression of HDAC2 (HYP7P.262)
Authors: Hansbro Philip , Kim Richard , Pinkerton James , Starkey Malcolm , Essilfie Ama-Tawiah , Mayall Jemma , Jones Bernadette , Haw Tatt , Keely Simon , Mattes Joerg , Adcock Ian , Foster Paul , Horvat Jay
Published: January 2023

BACKGROUND: Severe, steroid-insensitive (SSI) asthma is a substantial clinical problem. Effective treatments are urgently required, however, their development is hampered by a lack of understanding of the mechanisms that promote disease. SSI asthma is associated with respiratory infections and non-eosinophilic endotypes of disease, including neutrophilic asthma. OBJECTIVES: To develop and use mouse models of SSI neutrophilic asthma to investigate pathogenic mechanisms involving microRNA (miR)-21, phosphoinositide-3-kinase (PI3K) and histone deacetylase (HDAC)2 in order to identify new therapeutic approaches. METHODS: Novel mouse models of respiratory infection and ovalbumin-induced, SSI neutrophilic allergic airway disease (SSIAAD) in BALB/c mice were developed. The roles of infection-induced miR-21 expression and PI3K-dependent signalling in the lung were examined using a specific miR-21 inhibitor (antagomir-21) and the pan-PI3K inhibitor LY294002. RESULTS: Infection induced a miR-21-dependent, PI3K-mediated signalling pathway that decreased nuclear HDAC2 levels and promoted steroid-insensitive neutrophilic inflammation and airway hyper-responsiveness (AHR) in AAD. Inhibition of miR-21 or PI3K suppressed nuclear pAkt levels and restored HDAC2 levels and steroid sensitivity. CONCLUSIONS: We have identified a novel role for a miR-21/PI3K/HDAC2 signalling axis in SSIAAD. Our data highlights miR-21 as a novel target for treating this form of asthma.
Characterization of peanut-reactive monoclonal antibodies (HYP7P.265)
Authors: Little Christopher
Published: January 2023

Peanut allergies are a rising concern in Western society. Incidence and prevalence of affected children has tripled from 1997 to 2010. The exact cause for this increase is unknown, but lack of exposure to pathogens and increasing hygienic conditions has been implicated. Exposure to peanuts causes the immune system to become sensitized to peanut proteins. Subsequent exposure to peanuts causes hypersensitivity reactions which may escalate to anaphylaxis and death. The only course of prevention is complete avoidance. Limiting peanut exposure is difficult as even trace amounts of peanut (100µg-50mg; 1 peanut ≈ 1g) consumed can cause a reaction. Immunotherapies aim to increase the tolerance of these severely allergic individuals. Current approaches are limited to certain populations and are potentially dangerous. Better therapeutics require thorough characterization of peanut proteins responsible for peanut allergies. In this study, C3H/HeJ mice were sensitized to crude peanut extract (CPE). The mice were then exposed to CPE to induce a hypersensitive reaction. Hybridomas were generated , characterized for antibody production, subtype, and screened for peanut-specific immunoreactivity, as well as specific peanut proteins believed to be involved with peanut allergies. Characterization of peanut-specific monoclonal antibodies (MAb) may aid in the identification of immunodominant epitopes and lead to/aid in the development of immunotherapeutic interventions.
The transcriptional co-factor PARP14 limits severity of allergic skin inflammation (HYP7P.274)
Authors: Krishnamurthy Purna , Serezani Ana , Kaplan Mark
Published: January 2023

Atopic dermatitis (AD) is a chronic relapsing inflammatory condition of the skin that is characterized by pruritic skin lesions, erythema, and swelling, resulting in an impaired skin barrier. Patients with AD and mice subjected to models of AD develop an inflammatory T helper 2 (Th2) immune response and reduced expression of epidermal differentiation complex (EDC) genes, which encode proteins needed for keratinocyte maturation and barrier integrity. To identify potential therapeutic targets, we are exploring the molecular controls regulating Th2 cells and keratinocytes in a mouse model of AD. Poly-ADP ribose polymerse-14 (PARP14) is a transcription co-factor that enhances Th2 differentiation. Correspondingly, PARP14-deficient mice have reduced lung inflammation in a model of allergic airway disease; however, its potential role in AD has not been explored. Surprisingly, PARP14 deficiency resulted in an increase in the severity and number of skin lesions in an experimental mouse model of AD. Disease severity corresponded with increases in Th2 cytokine secretion and antibody production (IgE, IgG1). PARP14 deficiency resulted in altered EDC gene expression in the skin. Thus, PARP14 serves conflicting roles in allergic disease: promoting pathogenic Th2 immune responses while potentially limiting inflammatory responses during skin inflammation.
Oncostatin M promotes epithelial barrier dysfunction and is elevated in eosinophilic mucosal disease (HYP7P.255)
Authors: Pothoven Kathryn , Norton James , Suh Lydia , Carter Roderick , Hulse Kathryn , Liu Mark , Gonsalves Nirmala , Peters Anju , Harris Kathleen , Shintani Smith Stephanie , Conley David , Grammer Leslie , Kato Atsushi , Kern Robert , Bryce Paul , Tan Bruce , Schleimer Robert
Published: January 2023

Epithelial barrier dysfunction is thought to play a role in many mucosal diseases including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis (EoE). Oncostatin M (OSM), was elevated in both mRNA (28.3 fold, p<.01, n=12) and protein (4.4 fold, p<.05, n=12-19) in the nasal polyps of CRS patients compared to control tissue. OSM protein was also elevated in bronchoalveolar lavage (BAL) of allergic asthma (AA) patients following segmental allergen challenge (SAC) vs. saline challenge (14 fold, p<.0001). Additionally, OSM mRNA was elevated in esophageal biopsies of EoE patients vs. controls, (3 fold, p<.01). OSM stimulation of differentiated airway epithelium induced barrier dysfunction, as measured by decreased transepithelial electrical resistance (63% reduction, p<.0001, n=11) and increased permeability to 10kD dextran (2.45 fold, p<.05, n=5). Staining for the tight junction protein, occludin showed that OSM stimulation of differentiated airway epithelium impaired the organization of the tight junctions compared to unstimulated controls (n=5). Levels of OSM protein in tissue lysates from CRS and controls correlated with levels of α2-macroglobulin, a marker of epithelial leak, in matched nasal secretions (r=.5055, p<.01). Additionally, OSM in the BAL following SAC in AA patients correlated with serum albumin, another marker of epithelial leak (r=.8062, p<.001). These results suggest that OSM may play a role in epithelial barrier dysfunction in mucosal disease.
Repeated challenge with the hapten oxazolone provokes persistent vulvar mechanical sensitivity and labiar T cell changes in previously sensitized ND4 mice (HYP7P.258)
Authors: Chatterjea Devavani , Benck Charles , Mengistu Hanna
Published: January 2023

Epidemiological evidence suggests a link between environmental allergies and the risk of developing vulvodynia—a chronic provoked vulvar pain condition affecting up to 8-16% of 18-60 year old women. We have previously shown that acute labiar exposure to the hapten allergen oxazolone provokes increased vulvar mechanical sensitivity in ND4 Swiss mice. Here, we show that repeated oxazolone challenge produces persistent vulvar mechanical sensitivity in sensitized mice for at least12 days after final allergen exposure. T cell amplification in the draining iliac lymph nodes resolves within 5 days after final challenge. Persistent sensitivity in the skin is accompanied by sustained changes in skin T cells; CD44+ CD8 and CD4 subsets remain elevated, T cell transcription factors T-bet and FoxP3 continue to be upregulated for up to 17 days after 10 allergen challenges. This is the first characterization of immune infiltrate accompanying sustained allergen-provoked mechanical sensitivity that persists after the resolution of overt inflammatory events following repeated allergen exposures. Our findings can be used to used to elucidate the underlying etiology of allergy-driven subsets of chronic pain conditions.
Novel insights into Th17 mediated airway inflammation (HYP7P.259)
Authors: Koralov Sergei , Fogli Laura , Fanok Melania , Durbin Joan , Cadwell Ken , Bajwa Sofia , Rajewsky Klaus , goel swati , Sundrud Mark , Segal Leopoldo
Published: January 2023

Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Yet the direct impact of IL-17 and IL-22 on the lung epithelium remains to be fully elucidated. We took advantage of conditional gene targeting to generate a novel animal model of spontaneous Th17-driven lung inflammation that exhibits many similarities to neutrophilic asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. We demonstrate that IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We used this model to investigate the effects of IL-17 and IL-22 activity on airway epithelium transcriptional program and identified several important changes that contribute to neutrophil recruitment. The neutralization of IL-17 and IL-22 greatly ameliorates airway inflammation and improves lung function, underscoring a key role of these two cytokines in promoting chronic airway inflammation. We believe this novel mouse model will be highly valuable for the development of novel therapies targeting Th17 cells in steroid resistant asthma and COPD. We are now taking advantage of this animal model to explore the role of T cell receptor specificity and antigen (microbial and enviromental) to Th17 mediated airway inflammation.
Tpl2 is required for neutrophil antimicrobial functions (INC6P.311)
Authors: Elmore Jessica , Acuff Nicole , Kuriakose Teneema , Rada Balazs , Watford Wendy
Published: January 2023

Tumor progression locus 2 (Tpl2) is a serine/threonine kinase that promotes inflammatory cytokine production by activating the MEK/ERK pathway. Tpl2 has been shown to be important for the antimicrobial properties of macrophages. However, there is relatively little known about Tpl2’s contribution to neutrophil effector functions. This is an important consideration, since neutrophils provide the first line of defense against infection in the innate immune system. Gene expression analysis revealed that Tpl2 is highly expressed in both human and murine neutrophils, suggesting a potential function role for Tpl2 within this lineage. Despite a significantly higher proportion of bone marrow neutrophils in Tpl2-/- mice compared to wild type mice, Tpl2-/- mice had a significantly lower proportion of circulating neutrophils. In response to infection, neutrophils secrete inflammatory cytokines and produce reactive oxygen species (ROS). Tpl2 ablation impaired neutrophil TNF production in response to LPS stimulation and superoxide generation in response to fMLP, a chemotactic peptide. These results implicate Tpl2 in the regulation of multiple neutrophil antimicrobial pathways, including inflammatory cytokine secretion and oxidative burst. These studies suggest that Tpl2 functions very early during infection to bolster neutrophil-mediated innate immunity against extracellular bacteria, and current studies are evaluating the regulation of neutrophil-mediated bacterial killing by Tpl2.
CD49a/b-based human natural killer cell subsets in the blood and the liver: phenotypic and functional characteristics (INC6P.312)
Authors: Song Youkyong , Kim Jong Hoon , Shin Eui-Cheol , Yoon Joo Chun
Published: January 2023

Natural killer (NK) cells in the mouse liver sinusoid are divided into CD49a+DX5(CD49b)- and CD49a-DX5+, whereas those in the blood are mostly CD49a-DX5+. We therefore investigated whether human NK cells are also separated into distinct subsets according to CD49a/b expression. Peripheral blood mononuclear cells (PBMC) and intrasinusoidal lymphocytes (ISL) from healthy human donors were used for phenotypic and functional characterization of NK cells. Primary human NK cells expressed CD49a and CD49b and could be split into four subsets: CD49a+CD49b-, CD49a-CD49b+, CD49a+CD49b+, and CD49a-CD49b-. Frequencies of human NK cell subsets, however, differed from mouse NK cell subsets, as majority of NK cells in human PBMC are CD49a-CD49b- or CD49a+CD49b-. NK cells in ISL expressed lower CD49b but higher CD49a than those in PBMC. In addition, CD49a+ subset showed higher affinity to collagen. CD49b+ NK cells expressed more activation marker CD69 and inhibitory receptor ILT2, indicating they are activated subsets. While degranulation capacity was not affected by CD49a/b expression, CD49a-b+ subset produced higher IFN-γ and CD49a+b+ subset expressed more TRAIL upon activation. In conclusion, human NK cells were divided into four subsets based on CD49a/b expression. It appeared that NK cells in the liver have higher capacity to bind to extracelluar matrix. Small amount of CD49b+ NK cell subsets with activated phenotype were found in the blood but healthy liver had few CD49b+ NK cells.
Function of M1-polarized macrophages in the pathogenesis of Parkinson’s disease (INC6P.300)
Authors: Qin Hongwei , Liu Yudong , Fox Thomas , Harms Ashley , Standaert David , Benveniste Etty
Published: January 2023

Parkinson’s Disease (PD) is an age-related, chronic neurodegenerative disorder. Activated microglia/macrophages is associated with the pathogenesis of PD. Accumulation of α-synuclein (α-syn) leads to microglial activation and neurodegeneration. We have recently shown that inhibiting the JAK/STAT pathway prevents inflammation and neurodegeneration by suppressing activation of immune responses, and iNOS, TNF-α and IL-6 expression in an a-syn overexpression PD model. Studies from patients and animal models suggest that M1-polarized macrophage activation promotes the loss of dopaminergic neurons. As such, we investigated the function of M1-polarized macrophages in PD, especially how aggregated a-syn affects M1 phenotype. Our results indicate that expression levels of IFN-γ-induced MHC Class II, CD40, iNOS, TNF-α and IL-6 are synergistically enhanced by α-syn stimulation, along with higher activation of STAT1 and STAT3. We have previously shown that macrophages deficient in SOCS3 have heightened STAT3 activation and M1 phenotype. Stimulation of SOCS3-/- macrophages with a combination of α-syn and IFN-γ promotes a more pronounced M1 phenotype that promote neuronal degeneration. We hypothesize that hyperactivation of JAK/STAT and M1 macrophages promotes more severe synucleinopathy, including neuroinflammation and neurodegeneration. The characteristics of PD pathology in SOCS3fl/fl and LysMCre-SOCS3fl/fl mice with α-syn overexpression is currently under investigation.
Increased expression of CD155 and CD112 on monocytes in septic patients (INC6P.327)
Authors: Yan Zhang
Published: January 2023

The poliovirus receptor CD155 and its family member CD112 (PRR-2 (poliovirus receptor-related family 2), also called nectin-2) are ligands for human and mouse CD226. Human CD112 and CD155 are broadly distributed on epithelial and endothelial cells in many tissues, and overexpressed on certain tumors. Several reports have shown that the interaction between CD155 and CD226 are important for the elimination of target cells by NK and cytotoxic T cells, however, it remains elusive how CD155 and CD112 are regulated in sepsis. In our study, we showed that the expression of CD155 and CD112 on monocytes was significantly upregulated in septic patients which all had a significant reduction in expression of HLA-DR on monocytes, an immunosuppression marker of sepsis, indicating the existence of negative correlation between CD155/122 and HLA-DR. Next studies were undergoing on the mechanisms underlying regulation of the expression of CD155 and CD112 in sepsis and whether signals by CD155/CD112 negatively regulated the expression of HLA-DR.
Alternatively activated brain-resident macrophages rapidly acquire inflammatory properties following CNS infection (INC6P.301)
Authors: Rua Rejane , McGavern Dorian
Published: January 2023

Classic (M1) and alternative (M2) polarization of macrophages has been broadly studied in vitro but its relevance in the context of meningeal inflammation is unknown. The meningeal and perivascular spaces of the central nervous system (CNS) are inhabited by specialized macrophages. To gain novel insights into these cells, we analyzed the naive and virally infected brain using intravital two-photon microscopy (TPM) and flow cytometry. Examination of the naive brain by TPM revealed that meningeal and perivascular macrophages are highly dynamic and constantly survey their immediate surroundings similar to microglia. Under steady state conditions, we uncovered that they are maintained in a M2 state, which likely facilitates brain homeostasis. Interestingly, during the development of fatal viral meningitis induced by lymphocytic choriomeningitis virus, these cells were directly engaged by infiltrating virus-specific CD8+ T cells following acquisition of viral antigen and conversion into an inflammatory M1 phenotype. Mechanistically, the M2 to M1 transition occurred in the absence of CD8+ T cells and relied instead on innate cytokine release. These data indicate that meningeal and perivascular macrophages are highly plastic cells that can rapidly convert from a M2 to M1 phenotype and participate in the antiviral defense against an invading pathogen. This rapid inflammatory transition may explain why most CNS immune responses first develop in the meningeal and perivascular spaces.
Specific deletion of TRAF3 in myeloid cells leads to spontaneous inflammation and tumor development in mice (INC6P.304)
Authors: Lalani Almin , Luo Chang , Morse Herbert , Xie Ping
Published: January 2023

TRAF3, a cytoplasmic adaptor protein, is used in signaling by the TNFR superfamily and pattern recognition receptors. A number of these receptors are constitutively or inducibly expressed by myeloid cells, the crucial players in innate immunity and inflammation. To investigate the in vivo functions of TRAF3 in myeloid cells, we generated myeloid cell-specific TRAF3-/- (M-TRAF3-/-; TRAF3flox/floxLysM+/Cre) mice. We previously showed that young adult M-TRAF3-/- mice have normal frequencies and numbers of lymphocyte and myeloid cell populations in lymphoid organs, suggesting that TRAF3 ablation did not affect the maturation or homeostasis of myeloid cells. Here we report that 15- to 22-month-old M-TRAF3-/- mice spontaneously developed chronic inflammation or tumors, often affecting multiple organs. Diseased M-TRAF3-/- mice exhibited drastically altered lymphocyte and myeloid cell populations in lymphoid organs. Furthermore, we found that M‑TRAF3‑/- mice with spontaneous inflammation or tumor development contained strikingly elevated serum levels of a variety of chemokines and cytokines, including G-CSF, CCL1, IL-17, IP-10, MCP-1, MCP-5, CXCL9, TIMP-1, and TREM-1. This suggests that dysregulation of myeloid cells actively contributes to the pathogenesis of spontaneous inflammation and tumor development observed in aging M‑TRAF3‑/- mice. Taken together, our findings indicate that TRAF3 expressed in myeloid cells inhibits spontaneous inflammation and tumor development in mice.
Critical roles for mTOR signaling in DC homeostasis and function (INC6P.306)
Authors: Sinclair Charles , Loebbermann Jens , Ravindran Rajesh , Johnson Matthew , Bommakanti Gayathri , Li Shuzhao , PULENDRAN BALI
Published: January 2023

Intricate overlap between environmental and pathogen derived signals regulate innate immunity, and the mechanistic target of rapamycin (mTOR) kinase is a key signal integrator. Recent reports show mTOR inhibition increases the proinflammatory activity of DCs in vitro, suggesting that DC-targeted mTOR inhibitors could adjuvant immunity. We deleted mTOR in DCs using the CD11c Cre/LoxP system (mTOR DCKO), and challenged mice with distinct immunological stimuli. In contrast to our expectations, we found no inflammatory enhancement, but rather saw anatomically specific defects in lung immunity following intranasal administration of Th1 promoting (attenuated-influenza) or Th2 promoting (house dust mite, HDM) stimuli. Remarkably, the modality of immunity to HDM was altered by mTOR DCKO, resulting in a Th17 skewed asthma with pronounced neutrophilia. Surprisingly, reduced homeostatic frequencies of specific lung APC populations underlie the defects in lung immunity. Moreover, we identified further defects in homeostatic accumulation of DC populations in several additional organs, whereas others were unaffected. The mTOR dependence of DCs did not correlate with described paradigms of DC subdivision, and we are characterizing metabolomic and transcriptomic profiles to gain further insight into this phenomenon. Together our data reveal an unexpected role for mTOR in DCs, identifying a novel mechanism of immunosuppressive action that may be mediated by mTOR inhibitors.
Inflammasomes are critical for the host protection during bacterial urinary tract infection (INM6P.328)
Authors: Kulkarni Ritwij , Singh Sanjay , Jeyaseelan Samithamby
Published: January 2023

Urinary tract infections (UTI) are the second most common infection in the US. Gram-negative uropathogenic E. coli (UPEC) is the principal causative agent of UTI. The main goal of our work is to gain molecular insights into the role of cytoplasmic pattern recognition receptors, namely inflammasomes, in the protection of urinary tract during the course of bacterial UTI. Inflammasomes are multiprotein complexes made of ASC adaptor protein that facilitates interaction between NOD-like receptors with procaspase-1. Stimulation of caspase-1 activates IL-1b and IL-18. Since, the rapid induction of caspases and IL-1b are important hallmarks of UPEC-induced UTI, we hypothesized that the inflammasomes are critical for protection of the urinary tract during UPEC infection. To address this hypothesis, we challenged female WT (C57Bl6/J) and ASC-/- mice with UPEC strain CFT073 via transurethral catheterization. We observed increased bacterial burden in the bladders of ASC-/- mice as compared to the WT. In comparison to WT mice, CFT073 infection did not induce IL-1b production in the bladders of ASC-/- mice. Taken together these observations suggest for the first time that ASC-inflammasomes play a critical role in the protection of urinary tract during UPEC infection by modulating important innate immune cascades.
Nrf2-mediated pentose phosphate pathway activity promotes macrophage inflammasome activation and bacterial killing in microbial sepsis (INM6P.330)
Authors: Wen Haitao , Li Xinghui , Gillette Devyn , Cairns Bruce
Published: January 2023

Classical activation of the innate immune cells such as macrophages and dendritic cells causes dramatic metabolic changes towards increased glucose uptake, glycolysis and pentose phosphate pathway (PPP) activity. However, the role and mechanism of this metabolic reprogramming in the regulation of macrophage function and its disease relevance is poorly understood. Here we report a critical role of PPP activity in inflammasome activation. Pharmacological inhibition of PPP resulted in a defective inflammasome activation. Macrophages with a mutation in G6pd, the gene encoding a rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase, showed decreased inflammasome activation. Mechanistically, PPP activity was required for the prevention of excessive reactive oxygen species generation and safeguarding an intact extracellular Ca2+ influx, a critical step for inflammasome activation. Nuclear factor E2-related factor-2 (Nrf2) regulates the expression of several key enzymes involved in PPP. Furthermore, Nrf2-/- macrophages showed defective caspase-1 activation after E. coli infection, which caused decreased phagosome acidification and bacterial killing. Nrf2-/- mice and mice with PPP inhibition exhibited an increased bacterial burden in E. coli-induced and cecal ligation and puncture-induced microbial sepsis. In sum, these findings identify a novel function of PPP in inflammasome activation and suggest PPP as a therapeutic target in the treatment of microbial sepsis.
NLRP3 inflammasome controls adipose tissue leukocytosis and inflammation during aging (INM6P.331)
Authors: Camell Christina , Youm Yun-Hee , Nguyen Kim , Ravussin Anthony , Spadaro Olga , Dixit Vishwa
Published: January 2023

Aging is associated with chronic inflammation and increased risk for degenerative disorders and diseases. In the elderly there is increased visceral adiposity and increased numbers of fat-associated lymphocyte clusters within adipose tissue (AT); however the role for AT in age-induced immune defects is unclear. Our prior studies have identified that the canonical inflammasome, NLRP3, is required for diet-induced AT inflammation and age-related inflammation in multiple organs. We hypothesized that NLRP3 is required for age-induced changes in AT immune cells and metabolic dysfunction. We found that aging increases AT lymphocytes; compared to 24month old control mice, Nlrp3-/- animals were protected from increases in AT leukocytosis. RNA sequencing analysis of AT macrophages and B cells from aged mice revealed distinct inflammatory signatures that were regulated by NLRP3. AT displayed immunological properties, as during infection, a higher frequency of antigen specific T cells are recruited to AT than to spleen. Our results show that NLRP3 is required for age-induced T follicular cell and B cell infiltration into visceral AT. Furthermore, our data suggests that pharmacological inhibition of NLRP3 activation and IL-1β signaling may improve AT inflammation and insulin resistance in aged mice. These data demonstrates that macrophage-expressed NLRP3 inflammasome controls age-related inflammation and AT microenvironment by secondary effects on T and B cell subpopulations.
Somatic MyD88 mutations and a germline IRAK2 variant contribute to lymphoma by differential effects on NF-κB (INM7P.354)
Authors: Wolz Oliver , Wang Hui , El Maadidi Souhayla , Fischer Janett , Flannery Sinead M. , Dickhöfer Sabine , Butterbach Katja , Nieters Alexandra , Bowie Andrew , Weber Alexander
Published: January 2023

Within innate immune signaling pathways, Myeloid differentiation (MyD) 88 and Interleukin-1 receptor associated kinases (IRAKs) fulfill pivotal roles downstream of multiple Toll-like receptors (TLR). Somatic MyD88 mutations in B cells were reported to affect lymphomagenesis by constitutive hyperactivation of NF-κB. We here show that oncogenic mutants augment spontaneous TIR domain oligomerization into so-called Myddosome signaling complexes driving NF-κB activation. Blocking of MyD88 oligomerization consequently induced the death of MyD88-mutated but not unmutated lymphoma cells. In line with the involvement of the MyD88 axis in lymphomagenesis we detected a genetic association of a coding germline variant of IRAK2, a MyD88 interactor, with increased risk (OR = 1.43, 95% confidence interval 1.06-1.93, p=0.018) to develop lymphoma in a German cohort (n=387). This variant is frequently found in 15-44% of individuals of different ethnic groups. Unexpectedly, this variant was associated with reduced NF-κB activation and cytokine responses to TLR agonists in healthy donors. Mechanistically, the IRAK2 variant retained intact binding to but impaired ubiquitination of TRAF6, a vital step in signal transduction. Thus our findings confirm the involvement of MyD88/IRAK in lymphoma but suggest that MyD88 and IRAK2 may contribute differently to oncogenic signaling. Additionally we highlight IRAK2 genetic variants as a critical factor and potentially novel biomarker for human lymphomagenesis.
Genomic networks of inflammatory response (INM7P.355)
Authors: Poltorak Alexander , Ilyuha Vladimir , Bagina Uliana , Belova Lubava
Published: January 2023

Mediated by a family of Toll-like Receptors (TLRs) responses lead to transcriptional activation and synthesis of many inflammatory genes. As an example, thousands of genes are up-regulated in response to LPS (lipopolysaccharide), with many of them undergoing specific program of activation and silencing. To investigate the breadth and specificity of LPS-induced gene expression in macrophages, we compared expressional activation in response to LPS in genetically diverse mice of subspecies M.m.musculus and M.m.domesticus using next generation (NGS) RNA-sequencing. To characterize the genetic basis of LPS-response in detail, we applied a systems genetics approach, using mapping of gene expression traits with on a panel of a second-generation F2 intercross mice. Specifically, RNA from LPS-activated F2 macrophages was RNA-sequenced and hundreds of activated genes were used as phenotypic read-outs for mapping the traits. We identified several hundreds cis- and trans-acting QTL contributing to expression variation (eQTL). Many trans eQTL cluster into major groups, some of which are confined to genomic loci that do not contain known regulators of innate immune responses. We identified 227 cis-expression quantitative trait loci (cis-eQTL) that control expression. Our data provide the first genome-wide map of major regulators of the inflammatory responses in macrophages.
The effects of circadian rhythms on Toll-like receptor responses in murine macrophages (INM7P.357)
Authors: Sidhu Kiran , Cox Emily , Yanez Amanda , Heipertz Erica , Walker Wendy
Published: January 2023

Circadian rhythms refer to the endogenous oscillations of biological processes with 24-hour periodicity in response to environmental cues. On a molecular level, circadian rhythms are accomplished through positive and negative feedback loops among the core Clock genes: Clock, Period, Cryptochrome and Bmal. These oscillations in gene expression regulate a myriad of cellular functions and activities. Recently, circadian rhythms have been found to promote comparable oscillations in components of the innate and adaptive immune system. Synchronization of the circadian clock can be induced in vitro via a process known as serum shock. Here, we took advantage of this model to investigate the effects of circadian oscillations on Toll-like receptor (TLR) responses in murine macrophages. Serum shock induced a 24h oscillation in mRNA expression for the clock gene Period 2, as well as for a period2-luciferase fusion reporter gene, confirming synchronization of the circadian clock. Serum-shocked cells were then stimulated with a panel of TLR agonists and cytokine secretion was quantified over time to investigate the impact of circadian rhythms on TLR responses. Our findings suggest circadian oscillation with regard to interleukin (IL)-12p40 production upon stimulation of TLR2 and TLR7. Suggestive oscillations were also observed with regard to IL-6 production upon stimulation of TLR2. These data suggest that circadian rhythms control TLR responses at the cellular level.
Src family kinase members Fyn and Lyn mediate type I interferon production by plasmacytoid dendritic cells. (INM7P.345)
Authors: Dallari Simone , Macal Monica , Hesser Charles , Zuniga Elina
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are the most powerful type I interferon (IFN-I) producing cells and are therefore important for antiviral defense and for autoimmune diseases such as systemic lupus erythematosus. However, the signaling factors that regulate pDC IFN-I production are not completely understood. Here we investigate the role of Src Family Kinases (SFKs), a family of non-receptor tyrosine kinases, in pDC responses to toll like receptor (TLR) stimulation. We first found that while human pDCs expressed mostly Lyn, mouse pDCs expressed both Lyn and Fyn, two members of SFKs. Importantly, we observed that pDCs from either Fyn or Lyn deficient mice exhibited impaired IFN-I and pro-inflammatory cytokine production after TLR-9 and TLR-7 stimulation, whereas no difference was observed in conventional DCs. Consistently, incubation of murine or human pDCs with a pan-SFKs inhibitor resulted in a dramatic ablation of pDC IFN-I and cytokine production in a dose dependent manner. Finally, treatment of a human pDC cell line (CAL-1) or pDCs from human peripheral blood with Bafetinib (a selective dual Bcr-Abl/Lyn inhibitor) profoundly ablated their IFN-I production after TLR stimulation. Taken together, our data indicate that the SFKs members Fyn and Lyn promote pDC IFN-I and pro-inflammatory cytokine production and suggest that Bafetinib could be considered as a therapeutic candidate for attenuating pDC responses in autoimmune diseases.
PTP1B preferentially regulates IRF7-ISRE-IFN pathway and negatively impacts host defense against Pseudomonas aeruginosa infection. (INM7P.348)
Authors: Lin Tong-Jun , Yue Lei , Xie Zhongping , Li Hua , Junkins Robert , Tremblay Michel , Chen Xiaochun
Published: January 2023

Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Toll-like receptors (TLRs) contribute to innate immunity against P. aeruginosa through activation of transcription factors IRF7/IRF3 and NFκB. However, mechanisms involved in the regulation of P. aeruginosa-induced TLR pathway activation remain incompletely defined. Here, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance which is accompanied with increased neutrophil infiltration and cytokine production. Interestingly, PTP1B-deficiency mainly up-regulates the production of IFN-stimulated response elements (ISRE)-regulated cytokines and chemokines including CCL5 (RANTES), CXCL10 (IP-10) and IFN-β production. Further studies reveal that PTP1B-deficiency leads to increased IRF7 activation. Importantly, PTP1B is physically associated with IRF7 in dendritic cells. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-ISRE pathway may have broader implications in TLR-mediated innate immunity.
Butyric acid inhibits a Toll-like receptor 2 agonist-mediated increase of Toll-like receptor 3-induced chemokine production by HT-29 intestinal epithelial cells (IRC10P.413)
Authors: Shastri Padmaja , Green-Johnson Julia
Published: January 2023

The intestinal epithelium interacts with varied microbial components and metabolites, integrating multiple signals that influence immunity. We examined the time and dose-dependent effects of a Toll-Like Receptor (TLR) 2 agonist lipoprotein Pam3Cys (P3C) and the bacterial fermentation metabolite butyric acid (BA) (10mM and 25mM) on TLR3 agonist (poly(I:C)) (pIC)-induced chemokine production by HT-29 intestinal epithelial cells (IEC). Pre-treatment of HT-29 IEC with P3C enhanced (p(IC))-induced IL-8 production. However, pre-treatment of HT-29 IEC with P3C and 25mM BA together inhibited p(I:C)-induced IL-8 production, suggesting BA blocked the enhancing effect of this TLR2 agonist on TLR3-induced chemokine production. This inhibitory effect was not observed when IEC were pre-treated with a lower concentration of BA (10mM). Co-incubation of IEC with P3C and p(I:C) did not enhance TLR3 agonist-induced IL-8 production, while co-incubation with 10mM or 25mM BA acid significantly reduced p(I:C)-induced IL-8 and CXCL-10 production by HT-29 IEC. In addition, a p38 blocker (SB203580) significantly inhibited p(I:C)-induced IL-8 production by HT-29 IEC, while co-incubation with SB203580 and 10mM BA showed no additive inhibition. These findings suggest that while TLR2 agonists can upregulate TLR3-induced chemokine production, butyric acid inhibits this process, and the effects of TLR2 agonists and BA are dependent on timing of contact with IEC relative to TLR3 agonist stimulation.
Apoptotic cell-treated dendritic cells induce immune tolerance through specifically facilitating development of CD4+ effector memory T cells (IRC10P.408)
Authors: Zhang Guang-Xian , Zhou Fang , Rostami A
Published: January 2023

CD4+ memory T cells play an important role in induction of autoimmunity and chronic inflammatory responses. However, regulatory mechanisms of CD4+ memory T cell-mediated inflammatory responses are poorly understood. Here we showed that apoptotic cell-treated dendritic cells inhibit development and differentiation of CD4+ effector and central memory T cells in vitro and in vivo. Simultaneously, i.v. transfer of apoptotic T cell-induced tolerogenic dendritic cells can block development of experimental autoimmune encephalomyelitis (EAE), an inflammatory disease in central nervous system of C57BL/6J mouse. However, our results imply that it is effector memory CD4+ T cells, not central memory CD4+ T cells which play a major role in chronic inflammatory responses in mice with EAE. I.v. transfer of tolerogenic dendritic cells induced by apoptotic T cells leads to immune tolerance through specifically blocking development of CD4+ effector memory T cells compared with results of EAE control mice. Our results reveal a new mechanism of apoptotic cell-treated dendritic cell-mediated immune tolerance in vivo.
Infectious tolerance of LAG-3+ regulatory T cells with attenuated T-bet induction (IRC11P.433)
Authors: Huang Ching-Tai , Dutta Avijit , Miaw Shi-Chuen , Drake Charles , Powell Jonathan , Pardoll Drew
Published: January 2023

Infectious tolerance describes the maintenance of tolerance by a small number of antigen-specific regulatory T (Treg) cells through converting naïve T cells into further cohorts of Treg cells in vivo. Thus a very small number of antigen-specific Treg cells can be so efficient in exerting long-term suppressive effect with self-perpetuating immune homeostasis in vivo. We have previously shown LAG-3+ CD4+ Treg cells are engaged in dominant immune regulation in vivo. Here we revealed that LAG-3+ CD4+ Treg cells pass on the suppressive activity to the naïve CD4+ T cells they suppress and enable them to become the next generation of Treg cells. The effector function of suppressed CD4+ T cells is modulated with diminished T-bet induction and interferon-γ production. However, they still expand, acquire LAG-3 expression and in turn suppress the activation of another batch of naïve CD4+ T cells in vivo. The suppressed cells express elevated level of active TGF-β but not IL-10. Suppressed cells with Foxp-3 expression acquire LAG-3, although the overall Foxp-3 expression is not increased. Our results imply activation with attenuated effector function as the major mechanism by which LAG+ CD4+ Treg cells propagates their suppressive abilities and the infectious tolerance.
Intravenous immunoglobulin drives regulatory B cell expansion in the absence of endogenous regulatory T cells (IRC11P.436)
Authors: Kaufman Gabriel , Xue Di , Beland Marianne , Massoud Amir , Mazer Bruce
Published: January 2023

We have shown that induced regulatory T cells (iTreg) are necessary and sufficient to rescue antigen-challenged mice in a murine model of allergic airways disease (AAD). Intravenous immunoglobulin (IVIg) treatment of wild-type mice sensitized and challenged to antigen induces Treg in a dendritic-cell (DC) dependent mechanism. In the present study, DEREG mice (which co-express diphtheria toxin (DT) receptor with Foxp3, allowing for selective transient depletion of Treg) were treated with IVIg after depletion of all endogenous Treg with DT. This induces a phenotypically distinct population of functional iTreg. This increase in iTreg is associated with significant augmentation of systemic regulatory B cells (Breg), which express IL-10 and are CD138-high Blimp-low plasmablasts. Under these conditions, all aspects of AAD are ameliorated. However, in animals with an intact endogenous Treg compartment, Breg are not significantly increased by IVIg treatment. We postulate a balance between regulatory cells which favors Treg, and may shift to Breg in the absence of endogenous Treg. Further work will delineate the Breg:Treg balance and how these regulatory subsets contribute to suppression of allergic inflammation.
Characterization of cannabidiol-induced increases in CD25 and FOXP3 expression on CD4+ T cells (IRC11P.421)
Authors: Dhital Saphala , Stokes John , L. Kaplan Barbara
Published: January 2023

Cannabis sativa-derived cannabinoid compounds are being investigated as potential immunosuppressive agents and important drug candidates for immune related diseases. Our previous data demonstrated that under conditions of suboptimal T cell activation, cannabidiol (CBD) could increase interleukin-2 (IL-2) production. Initially this result was surprising given that cannabinoids are immunosuppressive, but IL-2 can also contribute to regulatory T cell (Treg) induction. We tested the hypothesis that part of the mechanism for immunosuppression by CBD is induction of a Treg population in response to suboptimal activation. Splenocytes were treated with CBD or vehicle (VH; 0.1% ethanol) for 30 minutes, then stimulated with low concentrations of PMA and ionomycin (Io). CBD induced IL-2, TGF-β, CD25 and FOXP3 on CD4+ T cells in response to 4 nM PMA/0.05 µM Io after an overnight stimulation. Further characterization of this effect demonstrated that CBD also induced CD25 and FOXP3 on CD4+ T cells after a 5-day incubation with 1 nM PMA/0.01 µM Io. Interestingly, while CBD robustly induced IL-2 after an overnight incubation with 4 nM PMA/0.05 µM Io, the IL-2 in the culture supernatant dropped by day 5. Overall, these data suggest that the mechanism by which CBD is immunosuppressive might involve induction of Tregs in response to suboptimal activation of T cells.
ITK controls the functional stability of regulatory T cells by limiting pathogenic reprograming in inflamed mucosa (IRC11P.422)
Authors: Huang Weishan , Deol Simrita , Koylass Nicholas , August Avery
Published: January 2023

CD25hiFoxp3+CD4+ regulatory T cells (Tregs) are important immune regulators that promote immune tolerance, but can also form barriers to effective immunity against tumor and chronic infection. IL-2-inducible T cell kinase (ITK) is highly expressed in T cells and regulates T cell development and function. We find that ITK is required for full suppressive function of natural Tregs in preventing the expansion of pro-inflammatory CD4+ effector T cells and chronic pulmonary and colonic inflammation upon transfer into Rag-/- mice. In the absence of ITK, Treg homing to these inflamed mucosal systems is intact, however, a significant proportion is converted into pathogenic effector T cells with ability to produce IL-17A and/or IFN-γ in the inflamed lung and colon, but not in the spleen. Such loss of functional stability and gain of pathogenic reprograming are T cell intrinsic and can be rescued by re-expression of ITK in Itk-/- Tregs. Homeostatic expansion contributed to the initiation of Itk-/- Treg destabilization and pathogenic conversion but did not trigger pathogenesis. Stimulation through the T cell receptor in the presence or absence of various cytokines revealed that ITK is required for sustaining Foxp3 expression. We conclude that ITK signals suppress pathogenic conversion of Tregs by maintaining Foxp3 expression, and contributes to the functional stability of Tregs in the face of inflammation by limiting their reprograming towards the pathogenic effectors.
Highly focused TCR Vβ repertoire is associated with a large number of naive precursors and robust CD8 T cell responses specific for a Plasmodium antigen (IRM14P.450)
Authors: Van Braeckel-Budimir Natalija , Harty John
Published: January 2023

Here we describe, to our knowledge, the most TCRβ focused CD8 T cell response to date. This TCR is specific for a conserved Plasmodium-derived epitope in mice (GAP5041-48), presented by H-2Db. After various immunization regimens, >99% of epitope-specific CD8 T cells detected by tetramer staining expressed TCR Vβ8.1 (TRB). Interestingly, priming with dendritic cells coated with GAP5041-48 peptide resulted in the same TCR Vβ distribution, suggesting that the observed bias does not evolve with multiple antigen exposures. Additionally, DC-priming induced exceptionally strong responses only 4 days post-immunization (~20% of total CD8 T cells were tetramer positive), suggesting an unusually large antigen-specific naïve CD8 T cell repertoire. Currently, the upper limit for the size of antigen-specific naive CD8 T cell repertoire ranges from 600-1500 cells (VacV B8R, MCMV M45). We show that GAP5041-48-specific naïve CD8 T cell repertoire, with its size of ~2,200 cells, extends this upper limit by more than 40%. Finally, classical Ala-substitution screening of the peptide (SQLLNAKYL), showed that amino acids (aa) at positions 6 and 7 are critical for TCR recognition. This epitope could be used as a valuable tool for better understanding the relation between a TCR structure and thymic selection. Moreover, the unique property of GAP5041-48-specific response make it a powerful tool to study fundamental processes underlying CD8 T cell activation upon Plasmodium infection.
Distinct stage-specific function of T-box transcription factors in the antiviral NK cell response (IRM14P.446)
Authors: Madera Sharline , Sun Joseph
Published: January 2023

The T-box transcription factors T-bet and Eomesodermin (Eomes) instruct discrete stages of natural killer (NK) cell development. Their role in the response of mature NK cells against pathogen infection has not been investigated. Here, we use an inducible gene deletion system to elucidate the role of T-bet and Eomes in mature NK cells during the course of murine cytomegalovirus (MCMV) infection. We show that although both transcription factors are dispensable for maintaining NK cell numbers during homeostatic proliferation, T-bet and Eomes are necessary for the proliferation of virus-specific NK cells during MCMV infection. T-bet, but not Eomes, is upregulated in NK cells early after viral infection, through pro-inflammatory cytokine and STAT4 signaling. However, deletion of either T-bet or Eomes in mature NK cells during viral infection led to a diminished NK cell response in lymphoid and nonlymphoid organs. Interestingly, maintenance of memory NK cell numbers was dependent on T-bet, but not Eomes. These data uncover a non-redundant and stage-specific influence of T-bet and Eomes on the NK cell response against infection.