bluuebunny/crossref_metadata_2025 huggingface dataset:
Toll-like receptor 2 is associated with house dust mite-induced inflammatory response in human bronchial epithelial cells (91.1)
Authors: Teng Fang , Xu Jimmy Peining , San Mateo Lani
Published: January 2023

Exposure of the respiratory tract of an atopic individual to allergens such as D. pteronyssinus (DP) house dust mite is associated with the development of allergic asthma. In the study, we explored the role of Toll-like receptors (TLRs) in allergen-induced inflammatory responses of human bronchial epithelial cells. Our results showed that DP induced production of G-CSF, GM-CSF, CXCL1, IL-6, CXCL8, and CCL2 in BEAS-2B (immortalized human bronchial epithelial cells) in a dose-dependent manner. An inhibitory peptide against MyD88 significantly inhibited the allergen-induced cytokine response, while monoclonal antibodies (mAbs) against TLR2 showed some (but not significant) inhibitory effects. Analysis of mRNA transcripts by qPCR revealed an increased expression of MyD88 at 1 hr post-stimulation with DP. DP stimulation also induced increased expression of TLR1, TLR2, TLR3, TLR5, TLR8, and TLR9, as well as IFNβ. HKLM, a TLR2 agonist, along with DP showed a synergistic effect on cytokine response of BEAS-2B and the response was inhibited by anti-TLR2 mAb. HEK293 cells expressing TLR1/2, TLR2/6 or TLR4/CD14/MD2 showed increased cytokine response to allergens, while anti-TLR2 inhibited the response in HEK293 cells expressing TLR1/2 or TLR2/6. These data demonstrate a link between DP allergen-induced inflammatory response and TLR2-mediated signaling in airway epithelial cells and contribute to our understanding of the role of innate immunity in allergic diseases.
TLR4/MyD88-induced non-migratory myeloid cells suppress Th2 effector function in the lung (91.15)
Authors: Arora Meenakshi , Poe Stephanie , Oriss Timothy , Krishnamorthy Nandini , Yarlagadda Manohar , Billiar Timothy , Ray Anuradha , Ray Prabir
Published: January 2023

Bacterial lipopolysaccharide (LPS) is immunostimulatory at a low dose but immunosuppressive at a high dose in the respiratory tract as observed in animal studies. Relevant to LPS-mediated immunosuppression in the lungs of animals is the protective effect of high doses of LPS in allergic diseases such as asthma in humans. Our study investigated the impact of a high dose of LPS on cells of the innate immune system in the lung to elucidate the basis of LPS-mediated immunosuppression. A high LPS dose blunted house dust mite-induced airway eosinophilia and Th2 cytokines production. Conversely, adoptively transferred Th2 cells were unable to mount allergic airway inflammation in LPS-treated lungs. The high LPS dose promoted the development of a lung-resident myeloid cell type in a TLR4- and MyD88-dependent fashion that suppressed lung DC-mediated reactivation of primed Th2 cells. Adoptive transfer of these myeloid cells suppressed allergen-induced airway inflammation. These data suggest that LPS-induced non-migratory myeloid cells might contribute to the protective effects of LPS in allergic airways disease by dampening Th2 effector functions in the lung. RO1 HL060207 (P.R.), RO1 HL077430 (A.R.), RO1 HL084932 (A.R. and P.R.) and AHA 0865379D (M.A.)
The role of CD1d-restricted type II NKT cells in the pathogenesis of inflammatory bowel disease (93.10)
Authors: Liao Chia-Min , Zimmer Michael , Cho Hoonsik , Choi Hak-Jong , Cardell Susanna , Yu Hoon-Tsen , Wang Chyung-Ru
Published: January 2023

CD1d is an MHC class Ib molecule that is specialized to present lipid antigens to NKT cells. Two subsets of NKT cells have been identified: type I NKT cells express an invariant TCR α chain while type II NKT cells express a more diverse TCR repertoire. Recent studies have shown that a specific pattern of CD1d expression is required for proper development and function of type I NKT cells. However, little is known about the regulation and selection of type II NKT cells as there is no method for direct detection of these cells. To address whether changes in CD1d expression patterns can regulate the function of type II NKT cells, we have generated a double transgenic mouse model, CD1dTg/24αβTg, that expresses high levels of CD1d as well as a TCR isolated from the type II NKT cell clone 24αβ. Total numbers of 24αβ T cells are significantly reduced in CD1dTg/24αβTg mice, suggesting that type II NKT cells undergo negative selection when engaged by abundant self-antigen. The remaining 24αβ T cells exhibit an altered surface phenotype and produce increased levels of inflammatory cytokines compared to equivalent cells that develop in a wild type B6 background. Interestingly, CD1dTg/24αβTg mice spontaneously develop inflammatory bowel disease (IBD) characterized by extensive colonic inflammation. Our studies highlight the importance of CD1d expression levels in the regulation of type II NKT cells and reveal a potential role for type II NKT cells in the pathogenesis of IBD.
Low copy-number of complement C4A, the presence of HLA-DR3, and the presence of HLA-DR2 are independent and additive risk factors for human systemic lupus erythematosus (SLE) (93.13)
Authors: Wu Yee , Lundstrom Emeli , Liu Chau-Ching , Yang Yan , Gunnarsson Iva , Svenungsson Elisabet , Zhou Bi , Jones Karla , Nagaraja Haikady , Higgins Gloria , Spencer Charles , Brunner Hermine , Birmingham Dan , Rovin Brad , Tsao Betty , Ahearn Joseph , Hebert Lee , Padyukov Leonid , Yu C.
Published: January 2023

The major histocompatibility complex (MHC) in chromosome 6p21.3 has the strongest genetic association with human SLE. This study aims to dissect the roles of MHC genes HLA-DRB1 and complement C4 in SLE. The study population includes 744 SLE patients and 760 unrelated healthy controls of European ancestry. Genomic Southern blot analyses and qPCR were used to determine the gene copy-numbers of total C4, C4A and C4B. Two-digit genotypings for HLA-DRB1 were performed by SSP-PCRs. The copy-numbers for total C4, C4A and C4B vary from 2 to 6, 0 to 5, and 0 to 4, respectively. Lower C4A copy-number (p=2.4x10-13), and increased frequencies of HLA-DRB1*03 (DR3; p=1.5x10-10) and HLA-DRB1*15 (DR2; p=0.032) are associated with SLE. Zero, 1 and ≥2 copies of C4A strongly correlate with homozygous, heterozygous and non-DR3, respectively, in controls (r2=0.435,χ2=374.0; p=1.2x10-79) and in SLE (r2=0.381, χ2=463.9; p=4.2x10-99). Comparing to subjects with non-low C4A (i.e. ≥2 copies), non-DR3 and non-DR2, the odds for SLE with low C4A only (i.e. <2 copies) was 2.22 (95% CI: 1.11-4.43; p=0.031), DR3+ only was 1.81 (1.21-2.71; p=0.004), and DR2+ only was 1.65 (1.26-2.16; p=0.0002). Notably, the odds increased to 2.80 (2.09-3.76; p=3.6x10-12) among subjects with low C4A and DR3, and 2.71 (1.10-6.64; p=0.037) among subjects with low C4A and DR2. In conclusion, low C4A gene copy-numbers, the presence of HLA-DR3 or HLA-DR2 are each independent risk factors with additive effects for SLE.
Activating KIR genes act as risk factors for Crohn’s Disease (93.34)
Authors: Samarani Suzanne , Almalte Zaema , Iannello Alexandre , Debbeche Olfa , Amre Devendra , Ahmad Ali
Published: January 2023

Activating Killer-cell Immunoglobulin-like Receptor (KIR) genes constitute a subset of KIR family. The genes are expressed as activating receptors on the surface of NK cells as well as on a subset of T cells. The receptors interact with poorly defined ligands and decrease the activation threshold of these cells. Overall, they increase immune reactivity of the individuals. The activating KIR genes, like other KIR genes, are highly polymorphic. Furthermore, KIR haplotypes differ from each other with respect to the number of activating genes. Some haplotypes have no activating genes while others may carry upto seven such genes. Consequently, humans differ from each other with respect to the number of inherited activating KIR genes. Activating KIR genes have been associated with autoimmune diseases. However, little is known about their association with Crohn’s Disease. We have investigated the potential association of these genes with this disease in Canadian children of the French origin. Our results show that these genes act as risk factors for developing CD in these children. An increased number of inherited activating KIR genes correspondingly increased the risk for the disease. These results were also replicated in the Canadian (Caucasian) children of non-French origin. Collectively, our results suggest that activating KIR genes act as risk factors in developing CD in humans. The study was supported by the Crohn’s and Colitis Foundation of Canada (CCFC)
Self-organized criticality theory for the cause of autoimmunity (93.39)
Authors: Tsumiyama Ken , Miyazaki Yumi , Shiozawa Shunichi
Published: January 2023

Objective: We here investigated the cause of autoimmunity from different angle, i.e., the defect in immune ‘system’, and discovered that overstimulation of immune system beyond its self-organized criticality leads to autoimmunity. Results: Repeated immunization with antigen causes systemic autoimmunity in mice otherwise not prone to spontaneous autoimmune diseases. Overstimulation of CD4+ T cells by repeated immunization with antigen led to the development of a fully-matured autoantibody-inducing CD4+ T (aiCD4+ T) cell which had undergone T cell receptor (TCR) revision and was capable of inducing autoantibodies. The aiCD4+ T cell was induced by de novo TCR revision but not by cross-reaction to immunizing antigen, and subsequently overstimulated CD8+ T cells, driving them to become MHC class I-restricted, antigen-specific cytotoxic T lymphocytes (CTL). These CTLs could be further matured by antigen cross-presentation, after which they caused autoimmune tissue injury akin to systemic lupus erythematosus (SLE). Autoimmune tissue injury did not appear in CD8+ T cell-deficient mice. Further, inhibition of antigen-cross presentation by treating with chloroquine abrogated the generation of CTL and autoimmune tissue injury. Conclusion: Systemic autoimmunity appears to be the inevitable consequence of over-stimulating the host’s immune ‘system’ by repeated immunization with antigen, to the levels that surpass system’s self-organized criticality.
Bacille Calmette Guérin (BCG) can induce Kawasaki disease like features in Programmed death-1 gene knockout mice (93.8)
Authors: Chun Jin-Kyong , Kang Dong-Won , Jeon Bo Young , Kim Dong Soo
Published: January 2023

The pathogenesis of Kawasaki disease (KD) is not clear, but certain genetic defects of inhibitory immune processes after infectious stimuli have been suspected as a feasible factor. In the present study, we investigated the associative roles of programmed death-1 (PD-1) gene in the pathogenesis of KD. In order to induce KD-like manifestations in young PD-1KO mice, injection of the bacilli Calmette Guérin (BCG) was performed twice with a 4 week interval. After the second injection, PD-1KO mice showed KD-like clinical features including prolonged fever for more than 5 days, erythematous swelling on soles, anal desquamation, and gallbladder (GB) hydrops. Inflammation in at least more than one coronary artery was found in all PD-1KO mice whereas scanty coronary lesion was found in wild type (WT) mice. When the PD-1KO mice were challenged twice with HSP65 as same methods of BCG, coronary arterial lesions similar to those seen after BCG injection were observed. Inflammatory reactions in other organs including hepatic arteries, renal arteries, and biliary arteries were observed in PD-1KO mice. The levels of Interferon-γ and IL-6 were significantly increased in both sera and splenocytes of PD-1KO mice compared with WT mice (P < 0.05). These results suggest that KD may develop in genetically prone populations by microorganism containing HSP65, and PD-1 gene may be an important component among genetic predispositions of KD.
Crystal structure of NKp30 and identification of ligand binding site through peptide mapping and blocking antibodies (94.12)
Authors: Joyce M.Gordon , Colonna Marco , Sun Peter
Published: January 2023

Natural Killer cells are a group of innate immune cells which carry out continuous surveillance for the presence of virally infected or cancerous cells. These lymphocytes are activated without any prior immune priming and elicit their effects via a combination of both activating and inhibitory receptors ensuring a highly sensitive mechanism which allows the differentiation between healthy and transformed cells. NKp30 is known to be the dominant activating receptor responsible for the destruction of a number of specific tumor cell types. In addition, NKp30 has been shown to cause the activation and expansion of resting NK cells upon interaction with dendritic cells and in addition to cause the death of immature dendritic cells (imDCs). The precise mechanism of how NKp30 interacts with virus-infected, cancerous cells and imDCs initiating NK cell killing is an intriguing question and to date, a number of NKp30 interacting molecules have been proposed. These molecules are quite diverse, ranging from Heparan sulphate to a plasmodium falciparum molecule and most recently a B7 homolog protein. How NKp30 recognizes these diverse molecules is to date unknown. In order to address this question we have determined the crystal structure of the extracellular domain of human NKp30. In an attempt to understand how NKp30 binds diverse ligands, we have identified a putative ligand binding region on the surface of NKp30 through peptide mapping and competition with blocking antibodies.
Viral immune evasion: HSV-1 immediate early protein ICP0 inhibits TLR2-driven NF-kB activation (94.18)
Authors: Kurt-Jones Evelyn , vanLint Allison , Murawski Matthew , Fitzgerald Katherine , Finberg Robert , Knipe David
Published: January 2023

The recognition of Pathogen Associated Molecular Patterns (PAMPS) by conserved receptors of the innate immune system is a critical step in the development of anti-viral immunity. We have studied a class of innate immune receptors, the Toll-like receptors (TLRs), in the anti-viral response to viruses including Herpesviruses. We have previously shown that HSV-1 infection induces NF-kB dependent innate immune responses via TLR2. TLR2 activation by HSV-1 drives NF-kB-dependent inflammatory cytokine production which plays a critical role in the HSV-1 pathology particularly the development of lethal encephalitis. Here we describe studies of TLR2-HSV-1 interaction which reveal a complex interplay between viral gene products and the host innate immune response. Using various mutant HSV-1 viruses, we find that ICP0, an immediate early viral protein, significantly inhibited TLR2-driven responses. Further, ICP0 expression alone was sufficient to block TLR2 activation by both viral and non-viral stimuli. Expression of ICP0 reduced levels of MyD88 and Mal, two critical adaptors for TLR2 signaling. ICP0 did not affect TRIF- or TRAM-driven NF-kB, but did block MyD88- and Mal-driven NF-kB responses. Proteosomes were required to inhibit TLR2 activation as was the E3 ligase function of ICP0. Our results suggest that ICP0 promotes degradation of the TLR adapter proteins, MyD88 and Mal, thereby blocking TLR2-driven activation of NF-kB sensitive inflammatory target genes.
Role of Myd88-mediated signaling in the prevention of colon adenocarcinomas (95.10)
Authors: Salcedo Rosalba , Worschech Andrea , Cardone Marco , Jones Yava , Gyulai Zsofia , Dai Ren-Ming , Marincola Francesco , Trinchieri Giorgio
Published: January 2023

Signaling through the adaptor protein MyD88 promotes carcinogenesis in several chemically induced cancer models. In contrast, in the AOM/DSS model of chronic colitis induced cancer, we observed a protective role for MyD88 in the development of colitis-associated cancer (CAC). The inability of Myd88-/- mice to heal ulcers generated upon DSS treatment creates an altered inflammatory environment that induces profound early alterations in expression of genes encoding pro-inflammatory factors as well as pathways regulating cell proliferation and apoptosis. Additionally, AOM/DSS treatment induced alterations in DNA repair in MyD88 deficient mice, resulting in increased frequency of mutations and a dramatic increase in adenoma formation and progression to invasive phenotype. Others have reported that Tlr4 deficient mice share similar susceptibility to colitis as Myd88 deficient mice but, unlike the latter, are resistant to CAC. We observed that mice deficient for Tlr2 or IL-1R do not show a differential susceptibility to colitis or CAC. In contrast, AOM/DSS treatment resulted in increased susceptibility to colitis development and enhanced polyp formation in Il-18-/- mice. This indicates that the phenotype of Myd88 -/- mice is in part due to their inability to signal through the IL-18 receptor. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that differentially impact tissue homeostasis and carcinogenesis.
Mechanism of inhibition of colitis by indole-3-carbinol (96.19)
Authors: Busbee Phililp , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Colitis is an autoimmune disease characterized by acute or chronic inflammation of the large intestine. Currently there is no cure for patients suffering from colitis, and most treatments involve the use of immunosuppressive drugs that can have adverse side-effects or increased toxicity. In the current study, we investigated the effects of indole-3-carbinol (I3C), a component found in a number of cruciferous vegetables. The products of I3C activate the aryl hydrocarbon (AhR) receptor, which has been shown in our laboratory to trigger apoptosis in activated T cells. In the current study, we tested the effect of I3C on a colitis model induced by administration of dextran sodium sulfate (DSS). The data showed that I3C administration reversed the weight loss seen during DSS-induced colitis. I3C treatment also reversed the shortening of the colon caused by DSS, as well decreased the level of immune cell infiltration found in the large intestine of colitis-induced mice. Early analysis of cell populations from the mesenteric lymph node showed that I3C treatment reduced the effects the disease model had on immune cells including T regulatory cells, natural killer cells, and myeloid-derived suppressor cells. In all these immune cell subsets, I3C treatment caused a return of the proportion of these cell populations to a more normal state. Together, the current study suggests that I3C may a serve as a novel treatment modality against colitis.
In vivo Treg expansion by TNFRSF25 stimulation inhibits allergic lung inflammation (97.11)
Authors: Tsai Matthew , Schreiber Taylor , Wolf Dietlinde , Chirinos Jackeline , Gonzalez Louis , Levy Robert , Podack Eckhard
Published: January 2023

TNF receptor super family member 25 (TNFR25, DR3) is constitutively and highly expressed by CD4+FoxP3+ regulatory T cells (Treg). Here we have utilized a TNFR25 agonistic monoclonal antibody, 4C12, to study the effects of TNFR25 signaling on Treg in vivo. We have previously demonstrated that signaling through TNFR25, in the absence of TCR signals, induces rapid and selective expansion of natural Treg in vivo to 30-35% of all CD4+ T cells within 4 days. TNFR25-expanded Treg remained highly suppressive ex vivo. Here we demonstrate that although certain IL-2 antibody complexes expand Treg to a similar degree, only in vivo TNFR25-expanded Treg are protective against allergic lung inflammation, a model for asthma, by reversing the T-effector to T-regulatory ratio in the lung, suppressing IL-13 and Th2 cytokine production and blocking eosinophil exudation into bronchio-alveolar fluid. Unlike TNFR25 responses by T effector cells, which require TCR engagement, expansion of Treg by TNFR25 signaling is not dependent upon stimulation by exogenous antigens. Our studies define a novel mechanism for Treg control and important functions for TNFR25/TL1A in regulating auto aggression that balance its known role in enhancing auto immunity.
Immunocomplexes down-modulate the induction of food allergy in mouse. (97.12)
Authors: Alves de Araujo Claudia , Clay Bryan , Ganeshan Kirthana , Bryce Paul , Sperling Anne
Published: January 2023

Food allergy is an adverse Th2 response whose incidence has been increasing dramatically over the past decade. We have demonstrated that signaling triggered by immunocomplexes (IC) through CD16 (FcgRIII) in conjunction with TLR4 prime dendritic cells (DCs) to drive Th2 responses. To address our hypothesis that IgG plays a role in the pathogenesis of food allergy in a murine model, we investigated the contribution of CD16 signaling. Wild type (WT) or CD16-/- mice were sensitized by gavage for 8 weeks with SEB, SEB+OVA or SEB+OVA-IC and challenged on the 9th week. To analyze the systemic responses, blood eosinophilia and OVA-specific serum IgE and IgG1 levels were measured. We found that CD16-/- had reduced responses compared to WT, however, both SEB+OVA-IC-immunized WT and CD16-/- mice had lower responses compared to SEB+OVA immunized controls. To investigate the effect of IC in DC functions, naïve WT and CD16-/- BMDCs were stimulated with SEB with either OVA or OVA-IC, and DC maturation and survival were determined. Compared to SEB-OVA stimulation, SEB+OVA-IC-stimulated WT and CD16-/- BMDCs have similar maturation, but a decrease in late apoptotic cells. Our findings suggest that ICs down-regulate the allergic response in the gut of WT and CD16-/- mice leading to lower systemic responses, and yet preserves DC functions. Thus, IC might induce a regulatory network in the gut mucosal that allows sensitization, but fails to trigger an exacerbated inflammatory response.
Allergic Airway Disease is Inhibited by Transfer of Lung Tissue CD8+ T Cells from Tolerant Mice (97.4)
Authors: McNamara Jeff , Secor Eric , Guernsey Linda , Schramm Craig , Lefrancois Leo , Thrall Roger
Published: January 2023

CD8+ T cells can play both a pro and anti inflammatory role in asthma, which we have examined using a biphasic model of allergic airway disease (AAD). Acute delivery (7-14 days) of ovalbumin (OVA) aerosol to sensitized animals results in AAD, while continued delivery of OVA (42days) results in local inhalational tolerance(LIT). During AAD the number and percentage of antigen specific CD8+ T cells continued to increase in the bronchoalveolar lavage(BAL) and lung tissue past the peak of BAL eosinophilia. After the chronic delivery of allergen high percentages of OVA specific cells were still found in the BAL, whereas lower levels were identified in the lung tissue. There were phenotypic changes in the BAL and lung tissue cells at LIT, including lower CD27, CD127 and granzyme b expression and higher expression of the inhibitory NK2GA molecule. Negatively selected CD8+ T cells from the lung tissue from LIT animals were transferred into sensitized animals. Subsequently, these mice received 6 days of OVA aerosol and then were examined for disease. Mice receiving LIT Lung Tissue CD8+ T cells had significantly fewer eosinophils in the BAL (9±5 x105 eosinophils) than mice receiving saline (47±14), or LIT lung tissue CD8- T cells (37±7 p = 0.05 by ANOVA). These data suggested that CD8+ T cells from the lung tissue may play an important role in the establishment of tolerance in AAD. What role the altered phenotype of OVA specific cells play in this inhibition is yet to be determined.
Bypassing a relatively common deficiency in neurotransmitter synthesis blocks allergic inflammation (97.8)
Authors: Cook-Mills Joan , Mahadevia Riti , McCary Christine , Abdala-Valencia Hiam , Berdnikovs Sergejs
Published: January 2023

An unaddressed triad of symptoms in allergic patients includes: 1) allergic/asthmatic responses, 2) anxiety/depression, and 3) constipation/acid reflux. We proposed that there is a common mediator of these symptoms. It is reported that a deficiency in the neurotransmitter serotonin increases anxiety/depression and reduces peristaltic gastrointestinal movement. In addition, leukocytes synthesize serotonin and serotonin receptors are on leukocytes. Therefore, we proposed that a common mediator is a deficiency in synthesis of the serotonin precursor, 5-hydroxytryptophan (5HTP). A reduction in synthesis of 5HTP occurs due to common polymorphisms in the rate limiting enzyme tryptophan hydroxylase (TPH). TPH converts tryptophan to 5HTP and decarboxylases convert 5HTP to serotonin. To address 5HTP regulation of inflammation, we determined whether dietary supplementation with 5HTP reduces mouse allergic lung inflammation induced by OVA or by house dust mite (DerP1). C57BL/6 mice were used since they have some of the serotonin pathway polymorphisms. 5HTP-containing diets were started two days before antigen. Interestingly, 5HTP blocked lung inflammation 50-70% without affecting body weight, blood eosinophils, Th1/Th2 cytokines, or chemokines. In addition, 5HTP pretreatment of endothelial cells blocked leukocyte transendothelial migration, suggesting that leukocyte recruitment is inhibited. Thus, 5HTP and its metabolites are critical for regulation of allergic inflammation.
Vitamin E isoforms and doses differentially regulate experimental asthma. (97.9)
Authors: McCary Christine , Abdala-Valencia Hiam , Berdnikovs Sergejs , Cook-Mills Joan
Published: January 2023

We have reported that supplemental doses of the alpha and gamma tocopherol isoforms of vitamin E decrease and increase, respectively, lung inflammation in murine experimental asthma. Because asthma clinical trials are currently examining the effect of highly-elevated tocopherol doses in humans, we examined the regulation of murine experimental asthma by highly-elevated doses of tocopherols, defined as 2 mg tocopherol/day (equal to 10 times the supplemental dose). Mice treated with highly-elevated doses of alpha or gamma tocopherol had significantly higher tissue levels of tocopherols, decreased leukocyte infiltration into the lung, and decreased Th2 cytokine and chemokine production. The reversibility of tocopherols’ effects was also determined. At either supplemental or highly-elevated doses, the regulatory effects of alpha and gamma tocopherol were fully reversible, revealing no sustained anti- or pro-inflammatory functions of these isoforms. Overall, gamma tocopherol enhances inflammation at supplemental doses and ablates inflammation at highly-elevated doses. In contrast, alpha tocopherol decreases inflammation at all doses tested. Our results have clinical implications for tocopherol supplementation and alter interpretation of tocopherol studies reported in the literature.
Effects of carotenoids on differential expression of scavenger receptors and cytokines in human monocyte-derived macrophage subpopulations (98.19)
Authors: Wiener Doris , Gong Xiaoming , Marisiddaiah Raju , Rubin Lewis
Published: January 2023

Circulating monocytes differentiate into macrophage (MΦ) subpopulations in response to environmental signals. Carotenoids may reduce MΦ oxidant stress, alter the inflammatory response, and lower risk of atherosclerosis. These observations prompted us to determine the effects of β-carotene, lycopene, astaxanthin and lutein on differentiation of human monocytes into MΦs (M1 and M2) phenotypes. Monocyte-to-MΦ differentiation was driven by treatment for 6 days with GMCSF or MCSF ± individual carotenoids. M1 MΦs (+GMCSF) were activated with LPS+IFNγ; M2 MΦs (+MCSF) were activated with IL-4. We validated that M1 and M2 cells showed distinct morphologies and expression patterns (qRT-PCR) of scavenger receptors (SR-A, LDLR, CD36, SR-B1) and cytokines (IL-10, IL-12). M1 and M2 markers (CD14, CD16, CD36, CD80, CD163) and cytokines (PGE2, IL-6, IL-8, IL-10, IL-12, TNFα, IFNγ) were confirmed by flow cytometry. Expression of CD14, CD16, CD36, CD163, and IL-10 was higher in M2 than M1 MΦs. Lycopene and astaxanthin decreased SRA, CD36 and LDLR mRNA levels in M1 cells but had no significant effect on M2 cells. Lutein also reduced SRA, CD36 and IL-10 expression in M2. Conversely, β-carotene increased M1 and decreased M2 expression of SRA, CD36, IL-10 and IL-12. Results show individual carotenoids can regulate MΦ scavenger receptor and cytokine expression. We speculate that carotenoids influence MΦ polarization, thereby inhibiting cholesterol accumulation in MΦ-derived atherogenic foam cells.
ESTROGEN RECEPTOR SIGNALING PROMOTES MONOCYTE ACTIVATION AND DIFFERENTIATION UPON INFLAMMATION IN VIVO (98.20)
Authors: Bajana Sandra , Turner Sean , Kovats Susan
Published: January 2023

17-β-estradiol at physiological levels regulates GM-CSF-mediated dendritic cell (DC) differentiation in vitro by promoting the generation of CD11bintLy6C- DC from myeloid progenitors. In this model, estrogen receptor alpha (ER) signaling induces and sustains elevated levels of interferon regulatory factor 4 (IRF4), a transcription factor critical in this pathway. Such events may relate to the de novo DC differentiation that occurs in vivo upon inflammation during infection or autoimmunity, resulting in inflammatory DC that determine the induction of specific Th cell populations. The current work evaluates whether ER signaling regulates new DC differentiation from blood monocytes during inflammation in vivo, and if this is associated with elevated IRF4 expression. ERα+/+ or ERα-/- mice were immunized subcutaneously with complete Freund’s adjuvant. By day 2 post immunization, compared to ERα-/- mice, ERα+/+ mice harbored an increased frequency of CD115+CD11b+ blood monocytes expressing higher levels of IRF4 and MHC class II. On days 2 and 7, relative to ERα-/- mice, lymph nodes of ERα+/+ mice contained greater numbers of monocyte-derived Gr1+CD11b+ CD11clo inflammatory DC. These data show that during inflammation in vivo, ER signaling promotes monocyte activation, IRF4 expression and differentiation, which leads to increased numbers of a de novo inflammatory DC population in draining lymph nodes.
Use of Computer Aided Drug Design (CADD) to identify small molecule inhibitors of TLR2 signaling (98.24)
Authors: Schwarz Ryan , Laird Michelle , Toshchakov Vladimir , Green Shannon , MacKerell Alexander , Vogel Stefanie
Published: January 2023

Toll-like receptors (TLRs) are a family of transmembrane innate immune molecules that play a key role in the detection of microbial and self ligands released from damaged cells. At their C-termini, Toll/Interleukin-1 receptor (TIR) domains dimerize upon ligand binding and initiate a signaling cascade that leads to upregulation of inflammatory cytokines. Overexuberant signaling leads to a “cytokine storm,” causing extensive tissue damage that can be fatal. The ability to block TLR signaling may ameliorate such a scenario. Using CADD, a panel of ~150 low molecular-weight compounds was identified based on their predicted ability to bind a pocket on the TLR2 TIR surface adjacent to BB loop. We performed preliminary screening of these compounds in TLR2-HEK293T stable transfectants and secondary screening of promising candidates in thioglycollate-elicited peritoneal murine macrophages. The impact of compounds on signaling was determined using quantitative RT-PCR of cytokines in response to TLR2-specific agonists (P2C and P3C) and TNF-α, which does not utilize TIR domains for signaling. Compound cytotoxicity was assessed by lactate dehydrogenase (LDH) release. To date, we have identified 10 putative TIR-specific inhibitors, 1 TNF-α-specific inhibitor, and 4 inhibitors of both TIR and TNF-α signaling. This project may lead to the identification of potent small molecule inhibitors of TIR-specific TLR signaling.
Elf3 regulates dendritic cell driven T cell differentiation (99.1)
Authors: Kushwah Rahul , Wu Jing , Oliver Jordan , Hu Jim
Published: January 2023

Elf3 belongs to the family of epithelium specific Ets transcription factors and can be upregulated in non-epithelial cells upon cytokine stimulation. Elf3-/- mice have normal T cell development and T cell differentiation into different T helper lineages. Here we show that upon epicutaneous sensitization with ovalbumin(OVA) followed by OVA intranasal challenge, Elf3-/- mice display a defect in Th17 induction, though OVA specific IgE and IgG1 antibody titers are several folds higher and airway inflammation is more extensive compared to Elf3+/+ mice, indicating an exaggerated Th2 response. Furthermore, intraperitoneal sensitization of Elf3-/- mice with OVA followed by OVA intranasal challenge, confirmed that Elf3-/- mice mounted an exaggerated Th2 response. Since, dendritic cells (DCs) are key players in initiation of adaptive immune response, we analyzed DC migration upon OVA challenge and showed that pulmonary DCs underwent hyperactivation in Elf3-/- mice compared to Elf3+/+ mice and rates of DC migration to the airway draining lymph nodes were also significantly higher in Elf3-/- mice, indicating that DCs may mediate the exaggerated Th2 response observed in Elf3-/- mice. Further analysis revealed that Elf3-/- DCs are more prone to maturation than Elf3+/+ DCs and upon maturation, Elf3-/- DCs preferentially secrete chemokines/cytokines that are important in polarizing Th2 differentiation. Taken together, these findings reveal a role of Elf3 in DC driven T cell differentiation.
RNA-binding protein HuR coordinately regulates genes essential for Th2 polarization and function at the posttranscriptional level (99.7)
Authors: Gubin Matthew , Stellato Cristiana , Casolaro Vincenzo , Tartar Danielle , Magee Joseph , Chen Jing , Calaluce Robert , Atasoy Ulus
Published: January 2023

Posttranscriptional gene regulation controls the expression of genes implicated in a variety of processes, including those of the immune system. The RNA-binding protein HuR regulates many early response genes as well as cytokines such as IL-4 and IL-13. Therefore, HuR has been implicated in regulating Th2 polarization and cytokine production. GATA-3, a transcription factor that is essential for Th2 polarization contains a putative HuR binding site in its 3’UTR. Immunoprecipitation (IP) of ribonucleoprotein complexes with an anti-HuR Ab revealed significant enrichment for GATA-3 mRNA. HuR association with GATA-3 3’UTR was confirmed by biotin pull-downs. To understand the role of HuR in Th2 polarization in vivo we generated a transgenic HuR CD4+ T-cell mouse model. Th2-skewed cells over-expressing HuR displayed increases in both numbers of GATA3-expressing cells, and in overall GATA-3 expression. The frequency of Th2-polarized cells, but not of Th1, increased in CD4+ T cell HuR transgenic cells and the levels of secreted IL-4 and IL-13, but not IFN-γ, also increased significantly in both activated splenocytes and polarized cells. Furthermore, a knockdown of HuR in Jurkat cells decreased protein levels of GATA-3. Hence, HuR may be functioning to coordinately posttranscriptionally regulate genes essential for Th2 polarization and function. Better understanding of posttranscriptional regulation may elucidate control mechanisms of naïve CD4+ Th2 polarization.
Cellular sentinels for Heat Shock Protein-peptide complexes in vivo. (100.1)
Authors: Messmer Michelle , Binder Robert
Published: January 2023

Heat shock proteins (HSPs) released into the extra cellular environment via cell death or through vaccination are taken up by antigen presenting cells of the immune system. HSP-chaperoned peptides are then cross-presented for priming of specific T cell responses. Our goal is to identify the subset(s) of cells that sample HSPs in vivo and then initiate T cell priming. By introducing a fluorescent labeled HSP into mice, cells in the draining lymph node that engage extracellular HSP have been identified. Our data demonstrate that CD4+CD11c+ dendritic cells (DC) are the predominant cells that internalize the HSP, while CD8+CD11c+ DCs did not significantly internalize the HSP. The localization of these cells within the lymph node and their ability to prime T cells are examined. These studies illustrate how HSPs act to alert the immune system of cellular damage and will be useful for optimizing HSP-based vaccination regimes in clinical studies of cancer patients.
Listeria-derived recombinant proteins are processed by a unique MHC class I processing pathway (100.10)
Authors: Wolf Benjamin , Princiotta Michael
Published: January 2023

Studies examining antigen processing in infected cells suggest that the efficiency of peptide-MHC class I (pMHC) production can differ by as much as 100 to 1000-fold depending on the source of the protein from which the peptide determinant is derived. For recombinant proteins expressed from vaccinia virus (rVV), ~3000 protein molecules must be degraded to generate one surface pMHC complex. By contrast, the measured efficiency of pMHC production from native Listeria monocytogenes proteins is ~1 surface pMHC complex per 3.5 to 35 protein molecules degraded. What accounts for this 100 to 1000-fold difference in efficiency remains unresolved. One possible explanation is that processing efficiency is a function of the peptide and/or protein studied. It is also possible that exogenously derived proteins, such as those secreted by Listeria, enter a different processing pathway than host-cell derived proteins. To better understand these differences in processing efficiency, we engineered Listeria strains to express recombinant proteins similar to those used for the rVV efficiency studies. We found that surface pMHC production was 20-fold more efficient from recombinant proteins expressed from Listeria than from a rVV-derived recombinant protein. These data suggest proteins expressed from Listeria follow a distinct and highly efficient processing pathway relative to host cell derived proteins.
Combinatorial peptide library scans of multiple HLA A*0201 restricted CTL reveal high levels of degeneracy at MHCI anchor positions (100.12)
Authors: Wooldridge Linda , Edwards Emily , Ekeruche Julia , Miles John , van den Berg Hugo , Clement Mathew , Sewell Andrew
Published: January 2023

CD8+ cytotoxic T lymphocytes (CTLs) hold the key to the successful prevention and eradication of infectious and neoplastic disease. T-cells recognize ‘foreign’ peptide fragments often referred to as epitopes, in the context of ‘self’ major histocompatibility complex class I (MHCI) molecules. Epitope identification is essential to define targets for the effective control of disease. Peptides bind to MHCI molecules by anchoring at position 2 and the C-terminus of the peptide ligand. Techniques such as pool sequencing have been hugely instrumental in revealing HLA allele-specific peptide binding motifs. However, this approach only reveals the most dominant MHC anchor residues. The preferred binding motif identified by pool sequencing for the most common MHCI, HLA A*0201, is L/M at position 2 and V/I/L/A at the C-terminus. Here, we use an extremely sensitive combinatorial library screening approach to show that in the context of HLA A*0201, degeneracy at anchor residue positions is much higher than previously appreciated. We also examined the recognition of peptides with substitutions of all 20 amino acids at both anchor positions in three different clonal systems which confirm these observations. Indeed, in one clonal system, 9-mer peptides containing A, C, F, I, K, L, M, Q, S, T, V in position 2 and A, C, F, I, L, M, T, V at position 9 were recognized efficiently. These observations have implications for T-cell epitope prediction and future vaccine design.
ERAAP-deficiency differentially affects peptide presentation across multiple MHC I (100.15)
Authors: Lind Kristin , Escobar Hernando , Reyes-Vargas Eduardo , Delgado Julio , Shastri Nilabh
Published: January 2023

Genome-wide association studies in humans have linked polymorphisms in the ER aminopeptidase associated with antigen processing (ERAAP) as well as particular MHC class I molecules to autoimmune diseases. How ERAAP polymorphisms affect function of different MHC I molecules is not known. ERAAP normally trims antigenic precursor peptides to generate potential peptide-MHC I ligands that can be recognized by CD8 T cells. To detect changes in the peptide repertoires displayed by different MHC I molecules, we immunized wild-type B10.D2 (H-2d) mice with ERAAP-deficient (ERAAP-KO) cells and analyzed the specificity of the CD8 T cell response. In wild-type mice, anti-ERAAP KO T cells responded primarily to peptides presented by Kd, and not the other two MHC I molecules, Dd and Ld of the H-2d haplotype. We also assessed the MHC I bound peptide repertoire by mass spectrometry. This analysis revealed that Dd and Kd molecules were bound to large number of shared as well as unique peptides, some with N-terminal extensions. Remarkably, Ld bound peptides were not detected in ERAAP-deficient mice. Thus, changes in ERAAP expression can have a major influence on the peptide repertoire presented by different MHC I molecules as well as on CD8 T cell responses.
Beryllium-specific T cells adopt an unusual binding topology for antigen recognition (100.25)
Authors: Bowerman Natalie , Falta Michael , Mack Douglas , Kappler John , Fontenot Andrew
Published: January 2023

Unconventional antigens, such as metals, stimulate T cells in a very specific manner. To delineate the binding landscape for metal-specific T cell recognition, alanine screens were performed on a set of beryllium (Be)-specific T cell receptors (TCR) derived from the lung of a chronic beryllium disease patient. These TCRs were HLA-DP2-restricted and expressed nearly identical Vβ5.1+ chains coupled with different Vα chains. Site-specific mutagenesis of all amino acids comprising the complementarity determining regions of TCR Vα and Vβ chains showed a dominant role for Vβ5.1 residues in Be recognition, with little contribution from the Vα domain, suggesting an abnormal binding topology. To confirm these findings, solvent-exposed residues along the α-helices of the α- and β-chains of HLA-DP2 (i.e., potential TCR contact sites) were mutated to alanine. Two β-chain residues, located near the proposed Be binding site of HLA-DP2, played a dominant role in T cell recognition, while the α-chain mutations had no effect on T cell activation. These findings strongly suggest that Be-specific T cells recognize antigen using an unconventional binding topology, with the majority of interactions contributed by residues in the TCR Vβ5.1 domain and HLA-DP2 β1-chain. We propose that unusual docking topologies are not used exclusively by T cells recognizing self-antigens, but also for the recognition of metal antigens, such as beryllium. Supported by NIH grants: HL062410 and HL092997.
Deficiency in Ab response to insulin in gamma-IFN-inducible lysosomal thiol reductase (GILT) knock-out mice (100.29)
Authors: He Xiao , Wang Xiaomin , Jensen Peter
Published: January 2023

The γ-IFN-inducible lysosomal thiol reductase (GILT) is a unique thiol reductase with optimal activity at low pH, and is expressed in Ag presenting cells (APC). GILT is critical to processing protein Ag by catalyzing disulfide bond reduction, thus facilitating the unfolding of native Ag containing disulfide bonds for further processing. GILT-/- mice are defective in processing exogenous protein Ag. However the role of GILT in Ab response is not clear. Here we report that after immunization, GILT-/- mice failed to produce Ab to exogenous bovine insulin (bIns), which has A and B chains and contains both inter and intra-chain disulfide bonds, while Ab response to OVA was normal in both GILT-/- and wt B6 mice. GILT-/- APC was able to present bIns to an insulin B chain peptide-specific T hybridoma, although it produced less amounts of IL2, compared to when using B6 APC. IL2 production was MHC-II mediated (could be blocked by α-mouse, but not α-human, MHC-II Ab) and the reduced IL2 production of GILT-/- cells was bIns-specific since GILT-/- APC could not present HEL, another Ag containing disulfide bonds, to an HEL-specific T hybridoma to produce IL2. This data indicates that the T cell response to bIns is only partially affected in GILT-/- mice, which was further supported by the fact that CFSE labeled, in vivo bIns primed, GILT-/- CD4 cells proliferated equally well as wt B6 CD4 cells upon restimulation by bIns. Our data indicates a novel role of GILT in Ab response to insulin.
Microparticles released by Listeria monocytogenes-infected macrophages contribute to the generation of protective immunity: insight into interaction between macrophages and dendritic cells (100.33)
Authors: Huang Bo , Zhang Yi , Liu Jing
Published: January 2023

Mutual interaction among immune cells is of paramount importance to an efficient protective immunity against bacterial infection. However, the interplay between macrophages and dendritic cells (DC) in the processing and presentation of bacterial antigens for T cell immune responses remains poorly understood. In this report, using Listeria monocytogenes (Lm) infection model, we demonstrate that DCs require the support of macrophages to elicit the protective immunity against Lm infection. We found that DCs themselves were inefficient at taking up Lm but capable of taking up microparticles released by Lm-infected macrophages. These microparticles transferred Lm antigens to DCs, allowing DCs to present Lm antigen to effector T cells. Such microparticles-mediated Lm antigen transfer required MHC class I participation, since MHC class I deficiency in macrophages resulted in a significant reduction of T cell activation. Moreover, the vaccination of mice with microparticles from Lm-infected macrophages produced strong protective immunity against the challenge of Lm. Thus, we have identified an intrinsic antigen transfer program between macrophages and DCs during Lm infection, and emphasized that macrophages also play an essential role in DC-elicited Lm-specific T cell responses.
Obese concentrations of leptin enhances dendritic cell indution of T cell activation (100.37)
Authors: Radovich Abraham , Garza Kristine , Gonzales Amanda , Delgado Christine
Published: January 2023

The treatment with leptin has shown an increase in the functionality in DCs as they produce longer dendrites, however, the interaction with T-cells and the migration in vivo have not been studied. Therefore the relevance of the investigation conveys immunological responses. The increase in T-cell interaction with the dendritic cells, will lead to a quicker migration in vivo of the DCs to the high concentration areas. The decrease in time of activation will then optimize the immunological response. The treatments of the DC are to be performed by concentration seen in obese conditions, that way the interaction of leptin can be assured. The usage of KODAC imager will demonstrate the increase of T-cell/DC interaction. The experiments demonstrated an increase of T-cell interaction with the DC. Also, the leptin increase the functionality of the DC lines, as could be seen to have longer and more prominent dendrites. The profileration of DC was also seen as they were treated in leptin and LPS. The treatment of DC with leptin, was tested to enhance the T-cell/DC activation and the results correlated the fact that dendritic cells migrate faster in vivo as they are treated with leptin. The fact that it can be demonstrated that the DCs, involved in immunological responses, can be enhance will open the path to bettering the immunological responses in order to be more effective in less time.