bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel role of tissue transglutaminase (TG2) in chronic inflammatory diseases (48.12)
Authors: Cexus Olivier Nicolas Felix , Labrousse Delphine , Luciani Alessandro , Elliott Tim , Maiuri Luigi , Quaratino Sonia
Published: January 2023

Tissue transglutaminase (TG2) has a critical role in the pathogenesis of chronic inflammatory diseases. We have previously described the key role of TG2 in cystic fibrosis (CF), a genetic disease characterised by chronic lung infections and inflammation. In CF, mutation on the CFTR gene results in an increased TG2 expression and activity leading to functional sequestration of the anti-inflammatory PPARγ and increase of inflammation.

Here we tested whether in vivo inhibition of TG2 can reverse inflammation in chronic inflammatory diseases. To assess the importance of TG2, we injected cystamine, a potent TG2 inhibitor, in a transgenic mouse model of CF and in the TAZ10 transgenic mice that spontaneously develop autoimmune thyroiditis.

Intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the CF model, where it decreased TG expression and activity. The treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional PPARγ. Interestingly, cystamine injections could also block inflammation in the TAZ10 TCR transgenic mouse model, highlighting the pivotal role of TG2 in generating inflammation in two very different pathologies.

This work underlines the critical role of TG2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases.
A critical role of BTLA in peripheral T cell tolerance induction (48.18)
Authors: Liu Xikui , Alexiou Maria , Martin-Orozco Natalia , Chung Yeonseok , Nurieva Roza , Tian Qiang , Lu Sijie , Kollias George , Graf Daniel , Dong Chen
Published: January 2023

T cell activation and tolerance are delicately regulated by costimulatory molecules. Although B and T lymphocyte attenuator (BTLA) has been shown as a negative regulator for T cells activation, its role in peripheral T cell tolerance induction in vivo has not been addressed. In this study, we generated a novel strain of BTLA-deficient mice, and employed three different models to characterize the function of BTLA in controlling T cell tolerance. In an oral tolerance model, BTLA-deficient mice were found resistant to the induction of T cell tolerance to an oral antigen. Moreover, compared with wild-type OT-II cells, BTLA-/- OT-II cells were less susceptible to tolerance induction by high-dose Ova peptide administered intravenously. Finally, BTLA-/- OT-I cells caused autoimmune diabetes in RIP-mOVA recipient mice. Our results thus demonstrate an important role of BTLA in the induction of peripheral tolerance of both CD4+ and CD8+ T cells in vivo.
Distinct lineages of human T-cell clones, including Th17/Th1 cells, isolated at different stages of anti-factor VIII immune responses (48.26)
Authors: Ettinger Ruth A , James Eddie A , Liberman Joseph A , Kwok William W , Thompson Arthur R , Pratt Kathleen P
Published: January 2023

Approximately 25% of hemophilia A patients, who lack functional factor VIII (FVIII), develop neutralizing antibodies after receiving therapeutic infusions of FVIII. HLA-DRA-DRB1*0101-restricted T-cell clones that respond to FVIII2194-2213 were isolated from a hemophilia A subject (the proband) ~5 and 21 months following his development of a high-titer FVIII neutralizing antibody response, and from his brother who has not developed a clinically significant antibody response. Three clones obtained from the proband at 5 months were TH17/TH1-polarized and two were TH1/TH2-polarized. The 8 proband clones isolated at 21 months were TH2-polarized, indicating T-cell lineage changes over time. The 6 clones from the brother were TH1-polarized, indicating that B-cell tolerance to FVIII can be maintained even with circulating antigen-specific TH1-polarized cells. Several TCRBV-D-J junction sequences were identified suggesting differences in TCR interaction with the MHC class II-peptide complex. Current investigations are examining the response of clones to altered peptide ligands. This is the first evidence that TH17/TH1-polarized cells play a role in hemophilic immune responses to FVIII; it is also the first detailed characterization of antigen-specific TH17/TH1 clones isolated using standard culture conditions.

Research Support: Bayer Hemophilia Award, CSL Behring Hemophilia Research Award, NIH.
Alterations in both coagulation and fibrinolytic pathways mediate thrombosis in the skin during murine Graft-Versus-Host (GVH)-induced scleroderma (48.5)
Authors: Burwell Tim , Delaney Tracy , Chen Cindy , Connor Jane , Coyle Anthony J , Lemaire Raphael
Published: January 2023

Scleroderma is a systemic autoimmune disease characterized by fibrosis, inflammation and vasculopathy of the skin, kidney and lung. The vasculopathy features occlusion of small arteries due vasoconstriction, intimal proliferation and thrombosis. Thrombosis results from an imbalance between coagulation and fibrinolysis. Whether thrombosis in scleroderma is primarily due to alteration in fibrinolysis or coagulation remains unclear. In this study, we investigated thrombosis in a murine GVH model of human scleroderma exhibiting an occlusive vasculopathy. We looked at fibrinolysis and coagulation biomarkers in skin, kidney and lung using qPCR and IHC. GVH skin showed microthrombi in dermis blood vessels. This was associated with an increase in a number of key regulators of fibrinolysis (PAI-1, tPA and uPAR) and coagulation (Tissue Factor, Factor VII and X), suggesting that thrombosis in GVH skin is caused by alterations in both fibrinolysis and coagulation pathways. Unlike skin, kidney and lung did not show fibrinolysis and coagulation alterations, suggesting that vasoconstriction rather than thrombosis mediates the occlusive vasculopathy in these tissues, consistent with increased endothelin-1 levels in kidney VSMCs. In conclusion, our study suggests that the GVH mouse is a useful model to study the balance of fibrinolysis and coagulation leading to thrombosis in scleroderma vasculopathy in the skin.
Uptake of apoptotic dendritic cells by viable dendritic cells: a novel mechanism of inducing immune tolerance (48.7)
Authors: Kushwah Rahul , Wu Jing , Oliver Jordan R , Jiang George , Zhang Jinyi , Siminovitch Katherine A , Hu Jim
Published: January 2023

Dendritic cells (DCs) are professional antigen-presenting cells, which can be affected by the death of other cells in close proximity. Spontaneous DC apoptosis has been observed in patients with sepsis and breast cancer. Additionally defects in DC apoptosis have also been associated with development of autoimmune diseases. However, the mechanisms of how DC apoptosis affects immune responses are unclear. Here we show that apoptotic DCs can mediate induction of immune tolerance by converting immature DCs into tolerogenic DCs. The uptake of apoptotic DCs by viable DCs prevents LPS-induced activation and T-cell proliferation, and also results in the upregulation of TGF-beta2 gene expression and TGF-beta1 secretion, which mediates differentiation of naïve T cells into regulatory T cells (Tregs). Additionally, delivery of apoptotic DCs prevents LPS-induced DC maturation and migration to lymph nodes in mice. Moreover, intranasal delivery of apoptotic DCs to LPS-inflamed lungs results in rapid resolution of inflammation along with production of TGF-beta1 and expansion of Tregs. Delivery of apoptotic DCs followed by immunization with ovalbumin (OVA) in Complete Freund's Adjuvant (CFA) resulted in generation of OVA-specific Tregs in wild-type mice and expansion of Tregs in T-cell receptor transgenic (OT-II) mice. Taken together, our findings identify apoptotic DCs as potent inducers of immunological tolerance.
The soluble CTLA-4 splice variant affects the function of CD4+CD25+ regulatory T cells (49.10)
Authors: Kissler Stephan , Fischer Kay , Zheng Peilin , Rainbow Dan , Wicker Linda
Published: January 2023

Type 1 diabetes is a polygenic autoimmune disorder. One of the genetic polymorphisms linked to type 1 diabetes, as well as to other autoimmune diseases, is located within the CTLA-4 gene. This polymorphism is associated with a change in splicing frequency, altering the ratio between full-length and soluble CTLA-4 (sCTLA-4) in human. The role and function of sCTLA-4 are as yet unknown. To investigate the sCTLA-4 isoform in a model organism, we generated nonobese diabetic (NOD) mice in which expression of sCTLA-4, but not full-length or ligand-independent CTLA-4, is constitutively reduced by RNAi. We find that loss of sCTLA-4 significantly reduces the potency of regulatory T cells, providing a possible explanation for the effect of the human CTLA-4 polymorphism on disease susceptibility. Our study demonstrates the utility of RNAi for the study of single splice variants in vivo, and supports a role for sCTLA-4 in regulatory T cell function.
A polymorphism within interleukin-21 receptor (IL21R) confers risk for systemic lupus erythematosus and is associated with malar rash in lupus patients (49.17)
Authors: Sawalha Amr , Webb R , Merrill J , Kelly J , Sestak A , Kaufman K , Langefeld C , Ziegler J , Kimberly R , Edberg J , Ramsey-Goldman R , Petri M , Reveille J , Alarcón G , Vilá L , Alarcón-Riquelme M , James J , Gilkeson G , Jacob C , Moser K , Gaffney P , Vyse T , Nath S , Lipsky P , Harley J
Published: January 2023

Interleukin (IL) 21 is a member of the type I cytokine superfamily that exerts a variety of effects on the immune system including B cell activation, plasma cell differentiation, and immunoglobulin production. The IL21 receptor (IL21R) is expressed on B cells, T cells, NK cells, and monocyte-derived dendritic cells. The expression of IL21R is reduced in B cells from lupus patients, while IL21 serum levels are increased in both lupus patients and some lupus-murine models. We recently reported that polymorphisms within the IL21 gene are associated with increased susceptibility to lupus. Herein, we genotyped SNPs in the IL21R gene located on chromosome 16p11. We identify and confirm the association between rs3093301 and lupus in two independent European-derived and Hispanic cohorts (p=7.2X10-5). In addition, the presence of the homozygous risk genotype in rs3093301 (A/A) was associated with the development of malar rash in the European-derived female lupus patients (OR=2.83, 95% CI=1.56-5.13, p=0.00045).
Development of inflammatory arthritis in DR4 transgenic and wild type DBA/1J mice immunized with human fibrinogen (49.9)
Authors: Cordova Kristen N , Holers V. Michael
Published: January 2023

The human MHC allele DR4 is associated with susceptibility for rheumatoid arthritis (RA). Citrullinated proteins, derived from the conversion of peptidyl-arginine to peptidyl-citrulline, are present in the joints of RA patients, who also uniquely produce high levels of anti-citrullinated protein antibodies (ACPAs). ACPAs enhance damage in animal models of autoimmune arthritis. DR4 molecules have been shown to preferentially bind citrullinated peptides over arginine controls. Recently, the DR4 allele was shown to be essential to develop arthritis in C57Bl/6 mice immunized with human citrullinated fibrinogen, but these mice demonstrated delayed arthritis at relatively low incidence and severity as compared to other arthritis models.

To potentially obtain a more susceptible strain, we backcrossed the DR4 gene onto the DBA1/J strain. Following immunization with either human citrullinated or noncitrullinated fibrinogen, these mice developed inflammatory arthritis around day 24 regardless of whether they were DR4+ or DR4- littermates. Serum from both strains demonstrated ACPA generation as defined by reactivity to a citrullinated but not control peptide, as well as a splenic proliferative response to both citrullinated and noncitrullinated human, but not mouse, fibrinogen. The DR4 DBA/1J transgenic strain may provide a useful tool for the analysis of ACPA-related arthritis.

Grant U19 AI146374
Gender difference in PPARα regulation of inflammation and demyelination in EAE model of multiple sclerosis (50.1)
Authors: Kanakasabai Saravanan , Walline Crystal C , Bright John J
Published: January 2023

Multiple sclerosis (MS) is a neurological disorder that affects more than a million people worldwide. The disease usually begins in young adults and affects women more than men. About 30% of MS patients develop clinical paralysis and become wheel chair-bound for the rest of their lives. The etiology of MS is not known and there is no medical treatment that can cure MS. Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor transcription factors that have anti-inflammatory properties. We and others have shown earlier that PPARγ, α and δ agonists ameliorate MS-like disease in experimental allergic encephalomyelitis (EAE) model. In this study we demonstrate that the PPARδ deficient male mice develop an exacerbated EAE, while the PPARδ deficient female mice developed comparable disease to wild type C57BL/6 mice, following immunization with MOGp35-55 antigen. The PPARδ deficient male mice showed an augmented Th17 response, without affecting neural antigen-specific T cell proliferation, Th1 differentiation and CD25+Foxp3+ regulatory T cells. Our findings highlight that PPARδ is a physiological regulator of EAE and confirm the use of PPARδ agonists for the treatment of MS patients.
Macrophages are required for anti-CD19 mAb mediated B cell depletion in mice (50.16)
Authors: Gallagher Sandra , Wang Yue , Groves Chris , Tedder Thomas , Coyle Anthony J , Herbst Ronald
Published: January 2023

Anti-CD20 mediated B cell depletion is an effective treatment for rheumatoid arthritis. An anti-CD19 mAb is under investigation as a new B cell depleting reagent. Studies have demonstrated that the interaction between the mAb Fc and Fc? receptors on effector cells is important for efficient target cell depletion. To enhance effector function, the anti-CD19 mAb was engineered with BioWa's Potelligent® technology to remove fucose from the Fc portion. The fucose-free mAb has increased affinity to human FCγRIIIa and mouse FCγRIV as well as improved cytotoxicity. The fucose-free anti-CD19 antibody (anti-CD19-aFuc) depleted >90% of murine B cells in human CD19 Transgenic mice. Further studies were carried out to investigate the mechanisms of action of anti-CD19-aFuc. B cell depletion was not affected in mice which were treated with to deplete NK cells or neutrophils. Similarly, depletion of B cells was not impaired in mice in which complement activation was blocked. In contrast, B cell depletion by anti-CD19-aFuc was significantly inhibited in mice in which macrophages were eliminated. Furthermore, anti-CD19-aFuc showed robust activity in an antibody dependent cellular phagocytosis assay with freshly isolate mouse macrophages as effector cells. These results suggest that anti-CD19-aFuc depletes B cells in mice through a macrophage dependent mechanism. This study is funded by MedImmune, LLC.
Lipid rafts represent a novel therapeutic target in SLE (50.25)
Authors: Deng Guo-Min , Liu Lena , Tsokos George C
Published: January 2023

Lipid rafts play a crucial role in T cell receptor (TCR) signaling. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by abnormal TCR signaling. We found that lipid rafts are aggregated on the surface membrane of T cells from lupus-prone MRL/lpr mice. High levels of aggregated lipid rafts could also be reproduced on T cells of normal C57BL/6 mice treated with anti-CD3 plus CD28 antibodies. The aggregated lipid rafts peaked at the age of 6-7 weeks prior to the appearance of disease pathology in MRL/lpr mice. Depletion of lipid rafts with methyl-B-cyclodextrin (MBCD), which is known to disrupt lipid rafts, or atorvastatin, which is known to inhibit cholesterol biosynthesis delayed disease progression, whereas acceleration of their formation with cholera toxin B, which is known to induce lipid raft clustering, promoted disease progression in MRL/lpr mice. The aggregated lipid rafts harbor diverse molecules including TCR signaling, inflammatory, costimulatory, adhesion and Toll-like receptor molecules. Recruited diverse molecules have costimulation to enhance TCR-mediated response and production of IFN-gamma through intact lipid rafts. Further, crosslinking of lipid rafts promoted IFN-gamma production in vitro, a cytokine known to contribute to the expression of SLE pathology in mice. We propose that lipid rafts represent a novel therapeutic target in SLE.
A novel inhibitor of Smad-dependent transcriptional activation suppresses tissue fibrosis in model mice of systemic sclerosis (50.31)
Authors: Hasegawa Minoru , Matsushita Yukiyo , Horikawa Mayuka , Higashi Kiyoshi , Tomigahara Yoshitaka , Kaneko Hideo , Takehara Kazuhiko , Sato Shinichi
Published: January 2023

Tissue fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc). TGF-β is well known to be the principal factor that leads to tissue fibrosis. We tested a novel small compound HSc025, which is an antagonist of TGF-β/Smads signaling, would prevent tissue fibrosis in vitro or in mouse models of SSc. Human dermal fibroblasts were exposed at various doses of HSc025 in the presence of TGF-β, and the expression levels of collagen or fibronectin were determined. Administration of HSc025 (15 mg/kg/day, p.o.), halofuginone (1μg/mouse/day, i.p.) or placebo to tight skin mouse or to bleomycin-induced pulmonary fibrosis mice was performed for 14 days. Pretreatment with HSc025 prevented Smad-dependent promoter activation in a dose-dependent manner, however, HSc025 had no effect on TGF-β-induced phosphorylation of Smad3. The inhibitory effects of HSc025 on TGF-β-induced collagen or fibronectin expression were also confirmed in vitro. The oral administration of HSc025 dramatically reduced hypodermal thickness in tight skin mice, and markedly decreased hydroxyproline contents and histological score in the lungs of bleomycine-treated mice.

These results demonstrate that HSc025 is a novel inhibitor against TGF-β/Smads signaling, resulting in the improvement of skin and pulmonary fibrosis. Therefore, orally available HSc025 might be useful for the therapy of SSc.
Immunomodulatory effects of piperine on dendritic cell function (50.36)
Authors: Rodgers Gemma , Doucette Carolyn D , Spurrell David R , Hoskin David W , West Kenneth A , Liwski Robert S
Published: January 2023

Piperine is a major alkaloid in black and long pepper and has been shown to have potent anti-inflammatory properties. In this study we investigated the immunomodulatory effects of piperine on dendritic cells (DC), key initiators and modulators of T cell mediated immune responses.

C57BL/6 bone marrow derived DC were matured in the presence of 100 µM of piperine or vehicle control. DC maturation marker and chemokine receptor expression was assessed by flow cytometry. T cell proliferation was measured by CFSE dilution. Cytokine secretion was assessed by cytometric bead array assay. In vitro DC migration was assessed using CCL21 as the chemotactic stimulus.

Piperine treated DC showed decreased expression of CD40, CD86, class II MHC and CCR7 and increased expression of CCR5, consistent with an immature phenotype. DC production of IL-6, TNF and MCP-1 was significantly reduced following piperine treatment. Treatment with piperine also reduced DC migration and DC induced T cell proliferation. In addition, naïve T cells activated with piperine-treated DC secreted lower levels IFN-γ, IL-4, IL-17, and IL-2.

This study demonstrates that piperine inhibits DC maturation, migration and T cell stimulatory capacity. Further investigation of piperine mediated DC immunomodulation could lead to the development of novel therapies for the treatment of autoimmune disorders or allograft rejection.

This research is supported by CIHR.
Long term elimination of epitope-specific diabetogenic CD8+ T cells using toxin coupled tetramers in NOD mice (50.37)
Authors: Young Ellen F. , Vincent Ben , Buntzman Adam , Tisch Roland , Frelinger Jeffrey A. , Hess Paul
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease in which CD8+ T cells with help from CD4+ T cells and B cells kill the insulin-producing islet cells in the pancreas. Although islet transplants have been successful in treating T1D, the transplants may not persist because of the ongoing autoimmunity. Patients can be treated with immunosuppressants, however these drugs have serious side effects. Selective elimination of diabetogenic T cells should specifically eliminate islet autoimmunity. We previously showed MHC class I tetramers coupled to the toxin saporin could selectively kill transgenic virus specific T cells. We have now used saporin coupled NRP-V7 tetramers to target diabetogenic T cells in vitro and in vivo in non-obese diabetes-prone (NOD) mice. NRP-V7 is a high affinity mimotope that is recognized by the 8.3 T cell clone, a known diabetogenic T cell. Treatment with saporin coupled NRP-V7 tetramers acutely depleted NRP-V7+ CD8+ T cells expressing Vβ 13-3 and elimination appeared to be long term since very few NRP-V7+ T cells were detectable in the islets of toxic-NRP-V7 tetramer treated NOD mice up to 45 weeks later. Treatment with toxic NRP-V7 tetramers resulted in a delay of T1D with a mean onset of 24 weeks compared to 16 weeks for treatment with PBS. Toxic tetramers could potentially be used to treat T1D as well as other T cell mediated autoimmune disease. Supported by JDRF 5-2006-13 and 1-2008-24.
Nociceptin/Orphanin FQ antagonists ameliorate inflammatory bowel disease (50.45)
Authors: Alt Carsten , Shew Ken , Fujita Nahoko , Tran Thanh-Thuy , Zukic Maja , Polgar Willma , Toll Lawrence , D'Andrea Annalisa
Published: January 2023

Human inflammatory bowel disease (IBD), primarily Crohn's disease and ulcerative colitis, affect 1-1.5 million people in the United States, with estimated annual medical costs of about $18,500 per patient. Although the etiology of IBD is unclear, the disease is commonly thought to be mediated by an uncontrolled autoimmune response against the bowel. No cure is available for IBD. Nociceptin/Orphanin FQ (N/OFQ) is a neuropeptide that has been hypothesized to be a stimulator of inflammation, and the N/OFQ receptor is expressed at high levels in the gut. We evaluated the therapeutic potential of N/OFQ antagonists in C57Bl/6 mice in an IBD model induced by treatment for 5 days with DSS. N/OFQ antagonists were administered prophylactically. Our results indicate that treatment with N/OFQ antagonists dramatically ameliorated the development of colitis as observed by changes in body weight, disease activity index, fecal consistency, bleeding, and cytokine levels in the colon. Furthermore, our data suggest that N/OFQ affects the recruitment of leukocytes to the inflamed bowel. Additional studies are in progress to elucidate this complex mechanism of action of N/OFQ and N/OFQ antagonists in the context of IBD. Our results indicate that treatment of the animals with N/OFQ antagonists significantly ameliorates the DSS-induced colitis, suggesting that inhibition of N/OFQ may be an effective therapy for IBD.
Modulation of migration of immune cells and molecules into the central nervous system by adenosine receptor signaling (50.5)
Authors: Bynoe Margaret S , Mills Jeffrey H , Thompson Linda F
Published: January 2023

We have shown that mice lacking CD73, an extracellular enzyme of the purine catabolic pathway that catalyzes the breakdown of adenosine monophosphate (AMP) to adenosine, were resistant to EAE, the animal model of the central nervous system (CNS) inflammatory disease multiple sclerosis (MS). Furthermore, blockade of adenosine receptor signaling with an antagonist specific for the A2a adenosine receptor, which is expressed in the CNS, inhibited lymphocyte migration into the CNS and protected wild type mice from EAE. We now show that blockade of adenosine receptors with broad spectrum or a specific A2a receptor antagonist prohibits the entry of immune cells into the CNS of (SJL/J x PL/J)F1 mice (that develop a relapsing-remitting form of EAE), inhibits the development of EAE, and halts EAE relapse. Importantly, we have strong data showing that adenosine receptor blockade can inhibit the entry of molecules such as FITC-Dextran and Evans blue dye across CNS barriers. These data provide convincing evidence that modulation of adenosine receptors can have a profound effect on CNS mediated diseases such as MS and drug delivery across CNS barriers. This work was supported by NIH grants AI 57854 and NS 063011 (to MSB) and AI 18220 (to LFT).
Generation of SIINFEKL from ovalbumin is only partially inhibited by proteasome inhibitors (78.10)
Authors: Garg Manish , Srivastava Pramod K.
Published: January 2023

We have treated PIK23 cells ( P815 cells genetically modified to express ovalbumin in an inducible manner) with irreversible proteasome inhibitors AdaAhx3L3VS and lactacystin and analyzed (a) the ability of inhibitors to block proteasome activity by in-gel peptidase assay, and (b) the generation of new cell surface Kb-SIINFEKL complexes as measured by B3Z activation. Our results show that complete inhibition of proteasome activity results in only a modest inhibition in generation of new Kb-SIINFEKL complexes. Only a combined inhibition of proteasomes and other cytosolic proteases leads to a complete inhibition of generation of such complexes. These results point to existence of redundant pathways for generation of SIINFEKL.
Carbohydrate antigen processing is self-regulated and promotes conventional antigen processing (78.20)
Authors: Cobb Brian A. , Lewis Colleen J.
Published: January 2023

Processing of T cell-dependent carbohydrate antigens in professional antigen presenting cells (APCs) is driven by the production of nitric oxide (NO) via TLR2 stimulation. Published reports have shown that preservation of carbohydrate fragments containing at least 4 repeating units are required for class II MHC presentation and T cell stimulation, yet each repeating unit is equally susceptible to oxidative cleavage. These observations suggest that a mechanism exists to prevent complete degradation of key carbohydrate epitopes during the oxidative burst. Our results have revealed that oxidative breakdown of carbohydrates following endocytosis generates an acidic environment in a proton pump-independent fashion. This acid environment is the direct result of proton release from the oxidation reaction and serves to inhibit further carbohydrate degradation via a pH-driven feedback loop while promoting the activation of proteases within the conventional processing pathway. These findings suggest a novel mechanism within APCs in which endocytosis and oxidation of carbohydrate-encapsulated microbes generates a low pH environment that negatively self-regulates carbohydrate breakdown while facilitating protein antigen processing.
Modulating MHC Class II Antigen Processing in Dendritic Cells Prevents Diabetes in NOD mice (78.25)
Authors: Denzin Lisa K. , Yi Woelsung , Wucherpfennig Kai W. , Sant'Angelo Derek B.
Published: January 2023

The presentation of disease relevant self-peptides is clearly essential for type 1 diabetes (T1D). General inhibition of antigen presentation, however, would severely compromise a patient's immune system. The over-expression or complete loss of the MHC-like HLA-DO (DO) protein, which binds to and modulates the peptide loading ability of HLA-DM (DM; H2-M in mice), has not been shown to cause any significant deviation in immune function. Variations in the presentation of both antigenic and self-peptides have, however, been documented. Therefore, we sought to subtly alter MHC class II (MHCII) Ag processing and presentation in non-obese diabetic (NOD) mice by over-expressing the DM-modulator, DO specifically in dendritic cells (DCs).

Our studies show, that NOD mice over-expressing DO (NOD.DO) in DCs never develop T1D. DO expression in DCs, prevented the infiltration of lymphocytes into pancreatic islets (insulitis), however, NOD and NOD.DO mice had similar levels of peri-islet lymphocytic infiltration. Remarkably, DO-over expression was required in only half of NOD DCs to mediate protection. NOD.DO mice were also partially protected from T1D after challenge with diabetogenic T cells from newly diabetic NOD mice. In stark contrast to T cells from NOD mice, NOD.DO T cells did not promote the rapid development of T1D when transferred into immunocompromised NOD.scid recipients supporting that NOD.DO T cells have not been primed, most likely due to limited antigen availability. Studies are underway to determine the mechanism by which DO over-expression in NOD DCs mediates protection from T1D.
Molecular modeling and simulation studies of CD1 binding proteins (78.3)
Authors: Faraldo-Gómez José D , Garzón Diana
Published: January 2023

The presentation of small protein fragments, both foreign and native, on the surface of specialized cells is one of the cornerstones of our immune system. In complex with MHC binding proteins, these protein antigens will be recognized by T-cell membrane receptors either as self or non-self, and an appropriate response will be initiated. In recent years it has become apparent that immune responses may also be induced by a diverse array of lipid antigens. The presentation of these antigens involves a class of binding proteins referred to as CD1, analogous to MHC proteins. The CD1 family includes 5 isoforms of distinct specificity, of which CD1a, CD1b and CD1d have been characterized structurally in complex with diverse lipid antigens. Here we report the results of extensive computer simulation studies of this family, from which we gain insights into the mechanism of lipid binding. We also present a molecular model of CD1c, and based on this, rationalize its specificity for branched mycobacterial lipids.
IVIg decrease antigen-dependent T cell activation by reducing the FcγR-dependent antigen presentation ability of APC (78.36)
Authors: Aubin Eric , Paquin Proulx Dominic , Lemieux Real , Bazin Renee
Published: January 2023

Intravenous immunoglobulins (IVIg) have been shown to have anti-inflammatory effects in a variety of autoimmune diseases, but their mechanisms of action remain unclear. In autoimmune diseases, activated self-reactive T cells invade specific tissues and activate local APC which in turn present autoantigens on their surface, leading to T cell restimulation in a positive feedback loop. Although it was shown that IVIg could reduce T cell activation and modify their cytokine secretion pattern, it is still not clear whether this effect occurs following a direct interaction of IVIg with T cells or through an interference of IVIg with the ability of APC to present autoantigens. To address this question, we used an in vitro antigen presentation system using ovalbumin as model antigen, to study the effects of IVIg on antigen-mediated T cell activation. The results obtained showed that IVIg inhibited T cell activation but that this effect was the indirect consequence of a reduction in the antigen presentation ability of APC. We further showed that this inhibitory effect was not due to modulation of expression of MHC II or CD80/86 costimulatory molecules on the surface of APC and was independent of inhibitory FcγRIIb. Finally, we showed that F(ab')2 fragments of IVIg had no effect on antigen-mediated T cell activation, suggesting that IVIg must interact with activating FcγRs to mediate their inhibitory effect.
Listeriolysin O is strongly immunogenic on multiple MHC backgrounds (78.42)
Authors: Carrero Javier A , Vivanco-Cid Hector , Unanue Emil R
Published: January 2023

Listeriolysin O (LLO) is a key virulence determinant of the intracellular microbial pathogen Listeria monocytogenes. Previous work has shown the ability of LLO to induce a wide range of biological effects, including cytokine secretion, MAP kinase signaling, cellular lysis and apoptosis. LLO is also the source of immunodominant antigenic determinants for both CD4 and CD8 T cell responses against L. monocytogenes on multiple MHC backgrounds. We have examined the presentation of LLO protein delivered as a soluble antigen and after bacterial infection. LLO is a powerful immunogen, eliciting T cell responses at femtomolar levels of presentation in vitro. In vivo immunization with LLO in incomplete Freund's adjuvant led to the induction of a robust CD4 and CD8 T cell response as detected by ELIspot. This effect can be shown with APC and T cells derived from C57/BL6 (H-2b), C.B-17/Balb/c (H-2d) and B6.g7/NOD (H-2g7) haplotypes. Interestingly, there is an immunogenicity cluster located in domains 2 and 3 of LLO which donates most of the MHC-II epitopes for presentation. We have engineered non-hemolytic mutants of LLO and can show that they are also presented with great efficiency to CD4 T cells. Addition of ectopic antigens derived from ovalbumin and hen-egg lysozyme to the amino-terminus of LLO allowed for robust presentation to CD4 T cells directed to these antigens. Our initial data suggests that LLOs ability to be a potent antigen is based on a very fast and efficient uptake and catabolism by APC. We detected maximal presentation to T cells within 30 minutes of giving LLO to APC. APC also internalized LLO and catabolized it completely to TCA precipitable peptides within 6 hours of treatment. We conclude that LLO is a good candidate for delivering antigenic peptides in vivo and in vitro.
Transforming Growth Factor β-Dependent Inhibition of CD1d1-Mediated Antigen Presentation (78.6)
Authors: Bailey Jennifer C. , Renukaradhya G. J. , Brutkiewicz Randy R.
Published: January 2023

CD1d is an antigen presenting molecule structurally similar to MHC class I and presents lipid antigens to natural killer T (NKT) cells. We have previously shown that the mitogen-activated protein kinases (MAPK), p38 and ERK1/2, reciprocally regulate CD1d-mediated antigen presentation--p38 activation leads to its inhibition, whereas ERK1/2 activation leads to its promotion. Transforming growth factor β (TGFβ) is a strong regulator of immune responses as well as cell proliferation, apoptosis and differentiation, and can induce the activation of p38. Our studies have shown that CD1d-mediated antigen presentation is inhibited by TGF β in murine LMTK fibroblasts expressing CD1d. Similar inhibition occurs after TGF β treatment of bone marrow-derived dendritic cells (BMDC). Our studies are aimed at dissecting the mechanisms by which TGF β influences CD1d-mediated antigen presentation. This work is supported by NIH grant PO1 AI056097 to RRB.
NKT cells are not required for development of allergic airway disease in allergen-immunized mice (79.1)
Authors: Perkins Charles , Orekov Tatyana , Yanase Noriko , Mattner Jochen , Finkelman Fred D.
Published: January 2023

The role of NKT cells in the pathogenesis of asthma and animal models of asthma is controversial. Although the initial mouse model study failed to identify an NKT cell requirement for induction of allergic airway inflammation or airway hyperresponsiveness (AHR), 2 subsequent mouse studies found both phenomena to be highly NKT cell-dependent. Some studies of human asthma patients also suggest strong involvement of NKT cells in airway inflammation, while others dispute this finding. To re-visit this issue at its origin, we evaluated the ability of ovalbumin (OVA) to induce allergic airway disease in BALB/c wild-type and NKT cell-deficient (CD1-deficient) mice, using a protocol in which mice were initially primed i.p. with OVA/alum, then inoculated intratracheally (i.t.) with OVA, as well as a protocol in which mice were inoculated i.t. with dust mite allergen without priming. Results from both studies demonstrated that wild-type and NKT cell-deficient mice develop similar airway eosinophilia, IL-4 and IL-13 responses, goblet cell hyperplasia and AHR (determined by both invasive and non-invasive methods). Flow cytometric analysis of lung and BAL lymphocytes from saline-, OVA-, and dust mite Ag-inoculated mice failed to demonstrate any specific staining for invariant NKT cells. We conclude that NKT cells are not required for allergen-immunized mice to develop full-blown allergic airway disease.
Expression of IL-19 in Airway Epithelial Cells During Asthmatic Response (79.11)
Authors: Hung Li-Yin , Nguyen Tiffany T , Wu Reen
Published: January 2023

IL-19 is a proinflammatory cytokine that belongs to the IL-10 cytokine family. In addition to its known connection with psoriasis, previous work in our lab, along with others, has also shown its association with asthma. Using cultured primary mouse airway cells, we demonstrated that Th2 cytokines such as IL-4 and IL-13 can induce the expression of IL-19 mRNA. Despite its ability to stimulate IL-19 production in human cells, IL-17A appeared not being involved in this process in the mouse model. With an OVA-induced mouse asthma model, we showed that IL-19 expression is increased in the asthmatic lung tissues at the early stage. The induction is specific to IL-19 rather than other IL-20 family members such as IL-20, IL-22, or IL-24. We further identified that airway epithelial cell, rather than the alveolar macrophage, is the source of IL-19 in the asthma model. These results demonstrate that IL-19 is produced by airway epithelial cell at the early stage of asthmatic response, and may further amplify the Th2 response through acting on T cells at the later stage of asthma.
Annexin-1 deficient mice exhibit enhanced antibody production in a mouse model of asthma (79.18)
Authors: Lim Lina HK , Biswas Subhra K , Mustafa Fatimah B , Kajiji Tasmeen S , Tan Yuwen , Ng Florence SP
Published: January 2023

Allergic asthma is a complex disorder characterized by airway inflammation and reversible airway obstruction and airway hyperresponsiveness. Glucocorticoids are currently the most effective anti-inflammatory therapy for asthma. Annexin-1 is an endogenous anti-inflammatory protein responsible for many anti-inflammatory effects of glucocorticoids. Here we show in a mouse model of asthma, that annexin-1 deficient mice do not exhibit enhanced inflammation, such as eosinophil infiltration, but exhibited higher antibody production. In particular, allergen-specific and total IgE levels, as well as allergen-specific IgG1, IgG2a and IgG2b serum levels were significantly higher in annexin-1 deficient mice. Our results in annexin-1 deficient mice demonstrate that annexin-1 may play a regulatory role in the intrinsic development of allergy.
Thymic Stromal Lymphopoietin (TSLP) Modulates the Balance between Th1 and Th2 Responses in the Skin (79.4)
Authors: Larson Ryan P , Ziegler Steven F
Published: January 2023

TSLP is an IL-7 related cytokine produced by epithelial cells in response to tissue damage, inflammation, or TLR ligation. Overexpression of TSLP in mouse skin or lung leads to the development of atopic dermatitis and asthma, respectively; however, these models fail to elucidate which cell types are acted on by TSLP. TSLP has also been hypothesized to inhibit aberrant Th1 responses by inhibiting the production of IL-12 family members by DCs. TSLP receptor deficiency significantly inhibits the development of Th2-type contact hypersensitivity (CHS) response induced by the irritant dibutyl phthalate in combination with the hapten fluorescein isothiocyanate (FITC). Specifically, FITC-sensitized TSLPR-/- mice do not experience swelling or eosinophilia at the site of challenge; nor do they experience increased serum IgE, all hallmarks of Th2-type CHS observed in WT mice. The reduced CHS response is due in part to the significant reduction in frequency and number of skin-derived antigen-bearing FITC+CD11c+ DC in skin-draining lymph nodes 24 hours post FITC sensitization of TSLPR-/- mice. Furthermore, skin-derived TSLPR-/- FITC+CD11c+ DCs are reduced in their ability to drive proliferation of naïve antigen specific CD4+ T cells compared to WT counterparts. In addition, we have found that there is no requirement for CD4+ T cells to respond to TSLP for the development of FITC-induced CHS. Interestingly, TSLPR-/- mice experience an enhanced Th1-type CHS response to the Th1-inducing contact sensitizing agent 2,4-dinitrofluorobenzene, compared to WT. These results support a role for TSLP acting on DC in vivo resulting in the development of Th2 allergic response and potentially playing a regulatory role by inhibiting Th1 CHS responses.
Coordinate posttranscriptional regulation of asthma genes by the RNA binding protein HuR (79.6)
Authors: Gubin Matthew M , Hollingsworth John D , Calaluce R , Magee Joseph D , Schwartz David A , Atasoy Ulus
Published: January 2023

Traditional microarray analysis has uncovered a variety of genes involved in diseases such as asthma. However, it may potentially miss important genes due to the discordance between steady state mRNA levels and protein products. Posttranscriptional events conferred by RNA binding proteins (RBPs) contribute to this phenomenon. We have developed novel techniques known as RIP-on-Chip (microchip) which allow for the immunoprecipitation and identification of in vivo mRNA targets bound to RBPs. We study the RBP HuR, which regulates many early response genes including cytokines and chemokines. Using RIP-on-Chip, HuR has been demonstrated to associate and regulate many asthma relevant genes including recently identified genes IL-4 and IL-13.

We have extended this assay to solid organ tissues. Employing the standard ovalbumin model of allergic airway inflammation, coupled to genome-wide arrays (47,000 genes) we have identified in vivo HuR targets in lungs of naïve, alum only or ova challenged mice. Remarkably, there are only 12 HuR targets (2 fold or greater) up regulated in ova and alum immunized over alum alone mice. Some are known HuR target genes such as TLR4 and IL-13; others are potentially novel asthma genes. Furthermore, we have created a transgenic mouse which over expresses HuR in CD4+ T cells. In the allergic airway inflammation model these mice have an increase in lung lymphocyte infiltration when compared to wild-type littermates, further implicating coordinate posttranscriptional gene regulation and HuR in allergic airway inflammation.
Selective stimulation of IL-4 receptor on smooth muscle induces airway hyperresponsiveness in mice (79.9)
Authors: Finkelman Fred D. , Perkins Charles , Smulian George , Gildea Lucy , Orekov Tatyana , Potter Crystal , Brombacher Frank , Wills-Karp Marsha
Published: January 2023

Hyperresponsiveness to cholinergic stimulation of increased airway resistance is a defining characteristic of asthma. Both IL-4 and IL-13 activate Stat6 via IL-4Rα to induce airway hyperresponsiveness (AHR) in mouse models of asthma and increase mouse and human smooth muscle contractility in vitro. However, the relatively small size of mouse vs. human airways, differences in airway smooth muscle cell distribution between mouse and human, and observations that selective IL-13 stimulation of Stat6 in airway epithelium is sufficient to induce AHR in mice have raised questions about the importance of direct IL-4/IL-13 effects on smooth muscle in murine models of allergic airway disease and whether these models are appropriate for studying AHR in human asthma. To determine whether direct effects of IL-4/IL-13 on smooth muscle contribute to murine allergic airway disease, we produced mice that express IL-4Rα only on smooth muscle and evaluated the effects of intratracheal IL-4, IL-13 and allergen on their airways. We demonstrate that IL-4 and IL-13 induce AHR in these mice, without stimulating goblet cell hyperplasia or airway eosinophilia. Similar results were observed following airway inoculation of these mice with a potent allergen. Thus, allergen-stimulated IL-4/IL-13 can induce AHR in mice through direct effects on airway smooth muscle.
Aspergillus-induced eosinophil recruitment to murine lung is dependent on cell wall polysaccharides (80.9)
Authors: Van Dyken Steven , Reese Tiffany A. , Huang Xiaozhu , Corry David B. , Locksley Richard M.
Published: January 2023

Development of allergic inflammation in response to fungal organisms involves innate immunity, but initial molecular recognition events remain unclear. We sought to identify elements of Aspergillus essential to initiating allergic inflammation using IL-4-GFP reporter (4get) mice. Intranasal administration of an Aspergillus preparation resulted in robust recruitment of innate immune cells to the lungs of 4get mice, independent of protease activity, implicating cell wall components. The major cell wall polysaccharides of Aspergillus include chitin, along with linear and branched β-glucan polymers, both of which can initiate innate immune responses in mammals. Enzymatic degradation of either chitin or β-glucans led to significant reductions in the magnitude of eosinophil recruitment to the lungs of 4get mice after intranasal Aspergillus administration, an effect that was enhanced additively by degradation of both polysaccharides. The specific contribution of chitin was examined using 4get-SPAM mice, which express elevated levels of acidic mammalian chitinase in the lungs and thus possess an enhanced ability to degrade chitin in vivo. After Aspergillus challenge, lung eosinophils numbers in 4get-SPAM mice were significantly diminished compared to 4get-WT littermates, in both acute and extended dosing regimens, while eosinophils remained at WT levels when challenged similarly with ovalbumin. These data reveal a previously unidentified link between specific polysaccharide recognition and eosinophil recruitment during Aspergillus-induced allergic lung inflammation.
Ad5 delivery of IL-15 enhances porcine natural killer cell activity (81.11)
Authors: Patch Jared R. , Toka Felix N. , Zarlenga Dante , Golde William T.
Published: January 2023

Foot-and-mouth disease virus (FMDV) is a highly infectious picornavirus that causes disease in cloven-hoofed animals, and remains an international economic threat to the livestock industry. Although a protective killed-virus vaccine exists, its utility is limited by the window of susceptibility present between vaccination and the induction of adaptive immunity. Natural killer (NK) cells play an important role in innate immunity by eliminating virus-infected cells. We have previously shown that interleukin-15 (IL-15) plays a critical role in the development, maintenance, and activation of NK cells in swine, and represents a potential method for improving porcine NK cell killing activity. We have constructed a replication-defective human Ad5 vector expressing the gene for porcine IL-15 (Ad5-pIL-15). Culture supernatants of cells infected with Ad5-pIL-15 were capable of stimulating porcine NK cells in vitro as indicated by elevated target cell killing. This suggests that Ad5-pIL-15 has the potential to stimulate porcine NK cell killing activity in vivo. Further experiments will focus on Ad5-pIL-15 and whether it can inhibit FMDV pathogenesis in pigs.
Onset of Antibody Repertoire Diversification in the Rabbit Appendix (81.17)
Authors: Lanning Dennis K. , Knight Katherine L.
Published: January 2023

Unlike mice and humans, rabbits generate their primary antibody repertoire by somatically diversifying V(D)J genes in gut-associated lymphoid tissues (GALT). Rabbit V(D)J genes are diversified by somatic hyper-point mutation and somatic gene conversion, both of which require activation-induced cytidine deaminase (AID). To determine when repertoire diversification begins, we examined AID expression in neonatal rabbit appendix by in situ hybridization. We first detected AID mRNA expression at one week of age, in B cells in the basolateral region of appendix follicles. We asked whether AID+ B cells were undergoing V(D)J gene diversification by using laser capture microdissection (LCM) to isolate AID+ and AID- B cells from 1-week-old appendix follicles. We RT-PCR-amplified and cloned VDJ genes from the two B cell populations, and searched for evidence of somatic diversification by comparing their nucleotide sequences to those of the appropriate germline genes. We found somatic gene conversion events in the VDJ genes from AID+ B cells, but not in those from AID- B cells. These results demonstrate that diversification of the rabbit primary antibody repertoire begins around 1 week of age, in B cells residing at the basolateral region of appendix follicles. This work was supported by NIH Grant 1 RO1 AI49458-01A1.