bluuebunny/crossref_metadata_2025 huggingface dataset:
NNovel phosphoantigen for human Vγ2Vδ2 T cells produced by Staphylococcus aureus and other Gram-positive cocci using the mevalonate isoprenoid pathway (55.21)
Authors: Morita Craig , Workalemahu Grefachew
Published: January 2023

Vγ2Vδ2 T cells are innate lymphocytes that use their TCRs to recognize nonpeptide antigens thereby bridging innate and adaptive immunity and expand to large numbers during a variety of infections. Vγ2Vδ2 T cell responses to Apicomplexan protozoa and many Eubacteria are due to TCR recognition of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), a metabolite unique to the 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway. However, Staphylococcus aureus, Streptocococcus spp., and most other Gram-positive cocci (GPC) using the mevalonate isoprenoid pathway also stimulate Vγ2Vδ2 T cells. We now report that this stimulation is due to the production of a novel phosphoantigen distinct from HMBPP, isopentenyl pyrophosphate, and other mevalonate and MEP pathway intermediates when analyzed by ion-pairing reverse phase chromatography. Moreover, production of this phosphoantigen does not require mevalonate pathway enzymes. Although S. aureus and other GPC do not secrete the phosphoantigen, exposure to live S. aureus, Streptococcus pyogenes, and Enterococcus faecalis bacteria expands Vγ2Vδ2 T cells in vitro. In contrast, non-pathogenic Staphylococcus carnosus lack detectable bioactivity and minimally expand Vγ2Vδ2 T cells. Thus, rather than responding only to MEP pathogens, Vγ2Vδ2 T cells also respond to GPC using the mevalonate pathway and likely play roles in human immunity to this important class of pathogens.
Differential c-type lectin receptor expression correlates with the magnitude of pathology in murine schistosomiasis (55.4)
Authors: Ponichtera Holly , Shainheit Mara , Larkin Bridget , Stadecker Miguel
Published: January 2023

In murine schistosomiasis, immunopathology and cytokine production in response to schistosome eggs is uneven and strain dependent. Infected CBA mice develop severe hepatic egg-induced granulomatous inflammation associated with prominent Th17 and Th1 cytokine responses, whereas in BL/6 mice milder lesions develop in a Th2-dominant cytokine environment. The pathogenic Th17 response in CBA mice is largely dependent on IL-1β and IL-23 produced by schistosome egg-stimulated dendritic cells (DC); by comparison, this pro-inflammatory cytokine pathway fails to materialize in low-pathology BL/6 mice. Whereas requirements for Th17 cell differentiation elicited by CBA DC are apparent, the reason for such a strain difference in APC reactivity to live eggs is not known. Initial gene profiling disclosed a significant difference in C-type lectin receptor (CLR) expression between CBA and BL/6 bone marrow derived DC. CLR are pattern recognition receptors capable of binding carbohydrates, including those secreted by schistosome eggs. A significant increase in CLR expression, particularly murine homologues of human DC-specific ICAM-3-grabbing non-integrin (DC-SIGN), was documented by real-time PCR and flow cytometry on tissues from infected CBA mice, including liver, spleen and granuloma cells. Current work is aimed at elucidating the functional implications of differential CLR expression on T cell subset differentiation, activation and pathogenicity during the schistosome infection.
Mouse macrophage galactosa-type lectin (mMGL) is critical for the host resistance against Trypanosoma cruzi infection (55.7)
Authors: Rodriguez-Sosa Miriam , Vazquez Alicia , Juarez Imelda , Gomez-Garcia Lorena , Calleja Elsa , Camacho Griselda , Chavez Ana , Terrazas Luis
Published: January 2023

The C-type lectin receptor mMGL is expressed exclusively by myeloid APC such dendritic cells and macrophages (MΦ), mediates binding to glycoproteins carrying terminal N-acetylgalactosamine (GalNAc) residues. Parasite glycosylated molecules are recognized by mMGL. T. cruzi parasite expresses large amounts of mucins (TcMUC)-like glycoproteins, here we show by Jacalin lectin-blot that GalNAc moieties are also expressed on their surface. Then, our goal was to study the impact of mMGL in T. cruzi infection. Male mMGL knockout (KO) and wild-type (WT) C57BL/6 mice were infected via intraperitoneal (i.p.) with 10-5 trypomastigotes of T. cruzi (Queretaro strain). Following i.p. T. cruzi infection, mMGL-KO mice developed higher parasitemia load and higher mortality associated with poor production of Nitric Oxide (NO), IL-12 and TNF-α by MΦ in response to total soluble T. cruzi antigens compared to WT. Interestingly, whereas heart from WT mice infected with T. cruzi did not present amastigotes nest, mMGL-KO mice did. We conclude that mMGL have an important role during the invasion of T. cruzi to the host cell, also induce inflammatory cytokines and NO production by macrophages.
Lack of IL-21R on donor cells attenuates chronic GVHD but promotes humoral autoimmunity in acute GVHD (56.11)
Authors: Rus Violeta , Nguyen Vinh , Tegla Cosmin , Luzina Irina , Chen Qing , Rus Horea
Published: January 2023

IL-21 enhances CD8 maturation into cytotoxic T lymphocytes (CTL), promotes the differentiation of T follicular helper cells (TFH) cells and Th17 cells, downregulates Tregulatory (Treg) cells and exerts stimulatory activities on B cells, suggesting a complex role for IL-21 in promoting autoimmunity. To assess the role of IL-21/IL-21R interaction on CD4 and CD8 T cells in promoting lupus-like disease we used the parent-into-F1 model of acute and chronic graft versus host disease (GVHD) using IL-21R KO and wild type B6 mice as donors. cGVHD induced with CD8 depleted splenocytes from IL-21R KO mice exhibited an attenuated disease phenotype characterized by significant decrease in the number of donor (d)CD4 and dTFH cells, impaired help for B cells as determined by anti-ssDNA antibody levels and less severe lupus-like glomerulonephritis. However the balance between IL-17 producing dTh17 cells and FoxP3 dTreg cells was not altered. Acute GVHD induced with IL-21R KO splenocytes resulted in an attenuated disease phenotype as indicated by a significant decrease in dCD4 and CD8 engraftment, decreased host B cell elimination (90%±0.3) compared to wt injected mice (57%±6; p=0.03), decreased anti-host CTL activity and increased anti-ssDNA Ab levels. These results suggest that IL-21/IL-21R interaction on T cells promotes lupus-like cGVHD by sustaining TFH cell number yet it downregulates the humoral component in aGVHD by enhancing host B cells elimination by CTL.
Inhibition of Calcium/calmodulin-dependent protein kinase IV suppresses the autoimmunity in lupus-prone mice. (56.4)
Authors: Ichinose Kunihiro , Kawakami Atsushi , Tsokos George
Published: January 2023

OBJECTIVE: Systemic lupus erythematosus (SLE) is a chronic inflammatory disease associated with abnormal immune cell function.SLE T cells express high levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV). We have shown previously that pharmacologic (Arthritis Rheum. 2011) or genetic silencing of Camkiv (J Immunol. 2011) suppresses lupus nephritis in lupus-prone mice. The purpose of this study was to determine whether pharmacologic inhibition of CaMKIV would improve immune function abnormalities. METHODS: We treated MRL/lpr mice with KN-93, a CaMKIV inhibitor, starting at week 8 of age through week 16 and evaluated the presence of CD4+, CD8+, CD3+CD4-CD8- (double negative, DN) and CD62Llow T cells and proinflammatory cytokine production. We also determined the effect of inhibition of CaMKIV on proinflammatory cytokine production by human T cells and macrophages. RESULTS: CaMKIV inhibition in MRL/lpr mice resulted in significant suppression of DN and CD62Llow of T cells population and IFN-γ production by T cells. In human activated T cells and macrophages, pharmacologic inhibition of CaMKIV resulted in suppression of IFN-γ production and CD69 expression by T cells and IL-1β, IL-6 and TNF-α by macrophages. CONCLUSION: We conclude that pharmacologic inhibition of CaMKIV suppresses cell activation and cytokine production in lupus-prone mice. Our data justify the development of small-molecule CaMKIV inhibitors for the treatment of patients with SLE.
Combined Treatment with Neutralizing Antibodies to IL-1α / β and TNFα / PGE2 in a Late Therapeutic Mode Ameliorates Inflammation and Inflammatory Bone Loss in a mouse Collagen Induced Arthritis (mCIA) model of Rheumatoid Arthritis (56.6)
Authors: Syed Farhan , Bryant Shaughn , Mario Regina , Mathieu Suzanne , O'Connor Elizabeth , Erickson Jamie , Waegell Wendy , Murtaza Anwar , Wang Li Chun , Kamath Rajesh , Gu Jijie , Olson Lisa , Annette Schwartz
Published: January 2023

Rheumatoid Arthritis is characterized by chronic inflammation and synovial hyperplasia which leads to the destruction of cartilage and bone. We have shown earlier (Wu et al, 2007) that blocking IL1α/β provides better efficacy than monotherapy in a CIA model when treatment begins at first clinical sign of inflammation but not necessarily bone loss (therapeutic mode). These studies describe a late therapeutic mCIA treatment mode designed to evaluate the efficacy of anti-inflammatory drugs on inflammation and bone in animals with established bone lesions. To define the kinetics of bone loss, male DBA1/J mice with established arthritis were sacrificed at days 5, 7, 9 and 11 after onset of first clinical signs of disease. Day 7 was selected as the optimal point to begin treatment based on 28% decrease in tarsal bone volume and 27% increase in erosion by microCT and correlating bone histomorphometry endpoints. The efficacy of two pairs of anti-inflammatory mAbs was evaluated: 1) Anti-TNF alone and in combination with anti-PGE2 and 2) anti-IL1β alone and in combination with anti-IL1α. Treatment was initiated on day 7 and was continued for 2 weeks. For both antibody sets, the paired combination significantly reduced inflammation (by 40%) and maintained bone volume to a greater extent than anti-TNF or anti-IL1β dosed alone (37% and 22% increase). Our data demonstrate that a late therapeutic mCIA mode can be used to evaluate effects of anti-inflammatory drugs on bone loss.
Gingiva-derived mesenchymal stem cells-mediated therapeutic intervention for experimental arthritis through Foxp3+ Tregs (56.7)
Authors: Zheng Song Guo , Chen Maogen , Su Wenru , Wang Julie , Brand David , Ryffel Bernhard , He Xiaoshun , Le Anh
Published: January 2023

Current approaches are unable to cure rheumatoid arthritis. Accumulated evidence has revealed that manipulation of bone-marrow mesenchymal stem cells (BMSCs) has a potential promise to treat these diseases. Nonetheless, isolation and expansion of BMSCs for therapeutic usage is less feasible in the clinic. Here, we demonstrate that use of gengival-derived mesenchymal stem cells (GMSCs), mostly sharing similar phenotypic and functional characteristics with BMSCs, resulted in significantly therapeutic effects on the established collagen induced arthritis (CIA). Infusion of GMSCs to DBA/1J mice at day 0, 14 or day 28 after CII/CFA immunization significantly decreased the severity of arthritis and pathology scores, down-regulated inflammatory cytokine (IFN-γ, IL-17A). The role of GMSCs in controlling the diseases may partially depend upon the induction of CD4+Foxp3+ Tregs, and CD39/CD73 signals. Strikingly, GMSCs increase CD4+Foxp3+ cells in naïve and CIA inflammatory mice. GMSCs significantly inhibit humanized xeno-GVHD model, suggesting that GMSCs have clinical relevance. GMSCs can function as an immunomodulatory and anti-inflammatory component of the immune system in vivo and use of GMSC may provide a promising approach for the treatment of rheumatoid arthritis and other autoimmune diseases.
Pin1 attenuates Fas-mediated apoptosis through binding to FADD in activated eosinophils. (57.1)
Authors: Oh Jiyoung , Malter James
Published: January 2023

Pin1 is widely expressed peptidyl-prolyl isomerase (PPIase) that plays a key role in a variety of events including cell cycle progression, response to viruses, expression of cytokines and cell death. Eosinophils (Eos) rely on Pin1 to mediate prosurvival signaling induced by IL-5 or GM-CSF by binding to and inhibiting pro-apoptotic Bax. Here we show Pin1 also participates in Fas-mediated apoptosis of Eos. Fas activation suppressed Pin1 activity despite co-treatment with IL-5. This was accompanied by phosphorylation of Pin1 at Ser16 and increased nuclear, rather than plasma membrane localization. Anti-Fas alone or in combination with IL-5 rapidly triggered FADD phosphorylation at Ser194. Interestingly, only the combination of agonists induced a stable interaction between P-FADD and Pin1. These results suggested Ser194-Pro195 was the Pin1 binding site in FADD. Consistent with this, phospho-mimic Ser194Glu FADD mutants preferentially interacted with Pin1 in vitro compared to WT or Ser194Ala mutants. Introduction of FADD Ser194Glu mutants significantly accelerated Eos apoptosis compared to WT or S194A. Therefore, our data suggest that Pin1 participates in extrinsic apoptosis via an interaction with FADD at P-Ser194-Pro195.
A critical role for the kainate receptor in both IgE production and IgE mediated allergic lung inflammation (57.8)
Authors: Sturgill Jamie , Brooks Keith , Conrad Daniel
Published: January 2023

According to the American Lung Association, approximately 34.1 million Americans have been diagnosed with asthma. Despite the extremely high prevalence of this disease, there is still no preventative measure, only treatments to alleviate the symptoms. Thus, novel treatments for asthmatic disease are certainly warranted. Recently, our laboratory has discovered that glutamate signaling through the kainic acid receptor (KAR) leads to enhanced ADAM10 activity and as a result leads to increased production of soluble CD23 and IgE production in the human in vitro culture system. This finding is critical because it is one of the first to highlight the importance of a neurotransmitter receptor on B cells and that signaling by this receptor may play a role in IgE mediated disease. We have extended these studies to demonstrate that in the murine in vitro system, the KAR is also critical for the in vitro production of IgE and that signaling through this receptor also leads to increased ADAM10 activity, production of soluble CD23 release, and subsequent IgE production. Furthermore, we determined in vivo relevance for this receptor. Mice deficient in the KAR post antigen challenge produce significantly less IgE and display significantly reduced airway hyperreactivity in a murine model of allergic airway disease. Thus, KARs may serve as an innovative pharmacological target for the treatment of asthma and other IgE mediated disease.
High Throughput Sequencing of the Human Antibody Repertoire in Response to Influenza Vaccination (58.14)
Authors: Jiang Ning , He Jiankui , Weinstein Joshua , Penland Lolita , Saaki Sanae , He Xiaosong , Dekker Cornelia , Wilson Patrick , Greenberg Harry , Davis Mark , Fisher Daniel , Quake Stephen
Published: January 2023

Despite the many advances in personal genome analysis, the immunoglobulin repertoire of the genome, while central to human health, is in practice extraordinarily challenging to measure and analyze. There are several reasons for this, including the facts that each B cell contains a distinct antibody sequence encoded in its genome, that the antibody repertoire is not constant but changes over time, and the high similarity between antibody sequences. We have addressed this challenge by using high-throughput long read sequencing to perform immunogenetic characterization of expressed human antibody repertoires. Informatic analysis of large numbers of antibody heavy chain sequences from individual subjects allowed us to perform global characterizations of isotype distributions, clonal lineage structure of the repertoire and age-related mutational activity. With influenza vaccination as a specific stimulus, we used lineage analysis to measure the clonal structure and mutational distribution of individuals’ repertoires; analysis of this data showed that elderly subjects have a decreased number of lineages but an increased pre-vaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. These analyses may ultimately be useful as metrics to measure vaccine response and to further understand impaired immune function in aging.
What is (in)significant? The Pathway Semantics Algorithm reveals complex immune response changes over time. (58.18)
Authors: Csencsits-Smith Keri , McGuire Mary , Nguyen Trinh , Pascual David
Published: January 2023

Results obtained from hypothesis-driven research may not represent the complex interactions between cells and cytokines required for the development of an acquired immune response. We applied a novel algorithm to measure change in immune response over time in a mouse model of anti-Factor VIII (FVIII) antibody formation in hemophilia A. Splenocytes isolated from FVIII injected mice treated with PBS or an experimental tolerogen (CFA/1) were cultured for 72 hours and cytokine concentrations determined via multiplex analysis at days 7 and 14. The concentrations of 22 individual cytokines were compared between the control and CFA/1-treated groups, and 3 cytokines statistically differentiated the two groups at day 7 and 5 cytokines at day 14. Median cytokine concentrations/group of each significance set were converted to pmol/L and Ingenuity Pathway Analysis was used to generate likely biological network pathways. Network graphs were converted to matrices and linear algebra was used to compare treatment groups. Sixteen different molecules were predicted to occur in PBS vs. CFA/1 groups at day 7. We validated the presence of two predicted outcomes - expression of TLR 4 and secretion of IL-25. At day 14, the predicted outcomes were similar between the treatment groups, despite being evoked from a larger significance set, suggesting that statistical differences in cytokine secretion do not consistently represent biologically relevant changes in immune response over time.
Development of IL-17A promoter-based reporter cell line and activation of the cell line by exogenous expression of RORγ and RORγt. (59.12)
Authors: Singh Sujay , Lee Hyun-Ku , Singh Gita , Luo Yi Feng , Chitta Sriram , Rosenberg Jonathan
Published: January 2023

As one of the six known IL-17 family members, IL-17A is a proinflammatory cytokine secreted by innate and adaptive immune cells. Especially IL-17A has been characterized as a key cytokine produced in a new CD4+ T helper subset, Th17 cells, which play an important role in host defense against specific pathogens. Effect of IL-17A is also implicated with pathogenic role of Th17 cells in autoimmunity and the chronic activation of IL-17-producing T helper cells can lead to autoimmune diseases. Indeed, increased levels of IL-17 have been observed in patients with autoimmune diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and psoriasis as shown by researchers. Hence development of a targeted therapeutic means or molecule that can effectively control IL-17 induction is crucial to prevent the initiation of IL-17-producing cell-mediated autoimmune diseases. In response to this, we have developed the IL-17A Prom/LUCPorter™ HEK 293 cell line, in which Renilla luciferase reporter gene is under control of the inducible IL-17A promoter. This cell line has been validated by exogenous expression of RORγ or RORγt construct, which can induce the IL-17A promoter activity leading to expression of luciferase reporter protein. This cell line is suitable for screening of antagonists for IL-17A induction which can be utilized as the therapeutic target molecules to control IL-17-producing cells.
Identification of a novel secreted molecule expressed by Th17 cells: potential association with inflammatory diseases. (59.3)
Authors: Valle-Rios Ricardo , Burkhardt Amanda , Hevezi Peter , Zlotnik Albert
Published: January 2023

CD4 T cells (Th cells) play an important role in adaptive immune responses. These processes are controlled in part by a pattern of soluble molecules (cytokines) secreted by different types of Th cells. Here we describe a novel secreted molecule which we have called barrier tissue and activated T cell-associated molecule (BATAM). Using a comprehensive database of human gene expression that comprises more than 115 tissues or organs of the body, we identified a secreted protein that is produced by activated CD4 T cells as well as skin and various mucosal tissues. BATAM encodes a ~60 KD protein that contains a thrombospondin type 1 repeat (TSR) and the adhesion associated domain AMOP, which are known to mediate cell adhesion and are also present in various proteins involved in inflammation. We confirmed the BATAM expression pattern by qPCR in mouse tissues. The production of BATAM by activated spleen CD4 T cells is significant but not strong. We therefore hypothesized that BATAM may be preferentially produced by a polarized subset of Th cells. To investigate this possibility, we polarized CD4 T cells into Th1, Th2, Treg and Th17 in vitro. The results indicate that BATAM expression is highly associated with Th17 cells. We have also confirmed that it is a secreted protein. Using recombinant BATAM, we are currently probing its biological activity. We conclude that BATAM is a novel pro-inflammatory molecule that may be associated with diseases mediated by Th17 cells.
T helper cytokines expression in leprosy forms and reactional states: serum and in situ analysis (59.9)
Authors: Trombone Ana Paula , Belone Andrea , Guidella Cassio , Fachin Luciana , Ramuno Natalia , Soriani Michelle , Ura Somei , Soares Cleverson , Garlet Gustavo , Rosa Patricia
Published: January 2023

Leprosy is a chronic infectious disease of the skin and peripheral nerves characterized by a broad spectrum of clinical forms, namely tuberculoid (TT) and lepromatous (LL), the borderline forms (BB, BT and BL) and the reverse reaction (RR) and erythema nodosum leprosum (ENL) reactional states. Since the immunological basis of leprosy is still unclear, we analyzed the profile of T helper (Th1, Th2, Th17, Treg) cytokines, in serum (ELISA) and in situ (qPCR), of 72 leprosy patients and 10 healthy controls. ELISA data shows that the frequency of IL-10, IL-6 and IL-22 detection was significantly higher in patients than in controls, while TGF-b was detected in all the samples from both groups. IFN-g, IL-4 and IL-17 levels were not detectable in the serum of both controls and patients. Comparing the leprosy subgroups, IL-6 was higher in ENL patients when compared to paucibacillary (BT/TT) and multibacillary (BB/BL/LL); while IL-10 was higher in multi versus paucibacillary forms. qPCR analysis demonstrated that IL-17, IFN-g, IL-6 and TGF-b prevail in diseased sites versus controls, but no significant differences were found between the clinical forms. Our results suggest that a broader analysis of cytokine profile can contribute to a better characterization of leprosy forms than the traditional Th1/Th2 archetype, and a larger sample must be evaluated in order to confirm these findings.
Complexed BCR-TLR9 stimulation induces an activation associated cell death in B cells which is rescued by BLyS (60.12)
Authors: Oropallo Michael , Green Nathaniel , Kiefer Kerstin , Moody Krishna , Marshak-Rothstein Ann , Cancro Michael
Published: January 2023

Combined B cell receptor (BCR) and Toll-like receptor 9 (TLR9) signaling has been associated with the T cell Independent (TI) activation of autoreactive B cells in the presence of self-DNA. However, TLR9 knockout models of humoral autoimmunity have increased disease severity, suggesting the receptor has a more complex role. We have recently found that while both WT and AM14 RF producing B cells stimulated with complexed BCR-TLR9 agonists indeed proliferate, they die rapidly within 48-72hrs of initial stimulus, implying that combined BCR:TLR9 signaling has a role in limiting the T cell independent activation of B cells. Importantly, this activation associated cell death is unique to complexed BCR-TLR stimulation, as BCR agonist (anti-IgM) alone, TLR9 agonist (CpG ODN) alone, or both BCR + TLR9 agonists uncomplexed induces cells to continue to survive and proliferate robustly within this time frame. Further, this apoptosis involves the intrinsic mitochondrial cell death pathway, as it involves cleavage of caspase 9, occurs independently of caspase 8, and is rescued by the presence of a BCLxL transgene. Finally, we have found that addition of the survival cytokine BLyS rescues B cells proliferating in response to complexed BCR-TLR9 from cell death through BLyS binding to BR3, providing insight into how increased BLyS titers contribute to the onset of autoreactive B cell activation.
High salt induces pathogenic Th17 cells and exacerbates autoimmune diseases (60.13)
Authors: Kleinewietfeld Markus , Manzel Arndt , Wu Chuan , Titze Jens , Kuchroo Vijay , Linker Ralf , Muller Dominik , Hafler David
Published: January 2023

Interleukin (IL)-17 producing CD4+ helper T cells (Th17 cells) play a pivotal role in autoimmune diseases. Apart from their importance for the control of certain pathogens, IL-23 dependent Th17 cells have been shown to be critical for the development of experimental autoimmune encephalomyelitis (EAE). Moreover, genetic risk factors associated with multiple sclerosis (MS) are linked to the IL23/Th17 pathway. Besides genetic factors, the increased incidence of autoimmune diseases like MS in developed countries is also believed to be related to environmental risk factors. However, how environmental risk factors such as changes in diet, sun exposure or medications could directly influence the development of pathogenic Th17 cells is not well established. Here we show that increased salt (sodium chloride; NaCl) concentrations dramatically boost the induction of murine and human Th17 cells in vitro. Moreover, mice fed with a high salt diet develop a very severe form of EAE, dependent on the induction of highly pathogenic Th17 cells. The generation of Th17 cells under high salt conditions is well regulated on the molecular level and seems to be dependent on specific pathways. Thus, increased salt intake, which may represents one of the biggest contributors to changes in diet during the last half century and accounts for cardiovascular disease, might also represent an environmental risk factor for autoimmune diseases through the exacerbated induction of pathogenic Th17 cells.
Autoreactive CD4+ Th1 cells activate monocytes to support Th17 responses in inflammatory arthritis (60.8)
Authors: Simons Donald , Oh Soyoung , Garcia Victoria , Kropf Elizabeth , Caton Andrew
Published: January 2023

Th17 cells have been implicated in a number of autoimmune and inflammatory disorders, yet signals that can lead to a Th17 response in vivo are poorly understood. We have analyzed this question using a transgenic mouse model in which autoreactive CD4+ T cells recognizing a ubiquitously expressed surrogate self-antigen (influenza HA) induce inflammatory arthritis by an IL-17-dependent mechanism. Arthritis development is accompanied by an accumulation in the joint draining lymph nodes of inflammatory monocytes (iMOs) that have an enhanced capacity to support Th17 responses in vitro. The accumulation of iMOs in the LNs is preceded by an autoreactive CD4+ Th1 cell response, and by the upregulation of MHCII on iMOs in the spleens of pre-arthritic mice. Adoptive transfer of HA-specific CD4+ T cells into mice that expressed HA as a self-antigen similarly led to the formation of Th1 cells and of MHCII+ iMO that could support Th17 cell formation in vitro, and significantly, the accumulation of these iMOs in the LNs was blocked by IFNγ neutralization. Collectively, these studies provide a basis by which autoreactive CD4+ Th1 cells directed to a systemically distributed self-antigen can promote a regional inflammatory disease (arthritis) by promoting the formation of Th17-trophic iMO and driving their recruitment into the LNs.
Annexin A1 mediates the anti-inflammatory effects during the granulocytic differentiation process in all-trans retinoic acid-treated acute promyelocytic leukemic cells (63.2)
Authors: Hsu Hui-Chi , Tsai Wen-Hui , Chien Hong-Yu , Shih Chung-Hung , Lai Shu-Lien , Li I-Ting , Hsu Shau-Chieh , Kou Yu Ru
Published: January 2023

Annexin A1 (AnxA1) originating from neutrophils and their microparticles (MPs) plays an important anti-inflammatory role. The role of AnxA1 during the process of granulocytic differentiation is still unknown. All-trans retinoic acid (ATRA) can induce differentiation of acute promyelocytic leukemic(APL) cells into mature granulocytes. We investigated whether or not AnxA1 contributed to the anti-inflammatory properties of ATRA-treated APL (NB4; ATRA-NB) cells. We found that ATRA was able to enhance the surface expression of AnxA1 and its receptor (FPR2/ALX) and the release of AnxA1-containing MPs from ATRA-NB4 cells. Further studies demonstrated that exogenous AnxA1 could inhibit ATRA-NB4 cells in their transmigratory and adhesive activity. In addition, the transmigratory activity of ATRA-NB4 cells can be significantly enhanced by pretreatment with a FPR2/ALX neutralizing antibody, suggesting that endogenous AnxA1 may contribute to the anti-migratory effects. Finally, ATRA-NB4-derived MPs could also inhibit recipient cells in their transmigratory and adhesive activities and these anti-inflammatory effects could be inhibited by pretreatment of MPs with an anti-AnxA1 antibody. Further studies demonstrated that FITC-labeled AnxA1 could be transport from MPs to the membrane of recipient ATRA-NB4 cells. We conclude that biologically active AnxA1 may play a role in the anti-inflammatory properties of ATRA-APL cells during the process of granulocytic differentiation.
Double knockout of Id2 & Id3 in developing T cells promotes the development of CD4-CD8- αβ T cell (63.3)
Authors: Li Jia , Zhuang Yuan
Published: January 2023

αβ T cell development is regulated by sequential rearrangement and expression of TCRβ and TCRα genes at the DN (CD4-CD8-) and DP (CD4+CD8+) stage, respectively. Expression of TCRβ paired with preTα at the DN stage leads to αβ lineage commitment and differentiation into the DP stage. DP cells undergo TCRα gene rearrangement, resulting in expression and the selection of functional αβTCR. Id3 has been shown to be an important sensor downstream of both preTCR and TCR signals. However, Id3 knockout only leads to a partial block in αβ lineage development, suggesting functional compensation from other Id proteins. To further investigate functions of Id proteins at the preTCR and TCR checkpoints, we simultaneously deleted Id3 and the structurally and functionally related Id2 gene at the early DN stage of T cell development. We find that removal of Id2 and Id3 failed to block T cell development at the preTCR checkpoint although imposed a strong block at the TCR checkpoint. Furthermore, deletion of both Id2 and Id3 promoted development of an aberrant population of αβ T cells that bear DN (CD4-CD8-) phenotype. This genetic system revealed a novel function of Id proteins in regulation of alternative αβ lineages during T cell development. We will present the latest information on characterization of this CD4-CD8- αβ T cell lineage associated with Id2 and Id3 disruption in developing T cells.
The role of Dectin-1 in fungal asthma (64.5)
Authors: Lilly Lauren , Metz Allison , Dunaway Chad , Schwiebert Lisa , Steele Chad
Published: January 2023

Sensitization to fungi, such as the mold Aspergillus fumigatus, is increasingly becoming linked with asthma severity. Although long considered a Th2 dominated disease, emerging data indicates that IL-17A and IL-22 may also contribute to asthma pathogenesis. Our laboratory has identified protective roles for Dectin-1 mediated beta-glucan recognition and Dectin-1 dependent IL-17A and IL-22 production during invasive A. fumigatus infection. Unexpectedly, in an allergic model of repeated lung exposure to live A. fumigatus conidia, we show that the absence of Dectin-1 resulted in markedly improved respiratory mechanics in the presence of reductions in multiple allergy-associated and inflammatory mediators. Intriguingly, attenuated lung IL-17A and IL-22 production in Dectin-1 deficient mice, but not Th2 cytokines, correlated with a better outcome. IL-22 deficiency reproduced the phenotype observed in Dectin-1 deficient mice after repetitive A. fumigatus exposure. We further identified IL-33 as a Dectin-1 and IL-22 dependent pro-allergic mediator. Collectively, our data suggests that during repetitive fungal exposure, beta-glucan recognition via Dectin-1 results in the generation of IL-17A and IL-22 responses as well as to the induction of IL-33 leading to the initiation of the allergic cascade. These results provide a novel link between infectious agents (fungi), new players (IL-22) and immunopathogenesis (IL-33) in severe asthma.
Identification of a novel motif in MARCH1 that affects the conformation and the E3 ubiquitin ligase activity (65.10)
Authors: Bourgeois-Daigneault Marie-Claude , Thibodeau Jacques
Published: January 2023

Some members of the MARCH family of RING-type E3 ubiquitin ligases target important players of immune responses. Of these, MARCH1 prevents the cell surface accumulation of MHC class II molecules and regulates antigen presentation in response to IL-10 in human primary monocytes (Thibodeau et. al., Eur JI 2008). Structure-function analysis of mouse MARCH1 showed the presence of two functional tyrosine-based endosomal sorting motifs in the C-terminal cytoplasmic part of the protein. We investigated the importance of the corresponding motifs in human MARCH1. Site-directed mutagenesis of these tyrosines did not reduce the activity of MARCH1 in transfected cells. However, a reporter molecule fused to the C-terminal cytoplasmic domain of MARCH1 was expressed less efficiently at the plasma membrane. A series of deletions were made in the C-terminal part of MARCH1 and a new functional motif was discovered. BRET experiments revealed that this motif is important for the spatial organisation of the two cytoplasmic domains of MARCH1. Interestingly, this motif is also present in others RING-type E3 ubiquitin ligases such as Parkin and its alteration is linked to the development of the disease in certain patients.
Histamine 2 receptor mediates IL-4-dependent class switching to IgE in B cells (65.5)
Authors: Swartzendruber Julie , Bryce Paul
Published: January 2023

Histamine is a critical mediator of allergic disease and signals through four receptors. We recently showed that histamine 2 receptor (H2R) is essential for IL-4 mediated eosinophil recruitment and that H2R deficient mice are protected from allergic airway inflammation. Additionally, we observed an inability to generate IgE in these mice. This was not due to a sensitization defect, since the mice are capable of generating antigen specific IgG1. However, the functions of H2R on B cells are unclear. Here we show that H2R is not required for B cell development and proliferation and that H2R deficient mice are capable of generating antibodies to the T independent antigens NP FICOLL and NP LPS. However, H2R deficient B cells were unable to class-switch to IgE in vitro. This was further characterized by an absence of epsilon specific post-switch transcripts in H2R deficient B cells. The upregulation of CD23 in response to IL-4 was also impaired in H2R deficient B cells. STAT6 phosphorylation in IL-4 treated B cells from H2R deficient mice was also reduced. These findings suggest that histamine is vital in permitting IL-4 signaling to mediate STAT6 phosphorylation and for the switch to IgE that occurs in B cells.
CD200R1 regulates synovial inflammation through neutrophil LTB4 (66.3)
Authors: Vaine Christine , Primo Valeria , Nikolic Boris , Calderone Nicholas , Nigrovic Peter , Soberman Roy
Published: January 2023

Rheumatoid arthritis (RA) is a debilitating disease afflicting over 2 million Americans. The K/BxN model of synovial inflammation shares several key aspects of pathology in common with human RA, and is an accepted model of synovial inflammation. The 5-lipoxygenase (LO) pathway product leukotriene (LT)B4, generated by polymorphonuclear leukocytes (PMNs) and macrophages, plays a requisite role in this model by initiating and amplifying the recruitment and activation of PMN in joints. Mechanisms that down-regulate LTB4 synthesis have the potential to play a major role in inhibiting synovial inflammation and provide novel therapeutic targets. To explore the role of the inhibitory CD200:CD200R1 axis in this process, we developed CD200R1-/- mice and examined them in the K/BxN arthritis model. We observed that CD200R1-/- mice show increased severity as compared to CD200R1+/+ mice. Synovial fibroblasts were found to express CD200 and are in close proximity to CD200R1-expressing macrophages in inflamed ankle sections. Finally, CD200R1-/- PMN generated approximately 50% more LTB4 in response to C5a or to a combination of GM-CSF and C5a. Together, these observations indicate that CD200R1 plays a key role in regulating LTB4 synthesis and synovial inflammation in the K/BxN model of RA.
Reconciling heterogeneous differentiation of CD4+ T cells by systems-level modeling (66.4)
Authors: Hong Tian , Xing Jianhua , Li Liwu , Tyson John
Published: January 2023

CD4+ T cells have several subsets of functional phenotypes, which play critical yet diverse roles in the immune system. Pathogen-driven differentiation of these subsets of cells is often heterogeneous in terms of the induced phenotypic diversity. In vitro recapitulation of heterogeneous differentiation under homogeneous experimental conditions indicates some highly regulated mechanisms by which multiple phenotypes of CD4+ T cells can be generated from a single population of naïve CD4+ T cells. Here, we propose a systems frame work to demonstrate how heterogeneous differentiation can be initiated by a dynamic signaling motif composed of competing master regulators common to all subsets of CD4+ T cells. With this motif, a population of naïve CD4+ T cells can integrate the signals from their environment to generate a functionally diverse population with robust commitment of individual cells. Notably, two positive feedback loops in this network motif govern three bistable switches, which in turn, give rise to three types of heterogeneous differentiated states, depending upon particular combinations of input signals. This framework provides a mathematical basis for understanding the decision-making mechanisms of CD4+ T cell, and it can be helpful for interpreting experimental results. This mathematical model makes specific testable predictions that may that may advance our understanding of the complex molecular network regulating T helper cell differentiation.
Dectin-1 pathway activation is important to Cryptococcus neoformans killing (67.10)
Authors: Bocca Anamelia , Coelho Carolina , Casadevall Arturo
Published: January 2023

Cryptococcus neoformans (Cn) is an encapsulated yeast that causes life-threatening meningoencephalitis in immunocompromised patients. The infectious propagules are inhaled into the host’s lug and interact with alveolar macrophages. An adequate pattern of macrophage activation is crucial to the outcome of the infection. However, it has been very difficult to demonstrate macrophage fungicidal activity in vitro. The main goal of this work was to determine if differential activation of macrophages by the dectin-1 pathway could increase Cn killing and improve host survival. Peritoneal macrophages were stimulated with zymozan and co-cultured ex vivo with opsonized Cn (H99) to allow phagocytosis. Our results showed that after zymozan stimulation of macrophages, fungal killing was increased. Stimulation with zymosan was more effective in fungal killing than stimulation with depleted-zymosan. Blockade of dectin-1 receptor reverted fungal killing to control levels only in macrophages stimulated with depleted zymozan. The ROS production was lower than in control groups. There was also a higher acidification of endosomes in zymozan-stimulated groups. Taken together, the data suggests that dectin-1 pathway activation by zymozan contributes to Cn killing. However, for the interaction between encapsulated C. neoformans cells and macrophages dectin-1 stimulation may be inadequate because the polysaccharide capsule blocks access to cell wall structures that trigger the dectin-1 receptor.
Peptidoglycan recognition protein 3 (Pglyrp3) is associated with intestinal mucosal homeostasis and protection against colitis (67.12)
Authors: Jing Xuefang , Dziarksi Roman , Gupta Dipika
Published: January 2023

Peptidoglycan recognition protein 3 (Pglyrp3) is a bactericidal protein expressed in the epithelial cells of tongue, esophagus, stomach and intestine. Pglyrp3-deficient mice compared to wild type (WT) mice have increased sensitivity to dextran sodium sulfate (DSS)-induced colitis. The increased colitis is characterized by loss of epithelial cells, severe hyperplasia of the lamina propria and ulcers. The aim of this study was to determine whether Pglyrp3 plays a role in maintaining intestinal epithelial integrity by regulating proliferation and apoptosis of epithelial cells. We report that Pglyrp3-deficient mice treated with DSS have significantly higher TUNEL positive cells, increased Annexin V staining and higher levels of cleaved caspase 3 in the epithelial cells of the colon compared to WT mice. The number of bromodeoxyuridine (BrdU) positive cells, which indicates proliferating cells, is significantly lower in Pglyrp3-deficient mice compared to WT mice. Serum levels of FITC-dextran are significantly higher in DSS-treated Pglyrp3-deficient compared to WT mice, which indicates an increased loss of epithelial lining and loss of barrier function in Pglyrp3-deficient mice. Our results indicate that Pglyrp3 is important in maintaining the epithelial lining of the colon and loss of this protein results in increased apoptosis, decreased proliferation and increased loss of barrier function, which are important contributing factors to increased colitis in Pglyrp3-deficient mice.
Inflammatory macrophages in dengue virus infection pyroptosis, infectivity, and enhancement of endothelial permeability (67.5)
Authors: Hsieh Shie-Liang , Wu Ming-Fang , Chen Szu-Ting
Published: January 2023

Macrophages (Mϕ) are the major source of inflammatory cytokines during dengue virus (DV) infection It has been shown that macrophage differentiation under macrophage-colony stimulating factor (M-CSF) (denoted as M-Macrophage) is regarded as M2-like Mϕ, while GM-CSF-derived macrophage (denoted as GM-Mϕ) is regarded as M1-like Mϕ, or inflammatory macrophages. Compared to M-Mϕ, we found that GM-Mϕ is more susceptible to DV infection, and release higher amounts of proinflammatory cytokines and infectious viral particles when incubated with DV alone or DV-Ab immune complex. In addition, GM-Mϕ releases higher amounts of IL-1and IL-18 and is susceptible to DV-induced pyropsis. Moreover, culture supernatant from DV-infected GM-Mϕ causes more severe endothelial cell permeability change than that of M-Mϕ. It is interesting to note the differential expression of innate immunity receptors, including C-type lectin CLEC5A, were found between GM-Mϕ and M-Mϕ, and anti-CLEC5A mAb is able to inhibit the release of IL-1 and IL-18, capase I activation, and pyropsis in DV-infected GM-Mϕ and M-Mϕ.
Cyclophilin A restricts rotavirus infection by enhancing type 1 interferon response in infected epithelial cells (68.1)
Authors: Li Jintao , He Haiyang , Wu Yuzhang
Published: January 2023

Rotavirus (RV) infection is the main cause of acute dehydrating diarrhea in infants and young children below 5 years old worldwide. RV infection causes a global shutoff of host protein synthesis, however, previous studies have revealed that RV infection can also lead to the upregulation of host proteins, which could play crucial roles in RV infection. Using proteomic methods, we found that Cyclophilin A (CYPA), a peptidyl-prolyl cis-trans isomerase (PPIase), was upregulated in MA104 cells infected by human rotavirus (HRV). Following infection, CYPA was recruited to the viroplasm where it interacted with HRV structural protein VP2, to inhibit viral protein expressions, thus producing a repressive effect on HRV replication, which was dependent on its PPIase activity. We further show that CYPA was required for host type-1 interferon (IFN-I) response to HRV infection, and this effect was independent on its PPIase activity but dependent on JNK signaling pathway. Furthermore, we found that the expression of CYPA in enterocytes correlates to the period when BALB/c mice become resistannt to RV infection-induced diarrhea. Together, our data identify CYPA as a new host restrictive factor that confers protection against rotavirus infection.
A heterozygous S32I mutation in IκBα that causes EDID results in defective development of lymphoid organs and impaired B cell function (68.13)
Authors: Mooster Jana , Le Bras Severine , Manis John , Geha Raif
Published: January 2023

Autosomal ectodermal dysplasia with immunodeficiency (EDID) is caused by mutations in the inhibitor of NFκB α (IκBα), which is phosphorylated and degraded in response to several immune signaling pathways. We generated a mouse model of EDID by replacing one IκBα allele with a non-phosphorylatable IκBα, which resulted in decreased NFκB signaling. These mice have dysmorphic hair and teeth, as well as decreased serum immunoglobulins, and a severe decrease in their specific antibody response to T-dependent and T-independent antigens. The mice lack lymph nodes (LNs) and Peyers patches (PPs), and have a disrupted splenic architecture, with no marginal zone and fail to develop germinal centers (GCs). T cell function is intact but B cell function is deficient in vitro. Rag2 bone marrow chimeras formed proper lymphoid organs and had normal cutaneous hapten sensitivity but did not produce specific antibodies. This mouse model shows that autosomal EDID results in failure to develop LNs and PPs, disorganized splenic architecture, failure to develop GGs and an intrinsic B cell defect.
Repression of the H3K4 demethylase Jarid1b leads to activation of RSV-infected dendritic cells (68.18)
Authors: Ptaschinski Catherine , Mukherjee Sumanta , Petersen Bryan , Moore Martin , Kunkel Steven , Lukacs Nicholas
Published: January 2023

Respiratory syncytial virus (RSV) is a common pathogen in children, with nearly all children being infected before the age of 2. Severe infection requiring hospitalization has been linked to the development of asthma later in life due to an improper Th2 skewing of the immune response. Dendritic cells (DCs) are key cells involved in presenting antigen to T cells and polarizing the T cell response. It has been reported that RSV interferes with the initiation of the adaptive immune response by inhibiting the production of type I interferons (IFN), including IFNβ, thus leading to a Th2 environment in the lungs. The mechanism by which this occurs is not fully understood. Epigenetic modulation of gene transcription has been shown to be important in regulating inflammatory pathways. We found that bone marrow derived DCs (BMDCs) infected with the RSV clinical isolate A2001/2-20 upregulated expression of Jarid1b, an H3K4 demethylase. Jarid1b has been described as a transcriptional repressor in cancer cells. We therefore hypothesized that Jarid1b plays a role in suppressing inflammatory genes in RSV infected DCs. We used siRNA to downregulate Jarid1b in BMDCs. Loss of Jarid1b led to a 10-fold increase in IFNβ compared to cells transfected with non-specific siRNA, as well as an increase in IL-6 and TNF-α. DC phenotype was unaffected as determined by flow cytometry. Investigations into the role that Jarid1b-induced DC regulation has in directing the T cell response are ongoing.