bluuebunny/crossref_metadata_2025 huggingface dataset:
Obesity increases the production of pro-inflammatory mediators from adipose tissue T cells and compromises TCR repertoire diversity: Implications for systemic inflammation and insulin-resistance (87.32)
Authors: Youm Yun-Hee , Yang Hyunwon , Badanmagsar Bolormaa , Gimble Jeffrey , Greenway Frank , Mynatt Randall
Published: January 2023

Yun-Hee Youm, Hyunwon Yang, Bolormaa Vandanmagsar, Jeffrey M. Gimble, Frank Greenway, Randall Mynatt and Vishwa Deep Dixit Emerging evidence suggests that increase in activated T cell populations in adipose tissue may contribute towards obesity-associated inflammation and insulin-resistance. Here we asked whether, 1)the adipose-resident T cells(ARTs) produce cytokines in response to T cell receptor(TCR) ligation. 2)If the extralymphoid ARTs possess unique TCR repertoire compared to lymphoid-resident T cells and if obesity alters the TCR diversity in specific adipose depots. 3)Can specific elimination of T cells regulate inflammation and insulin-action in obesity? We found that obesity reduced the frequency of naïve ART cells in subcutaneous fat and increased the effector-memory cells in visceral fat. The purified ARTs from obese mice had higher frequency of IFNγ+, Granzyme B+ cells and upon TCR ligation, produced pro-inflammatory cytokines and chemokines. Importantly extralymphoid ARTs exhibited markedly restricted TCR diversity and obesity further compromised the TCR repertoire in adipose depots. Collectively, these data establish that ARTs have restricted TCR-Vβ repertoire and T lymphocytes contribute towards the complex proinflammatory microenvironment of adipose tissue in obesity. However,specific depletion of extralymphoid T cells in visceral fat alone is insufficient to reverse obesity associated feed-forward cascade of inflammation and insulin-resistance.
Exosomes mediate pregnancy-associated suppression of central nervous system inflammation in a murine model of multiple sclerosis (87.7)
Authors: Williams Jessica , Smith Kristen , Cox Gina , Shawler Todd , Whitacre Caroline
Published: January 2023

Multiple sclerosis (MS) is thought to be a cell-mediated autoimmune disease that ultimately leads to demyelination and severing of nerve axons within the CNS. It is known that pregnancy suppresses MS relapses, particularly during the third trimester. We have recapitulated this observation in murine experimental autoimmune encephalomyelitis (EAE). A serum factor is implicated as responsible for EAE suppression during pregnancy. We observed that serum exosomes, nano-sized facilitators of intercellular communication, are significantly more concentrated during late pregnancy in mice. To determine which proteins are expressed in pregnancy versus control-derived exosomes, we performed differential gel electrophoresis followed by mass spectrometry. Several identified proteins have important functions in both the immune and CNS compartments. Proteins enriched in pregnancy exosomes facilitate the local action of corticosterone, scavenge oxygen-derived free radicals, inhibit matrix metalloproteases, and provide survival signals to oligodendrocytes, the myelinating cell of the CNS. Thus, we explored the effects of exosomes on EAE and on immune and CNS cells, including analyzing inflammatory cytokine profiles of myelin-specific T cells and the proliferation and maturation of oligodendrocyte precursor cells. Determining the mechanism by which pregnancy exosomes suppress inflammation and examining their neuroprotective properties will aid in the development of novel MS therapies.
Non-Coding RNA regulation of TNFα (88.10)
Authors: Sullivan Kathleen , Song Li , Bagashev Asen , Fitzgerald Michael
Published: January 2023

Non-coding RNAs have been implicated in the regulation of expression of numerous genes. In many cases, the regulation occurs by miRNAs. Less well understood is the mechanism of transcriptional regulation by long non-coding RNAs. We observed bi-directional long non-coding RNAs upstream of the TNFα gene in a region where a transcriptional repressor, GCF2, is known to bind. We examined the role of these long non-coding RNAs by knocking down each strand. Knockdown of the antisense species led to de-repression of TNFα expression in a cell line not normally competent for expression. Knockdown of the antisense species displaced GCF2 from the promoter and using an RNA immunoprecipitation, we showed that GCF2 bound less of the antisense transcript after knockdown. Conversely, knockdown of the sense transcript led to increased binding of GCF2 at the promoter. To understand how these transcripts might be regulating TNFα expression, we examined the binding characteristics of GCF2 using a biosensor. GCF2 bound more avidly to dsRNA and DNA:RNA hybrids than to dsDNA. To define the target chromatin characteristics, we used ChIP analyses. Histone marks of repression as well as EZH2, Suz12 and argonaute bound to the upstream region of the TNFα gene in repressed cells. These data demonstrate that TNFα is regulated at the level of non-coding RNAs in the upstream promoter region and implicate GCF2 in the non-coding RNA-mediated repression of TNFα.
Th17-specific demethylation and methylation-sensitive STAT3 binding at a DNA element in the IL-17 promoter (88.13)
Authors: Thomas Rajan , Wells Andrew
Published: January 2023

CD4+ helper T cells can be instructed by non-antigen-specific signals to differentiate into functionally distinct subsets with exclusive patterns of cytokine production. Activation in the presence of TGFbeta and IL-6 results in the development of Th17 cells that produce IL-17, an inflammatory cytokine that is not expressed by Th1 or Th2 lineage cells. The promoter region of the IL-17A gene contains 12 CpG dinucleotides, leading us to test whether the IL-17 gene is regulated epigenetically by DNA methylation. In Th1 and Th2 cells, all CpG dinucleotides were heavily methylated (80-100%), with the exception of the -33 dinucleotide adjacent to the TATA element, which was 70% methylated in all cell lineages. The Th17 differentiation program was associated with a selective, 4-fold increase in demethylation (from 90% to 60%) at CpG dinucleotides -144 and +13 bp from the transcriptional start site. Interestingly, the -144 CpG is located within a consensus element (CCpGTCA) for STAT3, a transcription factor that is required for IL-17 gene induction and Th17 differentiation. We find that STAT3 from Th17 nuclear extracts binds with high affinity to this CCpGTCA element in vitro, but shows low affinity binding to the methylated CmeCpGTCA element. These results suggest that signals from the TGFbeta and/or IL-6 receptors lead to demethylation of a STAT3 element in the IL17A promoter, and epigenetically program the IL-17 locus for transactivation by STAT3 in the Th17 lineage.
Ikaros silences the ifnγ gene during T cell anergy and T helper polarization (88.14)
Authors: Thomas Rajan , Chunder Neelanjana , Chen Chunxia , Taylor Justin , Pearce Edward , Wells Andrew
Published: January 2023

Ikaros is a zinc finger transcription factor required for lymphocyte development, but recent studies indicate that this factor also regulates peripheral T cell function. Our previous studies showed that Ikaros is a major repressor of il2 gene expression during the induction of T cell anergy, and in the present study, we have examined the role of Ikaros in ifnγ gene expression in CD4+ T cells. We find that Ikaros binds to the endogenous ifnγ and tbx21 loci, an event associated with increased DNA methylation at the ifnγ gene. Loss of Ikaros function in vitro resulted in increased IFNγ production by anergic T cells and Th2 cells, and was reinforced in Th2 cells by dysregulated expression of Tbet. Loss of Ikaros function in vivo led to an inappropriate Th1 response to the Th2 parasite S. mansoni, and also led to increased ifnγ gene expression and fatal intestinal immunopathology in a DSS model of colitis. Our results show that Ikaros is an important transcriptional repressor of Tbet and IFNγ expression, and further establish a role for Ikaros in T cell tolerance and T helper polarization.
Transgenic mice with inducible Kallikrein expression as a tool to study immune-mediated and spontaneous lupus nephritis (88.24)
Authors: Shao Xinli , Yang Ru , Yan Mei , Zhou Jinchun , Ramin Indu , Wakeland Ward , Mohan Chandra , Igarashi Peter , Li Quan-Zhen
Published: January 2023

Kallikreins (klk) play important roles in anti-GBM autoantibody-induced glomerular nephritis (AGN) and potentially lupus nephritis (LN). In this study, we have established a transgenic mice strain with inducible klk expression specifically in kidney tubular cells. The double transgenic mice carry two sets of transgenes, the mklk1 gene located down-stream of a CAG promoter-driven LacZflox-/- and the CreERT2 which is driven by kidney specific promoter (KSP). We have detected the LacZ expression in the kidneys and other tissues of the transgenic mice without induction. After induction by Tamoxifen, the mice expressed high level of klk1 in the kidneys but not in other tissues. The kidney specific expression of transgenic klk1 was confirmed by Q-PCR, Western blotting and immunohistochemistry. The expression of klk1 was co-localized in kidney tubular cells with Cre. Several inflammation cytokines, including IL-12, IP10 and MIP-1, were identified to be increased in the kidney of transgenic mice along with the elevated expression of klk1. The anti-oxidant molecules, Oxr1 and GSTM2, were also increased. In order to evaluate the protective effect of klk against the spontaneous LN, the CreERT2/mKlk1 transgenes were breed onto B6.Sle1.Sle3 lupus mice. We have confirmed the inducible expression of klk1 in the kidneys of the CreERT2/mKlk1/Sle1.Sle3 mice. These mice will be aged to 6-8 months and the effects of klk1 on the development of lupus nephritis will be evaluated.
The chromatin remodeling landscape of T helper cell differentiation (88.6)
Authors: Wurster Andrea , De Supriyo , Precht Patricia , Wood William , Becker Kevin , Pazin Michael
Published: January 2023

T helper cell activation and differentiation occur in part through changes in gene expression. Dramatic changes in chromatin structure occur and are thought to play a role in regulation. Little is known about the remodeling enzymes responsible for these changes. Here, we asked how binding of the SWI/SNF remodeling subunit BRG1 changed genome-wide during T helper cell differentiation, using ChIP-seq. Globally, BRG1 binding was positively correlated with gene activity. We found BRG1 binding to promoter proximal regions but also found many distal BRG1 binding regions, including some known distal regulatory elements, suggesting a general role for BRG1 in long-distance gene regulation. BRG1 binding changed dramatically in response to both differentiation and activation. BRG1 binding was associated with DNase I Hypersensitivity and enhancer activity at newly identified regions. Moreover, many of these BRG1 binding regions are lineage or activation specific, a large number of them are greater than 10kb away from the nearest gene, and do not appear to be marked by a common histone code. Together, these findings suggest BRG1 integrates differentiation and activation signals to change chromatin structure. We speculate these events play a causal role in programming gene expression and cell fate. We also suggest that BRG1 binding may be a useful marker for identifying distal regulatory regions.
Heterogeneity of responses of cord blood mononuclear cells to lipopolysaccharide induced epigenetic modifications of the Foxp3 gene. (88.7)
Authors: Goldberg Michael , Elizur Arnon , Nadiv Orna , Kovalchuk Natalya , Katz Yitzhak
Published: January 2023

We previously demonstrated that a high proliferative response to lipopolysaccharide (LPS) in cord blood mononuclear cells (CBMC) correlates with a change from a Th2- to Th1-induced cytokine expression profile. Evidence from mouse models indicates that lipopolysaccharide (LPS) can induce and activate T regulatory (Tregs) cells. The expression of Tregs cells is at least partially controlled by epigenetic modifications in the promoter region of their transcription factor Foxp3. Our goal was to examine whether LPS can lead to the generation of Tregs in human CBMC by inducing epigenetic modifications in the Foxp3 gene promoter. CBMCs were treated with LPS (1ng/ml) or PHA (5 µg/ml). After 72 hours, genomic DNA was isolated, bisulfite treated and the 5’ promoter Foxp3 CpG island (-15 to -138) analyzed by direct sequencing of cloned PCR product and by real-time PCR utilizing methylation-sensitive primers. Treatment of CBMC with LPS increased unmethylated foxp3 promoter DNA in 4/12 patients. In contrast, PHA increased unmethylated promoter CpG islands in 9/12 patients. An increase of Foxp3+ Tregs were seen by FACS in response to PHA, but not to LPS. In conclusion, LPS can induce epigenetic modifications of the foxp3 promoter in a subset of infants, but additional signals are likely required for its induction of Treg cells. Future studies will evaluate whether these modifications alter the risk of developing atopy in susceptible infants.
Molecular basis of regulatory effect of medicinal plant components on the innate immune response through TLR4 signaling (89.1)
Authors: Honda Hiroe , Nagai Yoshinori , Miyake Kensuke , Takatsu Kiyoshi
Published: January 2023

The mammalian immune system consists of two branches: innate and acquired immunity. The innate immune system recognizes microorganisms via germline-encoded pathogen-associated pattern recognition receptors (PARRs) and plays a role in the first line of host defense against pathogens. Toll-like receptors (TLRs) belong to PARRs play indispensable roles for both innate and acquired immunity. One-third of the current top 20 drugs on the market are plant derived. Interestingly, anticancer drug Paclitaxel from Pacific yew tree interacts with human MD-2 and activates dendritic cells through TLR4. We examined effects of components from Glycyrrhizae Radix on the TLR-mediated innate immune response and NF-κB activation. Results: We found that Glycyrrhizic acid (GL, saponin) and Isoliquiritigenin (IQG, chalcone) from Glycyrrhizae Radix suppressed TLR4-mediated TNF-α and IL-6 production by lipid A-stimulated macrophage cells line. GL and IQG also inhibited NF-κB activation induced by lipid A. Western blot analyses revealed that both GL and IQG suppress lipid A-induced IκB-α degradation and ERK1/2 phosphorylation. Intriguingly, GL, but not IQG could inhibit the LPS binding to TLR4-MD2 detected by using anti-TLR4-MD2 mAb. Conclusion: Both GL (saponin) and IQG (chalcone) suppress TLR4-mediated inflammatory cytokine production and NF-κB activation. GL, but not IQG can inhibit TLR4 ligand binding to TLR4-MD-2, indicating that GL and IQG differentially regulate TLR-4-mediated signaling pathway.
Innate immune suppression by gas6 expressed by γδ T cells (89.12)
Authors: Hedges Jodi , Radke Jay , Kimmel Emily , Zickovich Julie , Jutila Mark
Published: January 2023

Gas6, a Vitamin K-dependent protein, is an agonist for the newly described suppressors of inflammation, the TAM receptor family. Transcripts encoding gas6 are unusually highly expressed in purified neonatal bovine γδ T cells, compared to αβ T cells and B cells. Vitamin K-dependent proteins are known for roles in blood coagulation and bone growth, as such there are drugs that affect Vitamin K pathways, the most common of which is Warfarin (Coumadin). We hypothesized that functional gas6 expressed by γδ T cells would be immunosuppressive and addition of Warfarin would be pro-inflammatory. Observations to date are consistent with these assumptions; addition of Vitamin K to bovine PBMC dampens the innate response of monocytes, and this effect is dependent on γδ T cells. Addition of recombinant human gas6 has a similar effect. Conversely, the removal of γδ T cells or the addition of Warfarin to longer term cultures enhances (monocyte-dependent) CD8+ T cell responses in an in vitro model of Mycobacterium avium ssp. avium infection. Also, consistent with expectations, preliminary data in mice suggest that very low dose oral Coumadin increased severity of chemically induced colitis, but decreased colony counts in Salmonella-induced enterocolitis. The expression of Vitamin K-dependent gas6 by γδ T cells that localize to mucosal surfaces may be significant considering the common usage of oral Coumadin and its known side effects.
Both VAMP4 and VAMP7 are indispensable for NK cell cytotoxicity: the requirement for two R-SNARE proteins in granule exocytosis. (89.24)
Authors: Krzewski Konrad , Gil-Krzewska Aleksandra , Watts James , Coligan John , Strominger Jack
Published: January 2023

Natural killer (NK) cells kill cancer and virus-infected cells through their cytolytic activity. The last step in NK cell cytotoxicity, fusion of lytic granules with the plasma membrane resulting in exocytosis of granule content, is still poorly understood. Proteins from the SNARE family mediate all membrane fusion events in the cell. Here we show that NK cells express all seven members of the R-SNARE subgroup. Two of these R-SNARE proteins, VAMP4 and VAMP7, are associated with lytic granules during cytotoxic interactions. However, only VAMP7 co-localizes with granules in non-activated cells. The differences in co-localization of these two R-SNARE proteins, with respect to perforin-containing cytotoxic granules, indicate that they have different functions in exocytosis. Using the tumor NK cell line, YTS, we show that RNAi-mediated disruption of expression of either VAMP4 or VAMP7 inhibits release of lytic granules and severely impairs NK cell cytotoxic activity. Furthermore, VAMP7 but not VAMP4 is involved in cytokine secretion and endocytosis in NK cells, indicating that VAMP7 is involved in many fusion processes and thus plays a more general function in NK cell activity than VAMP4.
Decreased production of neutrophil defensins and increased bacterial translocation in severely burned patients (89.33)
Authors: Kobayashi Makiko , Yoshida Shohei , Jeschke Marc , Inatsu Akihito , Herndon David , Suzuki Fujio
Published: January 2023

α-Defensins are antimicrobial peptides produced by human neutrophils (HNPs). Because severely burned patients are greatly susceptible to gut-associated infections, in this study, the role of HNPs on the resistance of burn patients to bacterial translocation was investigated. PMN were isolated from peripheral blood of severely burned patients, as previously described. These cells (2 x 106 cells/mouse) were inoculated to X-irradiated and decontaminated NOD-SCID IL-2rγ-/- mice (BP-chimeras) orally infected with 106 CFU/mouse of Enterococcus faecalis. The mortality rates of the chimeras were compared with those of chimeras created with healthy donor PMN (H-chimeras). Also, without any stimulation, PMN (1 x 106 cells/ml) were cultured for 18 hrs, and HNP 1-3 in their culture fluids were measured by ELISA. In the results, a 100% mortality rate was demonstrated in a group of BP-chimeras orally infected with 106 CFU/mouse of E. faecalis, while all of H-chimeras survived after the same infection. Healthy donor PMN produced 35.1 ng/ml of HNP 1-3. However, PMN from 5 out of 18 burned patients did not produce HNPs, and trace amounts of HNP 1-3 were produced by PMN from other 13 out of 18 burned patients. These results indicate that the abilities of PMN to produce HNPs are impaired in severely burned patients, and the susceptibility of thermally injured patients to enterococcal translocation may be influenced by the impaired HNP production.
Measurement of rare innate immune cell activation directly in whole blood using imaging flow cytometry. (89.36)
Authors: George Thaddeus , Hall Brian , Mordecai Scott , Friend Sherree , Kong Raymond
Published: January 2023

Signal transduction pathways regulate innate immune cell activation and thus have a critical influence on the effectiveness of host defense against pathogens. Binding to pathogen epitopes triggers signaling cascades that result in the translocation of transcription factors from the cytoplasm to the nucleus, which can be directly measured using microscopy. However, innate immune cells present significant challenges to image-based analysis due to their rarity and the need for simultaneous multispectral immunophenotyping. These challenges have historically required enrichment prior to imaging, even when using high speed imaging cytometry. Recent advancements in multispectral imaging flow cytometry have reduced these barriers, and in this study we show image-based measurement of NFkB activation in two innate immune cell types found in human whole blood. In the first set of experiments, we show dose and time kinetic activation of NFkB translocation specifically in monocytes by LPS and TNF-α. In the second set we show TLR-mediated activation of non-enriched circulating plasmacytoid dendritic cells, which represent less than 0.1% of periperal blood leukocytes. Our results in each of these systems uniquely demonstrate a method to perform image-base measurement of rare immune cell function directly in whole blood samples.
Size-dependant gold nanoparticles attenuation of CpG DNA-induced TLR9 signaling by modulation of lysosome (89.42)
Authors: Tsai Chiau-Yuang , Lei Huan-Yao
Published: January 2023

Gold nanoparticles (GNPs), also called colloidal gold, have first appeared in antiquity in China and Egypt for therapeutic purposes although its active mechanism remains poorly understood. To explore the immunological effects of GNPs in macrophage is of great interest since macrophages are essential phagocytic and antigen-presenting cells in the immune system. Toll-like receptors (TLRs) are the most important sensors for microbial infection by recognizing microbial components and nucleic acids in infected host cells. We are interested in the effect of GNPs on macrophages. We found that GNPs accumulated in lysosome after phagocytosis efficiently inhibit TNF-α and IL-6 production by CpG DNA (TLR9). TLR9-mediated pathways of NFκB, JNK, and ERK were also affected in GNPs-treated macrophages. Interestingly, impaired CpG DNA-induced TNF-α production by GNPs was size-dependant in RAW macrophages; small GNPs have high inhibitory effects. Otherwise, GNPs neither had cytotoxicity and pro-inflammetory cytokines production nor altering the uptake and intracellular transport of CpG DNA and lysosome acidification. Elevated lysosomal cathepsins expression and activities by GNPs suggests that GNPs is a cysteine protease modulator to attenuate TLR9 signaling in macrophages. We conclude that GNPs can attenuate CpG DNA-induced TLR9 signaling through modulation of lysosome and this effect is size-dependant.
Protein-DNA complex is the major malaria parasite component that activates dendritic cells (89.60)
Authors: Wu Xianzhu , Gowda Nagaraj , Kumar Sanjeev , Gowda Channe
Published: January 2023

Dendritic cells (DCs) are the key cells that sentinel pathogens, initiate and shape protective immunity to malaria. However, how malaria parasites trigger immune responses in DCs remains unclear. Here, we purified merozoites, food vacuoles and parasite membrane fragments released during the Plasmodium falciparum schizont burst to homogeneity and tested for their capacity to activate Flt3-ligand differentiated bone marrow DC (FL-DC). We found that merozoites were the only components that activate FL-DC by a TLR9-dependent pathway. Further characterization demonstrated that, a protein-DNA complex is the exclusive parasite component that activates FL-DCs. In addition, polylysine and chitosan, can also impart stimulatory activity to parasite DNA, indicating that complex formation involves ionic interactions. Merozoites and DNA-protein complex could also induce the production of inflammatory cytokines in human blood DCs. Hemozoin is neither a TLR9 ligand for DCs nor it functions as a carrier of DNA into cells. Further, while TLR9 is critical for DCs to help NK cells to produce IFN-γ in response to merozoites, this receptor is not required for NK cells to secret IFN-γ; cell-to-cell contact among myeloid DCs, plasmacytoid DCs and NK cells is required for IFN-γ production. Together, these results contribute substantially toward the understanding of malaria parasite recognition mechanisms, and have important implications for the development of vaccine against malaria.
A genomics approach to understand γ/δ T cell function in the gut epithelial compartment. (90.11)
Authors: Barrett Desire , Cheadle Christopher , Soloski Mark
Published: January 2023

The gut epithelial functions to provide a protective barrier from enteric pathogens as well as facilitate the absorption and distribution of needed nutrients. These functions are accomplished by the concerted action of a number of interacting cell types including epithelial cells, stromal elements and immune cells. We reason that cells of the immune system are an essential component for the functioning gut epithelia and are likely to play a role in the maintenance of epithelial integrity and function. We have used a genomic-based approach to understand how a novel subset of intraepithelial lymphocytes, γ/δ T cells, contributes to the functioning of the gut epithelial barrier. Using mouse strains deficient for TCR γ/δ, we examined the genes and pathways of the GI tract that are affected by the absence of the TCR γ/δ bearing subset. Our initial analysis revealed that the expression profiles of genes encoding proteins involved in maintaining epithelial integrity and tissue remodeling are regionally and selectively altered in TCR γ/δ deficient mice. In addition, we examined the affect of TCR γ/δ deficiency on the gene sets and pathways activated in the early stages of enteric infection with Salmonella enterica typhimurium. These results reveal abnormal regulation of the acute phase response implying a role for γ/δ T cells in regulating the response to injury. Collectively these studies reveal novel insights into the function of this novel immune cell subset.
Role of Muc1 in Helicobacter pylori gastric mucosal inflammation (90.15)
Authors: Guang Wei , Ding Hua , Kim K. , Blanchard Thomas , Lillehoj Erik
Published: January 2023

Helicobacter pylori (Hp) is a human pathogen that inhabits the stomach and duodenum. Hp causes chronic inflammation of the gastric mucosa (gastritis) that is strongly linked with the sequential progression to atrophy, metaplasia, dysplasia, and stomach cancer. However, greater than 80% of individuals infected with Hp are asymptomatic and it is unknown what factors influence the incidence and character of Hp-associated gastric disorders. Our previous studies demonstrated that the Muc1 epithelial mucin glycoprotein counter-regulates bacterial inflammation. Therefore, we investigated the role of Muc1 in controlling inflammatory responses to Hp infection. Muc1 knockout mice exhibited increased bacterial colonization of the stomach and greater inflammatory cytokine/chemokine responses compared with wild type mice following experimental Hp infection. Overexpression of Muc1 in human gastric AGS cells was correlated with decreased NF-κB activation and IL-8 production compared with cells expressing normal levels of Muc1. Conversely, knockdown of Muc1 expression in AGS cells was associated with greater activation of ERK1/2, increased phosphorylation of IκBα, augmented activation and nuclear translocation of NF-κB, and enhanced production of IL-8. Based on these results, we conclude that Muc1 plays an anti-inflammatory role in Hp gastric infection and that decreased Muc1 expression in a subset of Hp-infected individuals promotes the development of chronic gastritis and adenocarcinoma.
A small intestine trans-epithelial conduit system assists in lamina propria DC sampling (90.2)
Authors: McDole Jeremiah , Wheeler Leroy , Newberry Rodney , Miller Mark
Published: January 2023

The mechanisms by which lamina propria DCs sample antigens from the gut remains a crucial topic in mucosal immunology. DCs acquire antigen by several distinct processes including M cell transcytosis, specialized transport involving the neonatal Fc receptor, paracellular leak and by DC trans-epithelial dendrite extension. The continuous sampling of luminal contents by gut resident DCs could both contribute to rapid pathogen recognition and provide normal flora and food antigens for tolerance induction. We used two-photon microscopy, to examine DC sampling behavior in vivo and discovered a previously unreported small-intestine-trans-epithelial-conduit (SiTEC) system. We found that SiTECs could carry macromolecules across the epithelium to the lamina propria where they were taken up by resident DCs. SiTECs have a functional molecular weight cut-off of approximately 70kD based on dextran permeability and can pass small proteins as well. The SiTEC system presents a simple mechanism to deliver luminal antigens to lamina propria DCs for the maintenance of tolerance in the steady-state.
Education of intestinal DCs by lamina propria cells (90.6)
Authors: Vicente-Suarez Ildefonso , Park Yunji , Mucida Daniel , Bekiaris Vasileios , Koltsova Ekaterina , Ley Klaus , Ware Carl , Cheroutre Hilde
Published: January 2023

The intestinal mucosa is constantly exposed to a vast load of antigens derived from nutrients and commensal bacteria but also from potential pathogens. In this situation intestinal dendritic cells (DCs) play a central role in the decision between suppressing inflammation or promoting host defense. Intestinal DCs display specific features that include the ability to promote gut homing of lymphocytes, the preferential induction of Foxp3+T cells and the inhibition of Th1 and Th17 differentiation. Interestingly the majority of these functions depend on the DCs ability to produce a vitamin A metabolite known as retinoic acid (RA). How intestinal DCs acquire this ability is largely unknown but it has been suggested that tissue specific factors produced by the intestinal epithelia might be required, among them RA itself. Here, we report that the interaction with stromal cells from the lamina propia imprints splenic DCs with a mucosal phenotype. The stromal cells in this study show characteristics that identify them as stromal organizing cells named after their ability to organize the development of secondary lymphoid organs. Our data implies that intestinal DCs might develop their specific characteristics in gut associated lymphoid tissues (GALT)
Intestinal microbiota and innate signaling via MyD88 promote the intestinal Th2 response to Trichinella spiralis (90.9)
Authors: Blum Lisa , Nel Hendrik , Fallon Padraic , Gagliardo Lucille , Cameron Jody , Leifer Cynthia , Appleton Judith
Published: January 2023

The role of commensal intestinal microbiota in colitis, type 1 diabetes, and other immune-mediated diseases has been established recently, but an influence on Th2 immunity in parasitic disease to has not yet been reported. Larvae of the parasitic nematode Trichinella spiralis move rapidly through epithelial cells in the intestine, where they induce a potent Th2 immune response. Here we report that mice deficient in the signaling adaptor MyD88 and mice treated with antibiotics to diminish the intestinal flora demonstrate reduced intestinal Th2 cytokine responses to T. spiralis. Furthermore, infection caused an overgrowth of intestinal bacteria. Parasite extracts and purified glycoproteins did not cause direct TLR activation in vitro. IL-33 (ST2L), which signals through MyD88 and is expressed by intestinal epithelial cells, also promoted the Th2 response to T. spiralis. These results reveal a previously undescribed interplay between an intestinal nematode, the mucosal immune system, and the commensal microflora.
The role of IL-6 in T-bet (-/-) mice in an allergic asthma model (91.10)
Authors: Fassbender Sonja , Übel Caroline , Böhm Livia , Lehr Hans-Anton , Finotto Susetta
Published: January 2023

IL-6 is a pleiotropic cytokine which induces Th2 type cells. IL-6 binds to IL-6R alpha chain and then induces gp130 dimerization resulting in the intracellular activation of Janus kinase and signal transducers and activators of transcription (STAT) pathways. We previously demonstrated that patients affected by allergic asthma have an increased level of soluble IL-6R which correlates with Th2 CD4+ T-cells in their airways. In a murine model of this disease, we also demonstrated that IL-6 inhibits the development of T-regulatory cells (Tregs) and local blockade of IL-6R resulted in the expansion of Tregs as well as Th1 cells in the lung. T-bet is a Th1-specific transcriptional factor. Mice lacking T-bet show increased asthmatic symptoms. We thus want to study the influence of T-bet on IL-6 mediated inhibition of Tregs. To this aim an antibody against IL-6R was applied to T-bet deficient mice (T-bet (-/-)) during allergen challenge. In preliminary studies we found that local blockade of IL-6R in T-bet (-/-) mice resulted in a reduced AHR and decreased lung eosinophilia. Furthermore we observed selective expansion of the CD4+ CD25+ Foxp3+ Tregs in the draining lymph-nodes taken from anti IL-6R-antibody treated T-bet (-/-) mice, compared to T-bet (-/-) untreated mice. These results suggest that local blockade of IL-6R in the absence of T-bet in allergic asthma could result in amelioration of the disease hallmarks because of inducing Tregs in the local draining lymph nodes.
Cleavage of CD23 by ADAM proteins is regulated by beta-2 adrenergic receptor stimulation on B cells (91.14)
Authors: Padro Caroline , Sanders Virginia
Published: January 2023

The level of IgE is often associated with the severity of allergic asthma. Beta-2 adrenergic receptor (β2AR) agonists alleviate bronchoconstriction by targeting the β2AR on bronchiolar smooth muscle cells, triggering airway relaxation. The β2AR is also expressed on B lymphocytes. In vitro data from our laboratory showed that β2AR stimulation on a CD40L/IL-4 activated B cell increases the level of IgE produced per cell, without affecting class switch recombination. We also found that surface CD23 expression remained constant, while CD23 mRNA, total CD23 protein, and CD23 cleavage increased. We know that soluble CD23 positively regulates IgE production. Therefore, β2AR stimulation may worsen bronchoconstriction over time, while relieving it in the short term. We hypothesized that the primary sheddase of CD23, ADAM10, was regulated by β2AR stimulation on the activated B cell. However, we found that β2AR stimulation was unable to affect the level of ADAM10 protein or mRNA in an activated B cell as compared to activated cells alone. Our recent data indicated that ADAM8 and ADAM28 might instead be involved in the β2AR-associated shedding of CD23. The differential β2AR-associated ADAM protein cleavage of CD23 may provide a potential molecular target to prevent a β2AR agonist-induced increase in IgE, which may counteract the therapeutic effect to relieve bronchoconstriction. Funded by NIH-AI-37326 and T32-AI55411
Dual Roles of Inositol Phosphatase SHIP-1 in Active and Passive Systemic Anaphylactic Responses (91.16)
Authors: Zhu Zhou , Oh Sun-Young , Wu Fan , Zheng Tao
Published: January 2023

SHIP-1 is a negative regulator of signaling pathways triggered by antigens and cytokines. We reported that SHIP-1 knockout (KO) mice develop a spontaneous allergic pulmonary inflammation. To determine the roles of SHIP-1 in innate and adaptive immune responses, we examined IgE-dependent passive (PSA) and OVA-induced active systemic anaphylactic (ASA) responses in mice deficient in SHIP-1. Heterozygous SHIP-1 (hSHIP) mice were compared with C57BL/6 wild type (WT) mice. For PSA, 24 h after passive immunization with 5 µg anti-DNP IgE, mice were challenged i.v. with 1 mg of DNP-HSA. For ASA, mice were immunized i.p. once or twice with 50 µg of OVA/Alum on day 1 and 7 and then challenged with 1 mg of OVA on day 14 or 21. The core body temperature was monitored and blood was collected for serum immunoglobulins. Compared to WT controls, hSHIP mice developed significantly enhanced IgE-dependent PSA, but surprisingly, with one-time sensitization, hSHIP mice developed diminished ASA responses. Serum OVA-specific IgE and IgG1 were significantly lower but OVA-specific IgG2a was significantly higher in hSHIP mice. These studies demonstrate that SHIP-1 deficiency leads to enhanced PSA by increased mast cell response but diminished ASA, possibly due to impaired Th2 response to allergen and decreased production of IgE and IgG1. Thus, in contrast to its role as a negative regulator in innate immunity, SHIP-1 may play a critical role in the Th2 adaptive immunity as a positive regulator.
Involvement of HIF1α in mediating VEGF-dependent epithelial cell growth and overexpression in chronic rhinosinusitis with nasal polyposis (91.4)
Authors: Kim Jean , Lee Hyun
Published: January 2023

We recently reported that VEGF is overexpressed in chronic rhinosinusitis (CRSwNP) and functions to promote nasal epithelial cell hyperplasia, a key feature of sinus polyposis, by regulating autocrine epithelial cell growth. However, the upstream signals regulating this process remain unclear. Hypoxia-inducible factor-1α (HIF1α) is a known regulator of VEGF. Therefore we examined whether HIF1α may regulate VEGF dependent cell growth in nasal epithelial cells. Immunohistochemical analysis of sinonasal surgical tissue showed significantly increased staining for HIF1α in epithelium in polyps of CRSwNP subjects as compared to normal control sinus tissue (p< 0.001, n=5). Culture of human primary nasal epithelial cells (PNEC) from CRSwNP subjects demonstrated abundant expression of HIF1α by realtime PCR (Ct=19+/-2, n=5). To implicate a role of HIF1α in regulation of VEGF in CRSwNP, we performed siRNA knockdown of HIF1α in PNEC from CRSwNP subjects. This resulted in specific inhibition of VEGF mRNA by 77% (p<0.01, n=3). ELISA of PNEC cell supernatants confirmed inhibition of soluble VEGF expression by 47% (p<0.01, n=3). In addition, silencing of HIF1α resulted in 56% reduction of cell growth rate comparable to that of PNECs from normal control subjects, as measured by DNA binding growth assay (p<0.01, n=3). These data suggest that HIF1α regulates VEGF in perpetuating hyperplastic epithelial cell growth observed in CRSwNP.
The role of CCR7 in CD8 T cell egress from the lung during influenza A virus infection. (92.17)
Authors: Jennrich Silke
Published: January 2023

The migratory capacity determines T cell effector function in vivo. Memory/effector T cells efficiently enter extralymphoid sites from blood and subsequently leave the tissue via the afferent lymph. While exit of resting T cells from uninflamed extralymphoid tissue is regulated by their expression of the chemokine receptor CCR7, it is unclear whether effector T cells exit from acutely infected sites. Here, we found that IFN-g expressing in vitro polarized (CD8+) Tc1 cells expressed functional CCR7 and were cytotoxic in vitro. Importantly, upon adoptive transfer, Tc1 cells were able to rescue mice infected with a lethal dose of influenza A virus. Transferred Tc1 cells did egress from infected lungs and could be recovered from draining lymph nodes. The pulmonary egress of Tc1 cells during acute infection required their expression of CCR7, demonstrating that egress from the inflamed lungs is an active, regulated process. Our data support a model, in which T cell retention in and egress from infected tissue is controlled by the regulated expression of CCR7, thereby affecting local T cell effector function and the efficiency of host defense.
Investigating original antigenic sin responses to H5N1 influenza viruses (92.19)
Authors: Masseoud Feda , Skountzou Ioanna , Temperton Nigel , Jacob Joshy
Published: January 2023

H5N1 influenza A virus remains a global threat since its outbreak in 1997. Its re-emergence in 2003 spread from Asia to Europe, the Middle-East and Africa, raising concerns of a possible pandemic. While stockpiling of H5N1 prepandemic vaccines has been initiated, strain prediction is difficult due to the emergence of distinct clades/subclades. Antigenic variation between vaccine and pandemic strains may confer cross-protection or result in antigenic sin, in which the immune response is directed against the original strain rather than the novel protective antigenic determinants. Observed between strains that have undergone antigenic drift, this misdirected response may compromise protective immunity. This study was carried out to determine the extent to which antigenic sin interferes with immune responses to the potentially pandemic influenza H5 subtype. We immunized mice with a DNA vaccine encoding the full length HA from influenza A/Vietnam/1194 followed by a vaccine encoding HA of drifted H5N1 strains, ranging in genetic identity from 100%- 88%. We are currently assessing responses directed against either the primary or the drifted strain by microneutralization using a pseudotyped lentivirus bearing HA from the respective H5N1 strains. By examining antigenic sin due to H5N1 viruses, we can better understand the behavior of the virus as it circulates in the human population, and therefore improve preparedness in the context of H5N1 vaccine development and pandemic planning.
An inducible antigen system to study endogenous CD8 T cell tolerance (93.11)
Authors: Jellison Evan , Turner Michael , Lefrançois Leo
Published: January 2023

Peripheral tolerance induction is critical for deterrence of autoimmunity. Most self-reactive T cells are developmentally deleted in the thymus. This deletion process is not absolute and self-reactive T cells can escape. These T cells must either be deleted or regulated in the periphery to prevent autoimmunity. To study these processes, we generated mice in which SIINFEKL expression can be tightly controlled in small intestinal epithelial cells or dendritic cells by administration of doxycycline. Our system provides the unique ability to examine endogenous T cell responses to tissue-specific and developmentally regulated self antigen. Importantly, the transgenic mice are ignorant of antigen prior to induction. Antigen induction in either the small intestine or dendritic cells caused tissue-specific activation of transferred TCR transgenic OT-I T cells. Antigen induction also resulted in activation and expansion of endogenous SIINFEKL-specific CD8 T cells that expressed PD1, but did not express KLRG1 or CD127. Antigen induction impaired a SIINFEKL-specific CD8 response to concomitant VSV OVA infection. However, in the absence of antigen induction, SIINFEKL-specific effector and memory CD8 T cell responses were generated, but the resultant memory CD8 T cells did not respond to subsequent self antigen induction. Characterization of immune responses in these antigen-inducible mice will provide key insights into mechanisms controlling tolerance versus autoimmunity.
Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic neuroautoimmune response (93.19)
Authors: Fu Haiyan , Killedar Smruti , DiRosario Julianne , Popovich Phillip , McCarty Douglas
Published: January 2023

Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive lysosomal storage disease with severe progressive neurological manifestation. Detailed mechanisms of neuropathology of MPS IIIB are unclear, though the primary pathology is known to be the lysosomal storage of heparan sulfate. Recently, using a mouse model of MPS IIIB, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. Here, we show for the first time, that lymphocytes isolated from MPS IIIB mice cause a mild paralytic disease when they are injected systemically into naïve wild-type mice. This disease is characterized by mild tail and lower trunk weakness with delayed weight gain. The MPS IIIB lymphocytes also trigger neuroinflammation within the CNS of recipient mice characterized by up-regulation of IL2, IL4, IL5, IL17, TNFα, IFNα, and Ifi30, glial activation and intraparenchymal lymphocyte infiltration. Together, these data suggest that an autoimmune response, especially T-cell autoimmunity, directed at CNS proteins contributes to MPS IIIB neuropathology independent of lysosomal storage pathology.
Identification of specific single-nucleotide polymorphisms that modify alternative splicing in a lupus-associated haplotype of complement receptor 2 (93.26)
Authors: Lough Kara , Douglas Katherine , Homme Carissa , Ulgiati Daniela , Tsao Betty , Boackle Susan
Published: January 2023

A haplotype formed by the minor alleles of three single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene is associated with decreased risk of systemic lupus erythematosus (SLE). Two of these SNPs (rs1048971 and rs17615) are in exon 10 and the third (rs4308977) is in the alternatively spliced exon 11, which is differentially expressed on follicular dendritic cells (FDC). The minor alleles of these three SNPs and a fourth SNP in exon 11 (rs17616) decrease the splicing efficiency of exon 11 in vitro and ex vivo, but the specific SNP responsible for this is not known. An exon-trapping vector (pL53In) in which genomic DNA spanning introns 9 to 12 of CR2 had been cloned was modified by site-directed mutagenesis to generate vectors containing various allelic combinations of these SNPs. After transient transfection into the Raji B cell and HK FDC lines, the relative amount of vector-derived mRNA transcripts including or excluding exon 11 was measured by quantitative RT-PCR. The presence of the minor allele at rs17615, rs4308977, or rs17616 partially reduced splicing efficiency of exon 11, whereas the presence of the minor allele at both rs17615 and rs17616 reduced it to same extent as when all four SNPs were minor. These data suggest that the minor alleles at rs17615 and rs17616 modify the secondary structure of the CR2 pre-mRNA, interfering with splice site recognition by regulatory proteins that could serve as targets for therapeutic intervention.
IL-21/IL-21R interactions on host B cells but not on donor CD4 cells are critical for the development of autoimmune features in chronic graft versus host disease. (93.9)
Authors: Nguyen Vinh , Tegla Cosmin , Luzina Irina , Rus Horea , Rus Violeta
Published: January 2023

Studies in murine models of lupus have indicated increased production of IL-21 and attenuation of autoimmune features following IL-21 blockade. IL-21 exerts an autocrine effect on T follicular B helper cells (TFH) cells and also stimulates B cell proliferation, plasma cell (PC) differentiation and germinal center (GC) expansion. To determine whether IL-21 promotes systemic lupus through effects on TFH cells or B cells, we assessed the effect of IL-21/IL-21R signaling on B cells independent from the effect on CD4 T helper cells using IL-21R-/- or IL-21R+/+ mice as donor or hosts in the P-into-F1 and Bm12-into-B6 models of cGVHD. CGVHD induced by injection of IL-21R-/- CD4 cells from B6 mice into B6D2F1 hosts was characterized by a decrease in the expansion of donor CD4, TFH and GC cells. However, other parameters of cGVHD such as host B cell expansion, MHC class II upregulation, PC differentiation and anti-ssDNA autoantibody production did not differ between groups. In contrast, when cGVHD was induced by injecting Bm12 spleen cells into IL-21R-/- B6 mice, parameters of cGVHD were markedly attenuated compared to IL-21R+/+ hosts. Specifically, MHC class II upregulation of B cells decreased by 32%, PC differentiation by 66%, GC cells by 60% and anti-ssDNA antibody level by 81%. These results suggest that IL-21R signaling on host B cells and possibly myeloid cells but not on Ag-specific CD4 T cells is critical for the initiation and progression of systemic autoimmunity in cGVHD.