bluuebunny/crossref_metadata_2025 huggingface dataset:
Supressor T cell–derived miRNA that acts in part via attached antigen specific Immunoglobulin and Prostaglandin-E2 (PGE2) (89.10)
Authors: Askenase Philip , Szczepanik Marian , Majewska Monika , Hafner Markus , Tuschl Tom , Kleinstein Steven , Redegeld Frank , Ptak Wlodzimierz , Bryniarski Krzysztof
Published: January 2023

Soluble antigen (Ag)-specific T cell-derived suppressive factor (TsF) of contact sensitivity (CS) is a small RNA, not a protein, induced in suppressor cells of spleen and nodes by high dose Ag iv tolerance; and is not IL-10 nor TGF-beta. The RNA TsF inhibits effector Th1 cells that elicit classical cutaneous CS and in vitro inhibits responsiveness of T cell lines to rIL-2; both in a paracrine manner; like exosomes containing specific mRNA and miRNA (Nature Cell Biol 9:654, '07). The TsF obtained by phenol chloroform extraction of cell supernatants, is degraded by RNase A, and RNase III (specific for ds RNA like pre-miRNA), but not DNase. It is in the RNA fraction from a Quiagen DNA/RNA separation column and about 75bp size (like pre-miRNA) on denaturing electrophoresis.

The suppressive RNA TsF likely is associated with Ig (likely kappa light chain, Nat Med 8:694, '02) and PGE2 (Jo Im 150:2102, '93), that may account for Ag specificity and some suppression. The miRNA part of TsF, after uptake by target cells, may block translation via degrading mRNA encoding a protein in the IL-2 pathway, that is needed by CS Th1 effector cells.

Thus, an Ag-specific TsF is dsRNA, associated with Ig light chain and inhibitory PGE2. This complex, released by a suppressor cell mix, perhaps via exosomes acts in a paracrine manner to target the effector T cells of CS, to intracellularly degrade mRNA translating protein of the IL-2 pathway, needed for effector T cells to elicit CS. Supported by grants from NAID, NIH, the Yale Molecular Hematology program, and the Polish Ministry of Science and Higher Education.
Differential effects of galectin-9 ligation with TIM-3 expressed on Th1 and Tregs (89.12)
Authors: Sehrawat Sharvan , Hirashima Mitsuomi , Rouse Barry T
Published: January 2023

TIM-3 (T cells immunoglobulin and mucin -3) is a surface protein expressed differentially on terminally differentiated Th1 but not Th2 cells. Ligation of galectin-9 with TIM-3 on Th1 cells induces a lethal signal leading to apoptosis of these cells. We report here that same molecule i.e. TIM-3, when expressed on Foxp3+ Tregs, instead of delivering a lethal signal, promotes their proliferation and functional activity. We show that TIM-3 expression was upregulated on a significant numbers of CD4+ T cells including Foxp3+CD4+ T cells, after ocular HSV infection. Furthermore, 50% or more of T cells in ocular lesions (stromal keratitis) caused by HSV in mice express TIM-3 and blocking signals from its natural ligand with a monoclonal antibody results in more severe lesions. More importantly the provision of additional galectin-9 diminished the severity of SK lesions that was achieved by multiple mechanisms including apoptosis of the orchestrating effector T cells, an increase in the representation of two separate subtypes of regulatory cells. The molecular events induced in Tregs and Teffectors after galectin-9 ligation to TIM-3 would be discussed. Our results indicate that galectin-9 therapy may represent a useful approach to control HSV induced lesions, the commonest cause of infectious blindness in the Western World.

Work supported by AI 063365 and EY 05093
Induction of “regulatory” dendritic cells via AhR activation (89.15)
Authors: Bankoti Jaishree , Rase Ben , Shepherd David M.
Published: January 2023

Aryl hydrocarbon receptor (AhR) activation by its prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is shown to induce Tregs. Dendritic cells (DCs) are key antigen presenting cells governing T cell activation and differentiation. How AhR activation in DCs may induce Tregs is undefined. We hypothesize that AhR activation induces a novel population of inflammatory DCs (GM-CSF derived) that can generate T regs. To test this hypothesis, inflammatory BMDCs were generated in the presence of vehicle or TCDD. TCDD decreased CD11c expression but increased MHC class II, CD86 and CD25 expression on BMDCs. The effects were strictly AhR- dependent as no differences in the phenotypic markers were observed using TCDD-treated BMDCs from AhR KO mice. Induction of indoleamine 2,3-dioxygenase (IDO) and TGF-β3 has been implicated in the generation of Tregs following AhR activation. AhR activation by TCDD increased IDO1, IDO2 and TGF-β3 mRNA levels in BMDCs as compared to vehicle. TCDD upregulated LPS-and CpG-induced TNF-α and IL-6 production in these BMDCs. AhR activation is demonstrated to alter NF-kB signaling. TCDD downregulated LPS- and CpG-induced p65 levels and induced a trend towards upregulation of RelB levels in BMDCs. The results suggest that AhR activation can induce a regulatory phenotype in DCs, an effect that may govern generation of Tregs. The research was supported by grants ES013784 and RR17670.
Novel population of CD39-expressing T cells enriched in the inflamed site (89.21)
Authors: Moncrieffe Halima , Nistala Kiran , Wedderburn Lucy R
Published: January 2023

CD39-mediated hydrolysis of proinflammatory ATP is a newly described mechanism of Treg suppression. We describe the presence and function of a novel population of CD39+ Foxp3- CD4+ T cells enriched in the human inflammatory site. 36 patients with juvenile idiopathic arthritis (JIA) and 25 healthy controls were included in this study with full ethical approval (LREC). Mononuclear cells purified from peripheral blood (PBMC) and synovial fluid (SFMC) were analysed by FACS, functional suppression and ATP assays.

CD39+ Foxp3- CD4 T cells were significantly enriched (p<0.001) in the inflamed joint, compared to PBMC from patients or healthy controls (18.6%, 2.9%, 4.2%, respectively). Unlike CD39+Foxp3+ Treg, this novel population produced the proinflammatory cytokines IFNg and IL-17 and also IL-2. CD39+Foxp3- cells expressed lower CCR4 and higher levels of CD127 than classical Treg. Upon cell sorting, CD39+Foxp3- cells had comparable ATP hydrolysis activity to Treg, but crucially, failed to suppress cell proliferation in vitro.

Thus, the expression of Foxp3 with CD39 may be dissociated in the inflamed site and CD39 expression on CD4 T cells may not result in regulatory status. This will be important in understanding the complex effects of extracellular ATP in human inflammation.

This study was supported by SPARKS UK and the Big Lottery Fund. KN is supported by an ARC Fellowship (Ref. 17998).
Defective Tregs expression of the NTPDase CD39 in lupus (89.24)
Authors: Khan Islam U , Anderson A. Shane , O'Rourke Kenneth S. , Loza Matthew J.
Published: January 2023

The role of aberrations in the regulatory T cells (Tregs) pool for the development of systemic lupus erythamatosus (lupus) remains uncertain. Treg-mediated generation of adenosine, dependent on the ectonucleotidase CD39, is an important mechanism for suppression of T-cell responses. We studied 15 SLE patients along with 6 rheumatoid arthritis and 24 healthy controls. Levels of Treg phenotypic markers, including CD39 expression, were studied by flow cytometric analysis. Varying numbers of sorted Tregs cells were co-cultured with responder T (Tresp) cells, with proliferation assessed by 3H-thymidine incorporation. The proportion of Foxp3+ CD25+high CD127-/low Tregs was similar between lupus and control populations. CD39-expressing Tregs comprised 37 ± 13% of the Treg population in healthy controls and 36 ± 21% in lupus subjects using nonsteroidal immunosuppressants to control active disease, but was nearly absent in 5 of 6 lupus subjects with minimally active disease. In lupus subjects with the CD39 defect, Treg-mediated suppression was significantly reduced and, unlike in healthy control subjects, adenosine receptor antagonism could not reverse the observed suppression. These results suggest that functional defects in Tregs, rather than reduced Treg numbers, may be important for the loss of peripheral tolerance in lupus.

Supported by NIH RO1-AR39501,WFUSM Venture Funds.
TNF/TNFR2 signaling in T regulatory (Treg) cells induces a distinctive NF-kB-dependent cellular program that modulate their suppressive capacity (89.27)
Authors: Nagar Meital , Jacob-Hirsch Jasmine , Vernitsky Helly , Amariglio Ninette , Rechavi Gideon , Goldstein Itamar
Published: January 2023

Anti-TNF therapy has a proven high-degree of clinical efficacy in the treatment of selected T cell-dependent autoimmune disorders. In treated patients, TNF-blockade augments the number and suppressor function of Foxp3+ T regulatory cells (Treg), whereas TNF treatment, in vitro, has opposite effects. Thus, it was of interest to define in vitro the cellular programs induced by TNF in human Treg using various methods: gene expression arrays, RTqPCR, and protein expression analysis. Here we show that TNF/TNFR2 signaling induces within 2 hrs the transcription of distinct NF-κB dependent gene networks rather unique to Treg. Members of the following biological pathways were particularly induced by TNF in Treg: (a) TNF super family (TNFSF) signaling; (b) NF-κB cascade; (c) negative regulation of apoptosis; and (d) immune-response related receptors. No significant effect on the transcription of FOXP3 and other "Treg signature" genes was detected during the first 2 to 24 hrs after TNF treatment. The suppressive function of Treg was partially inhibited by TNF, and this effect was enhanced by co-treatment with 4-1BB ligand (TNFSF9). In summary, TNF induces a pro-inflammatory NF-κB dependent cellular program in Treg that modulates their suppressive function and renders them more susceptible to inhibition by other TNFSF members.
T-regulatory cells and IL-10 are important in maintaining control of germinal center reactions (89.29)
Authors: Alexander Carla-Maria , Waldschmidt Thomas
Published: January 2023

Previous work from our laboratory demonstrated the primary splenic germinal center (GC) reaction to display traits of a steady state, especially in regards to the ratio of IgM+ to switched GC B cells. The GC is a site of intense proliferation, class switching, apoptosis and selection, and as such must have strict regulation. Studies in our laboratory have shown that T regulatory cells (T-regs) play a pivotal role in regulation of the GC, as steady state dynamics were disrupted when T-regs were depleted or dysregulated by anti-CD25 and anti-GITR, respectively. Current studies are exploring the mechanism by which T-regs exert their control within the GC. Based on previous reports studying the means by which T-regs suppress their targets, we have tested whether granzyme B, perforin, Fas and IL-10 contribute to the homeostasis of GC responses. GC reactions in granzyme B-/-, perforin-/-, and B6.lpr mice were found to be normal after immunization, demonstrating that the granzyme B/perforin and Fas/Fas ligand pathways are unlikely to play a central role in controlling GCs. Treatment with a blocking anti-IL-10R mAb did result in loss of GC control however, suggesting that T-regs may be utilizing this cytokine to maintain the balance of IgM+ to switched B cells within the GC. Studies are continuing to determine if T-regs and IL-10 also affects other elements of B cell differentiation including affinity maturation and memory.
NEGATIVE FEEDBACK REGULATION OF T CELLS: INTERLEUKIN 2 AND FOXP3 RECIPROCITY (89.8)
Authors: Popmihajlov Zoran , Smith Kendall A
Published: January 2023

TCR activation induces T cell FOXP3 expression, thereby calling into question FOXP3 as a distinct marker for Regulatory T cells. We investigated the role of IL2 in the TCR/CD3-induction of FOXP3 expression by normal human and murine T cells in vitro. Freshly isolated human PBMCs were stimulated with soluble anti-CD3, and four-color flow cytometry was used to determine the expression of FOXP3. Anti-CD3-activation increased the % of cells expressing FOXP3 in both T cells subsets with a peak at 24h. Both anti-CD25 and anti-IL2 inhibited anti-CD3-stimulated FOXP3 expression. The addition of rIL2 restored and prolonged FOXP3 expression. When anti-CD3-activated PBMCs were monitored daily for 4 days, FOXP3 and IL2 expression remained mutually exclusive, indicating that FOXP3 restricts but does not suppress IL2 expression, even upon TCR/CD3-restimulation. By comparison, soluble anti-CD3/CD28 stimulation +/- rIL2 of murine splenocytes did not up-regulate FOXP3 expression. However, upon solid-phase anti-CD3 activation, the presence of both exogenous TGFβ and rIL2 induced FOXP3 expression by both CD4+ and CD8+ T cells.

Therefore, TCR-activated T cell FOXP3 expression is primarily regulated by IL2/IL2R signaling and occurs only transiently in a subset of both CD4+ and CD8+ T cells. Since FOXP3 functions to restrict TCR-induced IL2 gene expression, these results are consistent with an IL2-dependent FOXP3-mediated negative feedback loop that regulates IL2 production via FOXP3 at the level of transcription. Therefore, FOXP3 cannot be considered solely a phenotypic marker for T-Reg cells. Instead, FOXP3 plays a key role in the feedback regulation of IL2 availability, thereby affecting the proliferation and survival of T cells via a passive suppressive mechanism.
Ndfip1 Regulates Adaptive and Innate Immune Responses in a Model of Inflammatory Bowel Disease (90.17)
Authors: Ramon Hilda E , Riling Christopher R , Hakonarson Hakon , Oliver Paula M
Published: January 2023

Ndfip1 is an adaptor protein that regulates the E3 ubiquitin ligase Itch. Ndfip1 promotes Itch ubiquitination of JunB, a transcription factor involved in TH2 differentiation. Ndfip1-/- mice develop a TH2-mediated inflammatory disease in the skin and lungs. We now show that Ndfip1-/- mice also develop gastrointestinal (GI) inflammation, characterized by the infiltration of eosinophils. The GI inflammation is accompanied by weight loss and premature death. We have found that Ndfip1-/- T cells are both necessary and sufficient to recruit eosinophils into the GI tract. Furthermore, we have found that IL-5 plays a major role in disease induction. Ndfip1-/- mice show increased IL-5 levels in the blood and Ndfip1-/- T cells secrete IL-5 when activated ex vivo. Taken together, our results indicate that the eosinophilic inflammation of the GI track in Ndfip1-/- mice is caused by T cell activation and consequent secretion of IL-5. A similar mechanism could lead to Inflammatory Bowel Disease (IBD) in humans, in particular, TH2-mediated forms of the disease. Single Nucleotide Polymorphisms (SNPs) were analyzed within the Ndfip1 gene. Comparing patient populations with or without IBD, significant differences were found (p-value <0.001) in several SNPs. Thus, Ndfip1-/- mice are a novel genetic model of TH2-driven gastrointestinal inflammatory disease that could provide novel insights into IBD.

NIH training grant: T Lymphocyte Development and Activation.
Construction and characterization of three chimeric proteins of CD38 (90.29)
Authors: Vences-Catalán Felipe , Cedillo-Barrón Leticia , Santos-Argumedo Leopoldo
Published: January 2023

CD38 is a surface receptor as well as an enzyme present in various cell types, including B and T lymphocytes. It has been implicated in several biological functions such as chemotaxis, cell activation and proliferation, rescue or induction of apoptosis. It is not known how this molecule has different functions in different cells. CD31 was identified and characterized as a possible ligand; however, these data are still controversial. In the mouse there is less information, since only two molecules have been identified (without a definitive characterization) as potential ligands. In order to identify CD38 ligand(s) in mice, we designed three soluble chimeric proteins of CD38. One fused with CD8α, a second one fused with the Fc from the IgG1 immunoglobulin, both from human, and a third one containing only a V5 paramyxovirus epitope and a histidine tag. The proteins were characterized by Western blot and enzymatic activity assays. The results showed that all the proteins formed soluble dimers and exhibited enzymatic activity. With these tools, we obtained data suggesting the existence of a possible ligand present on the surface of CD11c+ cells from mice′s spleen. In conclusion the chimeric proteins can be useful tools to continue the characterization of potential ligands of CD38. Supported by CONACyT 56836.
Identification and Characterization of Vstm3 as an inhibitory member of the CD28 family (90.31)
Authors: Levin Steven D , Brandt Cameron S , Howard Edward D , Johnston Janet , Chadwick Eric , Hammond Angela , Taft David W , Hebb Luanne , Wolf Anitra , Bukowski Thomas R , Rixon Mark , Ostrander Craig D , West James W , Fox Brian , Gao Zeren , Lewis Katherine , Xu Wenfeng , Ramsdell Fred
Published: January 2023

Members of the B7-CD28 family play critical roles in the response of T cells. We have identified the transmembrane protein Vstm3 as a member of the CD28 family based on gene structure. It has two immunoreceptor tyrosine inhibitory motifs in its cytoplasmic domain and is expressed on NK cells and a subset of T cells. Antibodies against VSTM3 antagonize multiple aspects of T cell activation including proliferation and cytokine production indicating it plays an inhibitory role in T cell activation. We have also identified two counter-structures for this protein, the previously identified proteins CD155 and CD112, which are expressed on activated antigen presenting cells. CD155 and CD112 also bind to the activating receptor CD226 and hence this extended family of interacting proteins forms a network of molecules similar to the well characterized CD28-CTLA4-CD80-CD86 network. A soluble form of Vstm3 binds to CD155 and CD112 and disrupts their ability to trigger an activating response through CD226, and hence antagonizes immune responses in vitro and in vivo. On the other hand, an antibody that specifically blocks Vstm3 interaction with CD155 and CD112 tends to enhance T cell responses in vitro and in vivo. Finally, mice that lack Vstm3 are more sensitive to the development of autoimmune diseases. Our data indicate that this novel member of the CD28 family negatively regulates T cell responses.
Reciprocal control of IL-4 and IFN-Lambda (IL-29); mechanisms for the control of Th2 polarization (90.32)
Authors: Megjugorac Nicholas J , Gallagher Grant E , Gallagher Grant
Published: January 2023

The newly-described Type III IFN family is comprised of three members: IL-29 (IFN-α), IL-28A (IFN-β) and IL-28B (IFN-γ). All three signal through the same receptor complex, which consists of IL-28Rα (a unique binding and signaling subunit) and IL-10Rβ. While much of the early work describing the activity of IFN-λ1 has focused on its antiviral function, IFN-λ has also been shown to exert powerful immunomodulatory effects, by antagonizing the effect of IL-4 on T cells and inhibiting production of Th2-associated cytokines. To determine whether Th2 cytokines exert reciprocal control over IFN-λ production, PBMC were treated with IL-4 and IL-13 during stimulation. Interestingly, these cytokines significantly enhanced IFN-λ1 production in response to stimuli such as herpes simplex virus (HSV). Intracellular flow cytometry and magnetic bead depletion were used to identify the IL-4-sensitive, IFN-λ -producing cells in peripheral blood. While plasmacytoid DC are a major HSV-responsive cell, they are not responsible for enhanced IFN-λ production. Instead, myeloid cells mediate this response. This identifies a novel mechanism for the regulation of T cell responses.
Talin is required for LFA-1-induced natural killer cell polarization and cytotoxicity (91.1)
Authors: Mace Emily M , Takei Fumio
Published: January 2023

The purpose of this study is to elucidate the role that the cytoskeletal adaptor protein talin1 plays in the function of the integrin leukocyte function associated antigen-1 (LFA-1) in NK cells. It has been proposed that LFA-1 not only mediates adhesion of NK cells to target cells but is also an important activating receptor for NK cell cytotoxicity. Talin is known to act as a link between integrins and the actin cytoskeleton and to regulate ligand-binding function of LFA-1. We generated NK cells in vitro from talin1 knockout (KO) embryonic stem cells, tested the effects of talin deficiency on these NK cells, and found that talin plays a dual role in NK cell cytotoxicity. First, it is required for LFA-1 mediated adhesion to the target cell. Second, it is required for the accumulation of actin and actin polymerization machinery, including vinculin and WASP, following binding of LFA-1 to its ligand ICAM-1.This LFA-1- and talin-dependent actin accumulation is a critical for further steps leading to NK cell cytotoxicity. Although talin-KO NK cells adhere to target cells in the presence of Mn++, they are unable to kill the prototypic NK target YAC-1. Wild-type, but not talin-KO, NK cells polarize cytotoxic granules following engagement of both LFA-1 and the activating receptor NKG2D. These data show that LFA-1 mediated actin accumulation precedes and is required for subsequent signalling that leads to cytotoxicity.

This work is supported by a grant from the Canadian Institutes of Health Research (CIHR).
Mechanism of a2V-ATPase in Sterile Inflammation (91.15)
Authors: Kwong Christina , Gilman-Sachs Alice , Beaman Kenneth
Published: January 2023

Key to our understanding the immune response during cell injury is the recognition of the unique mechanism used by the body to detect and respond to cell destruction. We show data that a vacuolar ATPase (a2V) is responsible for detecting aseptic cell injury and directs the activities which can lead to both tissue repair and disease pathogenesis. Rapid clearance of cellular debris is a crucial event to prevent host proteins from being recognized by immune cells to prevent unnecessary inflammation. a2V is a key protein that is up-regulated during sterile inflammation. After membrane receptors detect proximal cell death, a2V migrates to the plasma membrane and the N-terminus (a2NTD) is cleaved and secreted into the extracellular space. a2NTD stimulates primary human monocytes to secrete IL-1β. a2NTD also stimulates THP-1 cells in a concentration-dependent manner to up-regulate pro-inflammatory genes including IL-8, MCP-1 and MMP-9. We show a2NTD binding to monocytes cell surface that is reversible in a competition binding assay via flow cytometry and fluorescent microscopy. These data show involvement of CD14 with a2NTD binding and possible interaction between a2NTD's receptor and the inflammasome. We describe a new pathway by which monocytes respond to tissue destruction. Secretion of a2NTD represents a vital pathway utilized by tissues to initiate immune responses after cell injury.
Mechanisms of social disruption stress induced glucocorticoid resistance (91.16)
Authors: Stiner LaTonia , Padgett David , Sheridan John
Published: January 2023

Social disruption (SDR) stress increases splenic CD11b+ myeloid cells, which produce high levels of proinflammatory cytokines upon Toll-like receptor ligation and show impaired nuclear translocation of the GC receptor (GCr) after corticosterone (cort) stimulation. To uncover the mechanism of GC resistance, p38 MAPK (p38) expression and phosphorylation were investigated along with phosphorylation of the GCr. To examine the mechanism(s) of enhanced proinflammatory cytokine responses, CD11b+ splenocytes from control (HCC) and SDR mice were stimulated with lipopolysaccharide in the presence of cort. SDR and HCC CD11b+ cells show similar levels of unphosphorylated p38 protein. CD11b+ cells from SDR mice exhibited an increase in phosphorylation of p38 and of the GCr. Since phosphorylation of p38 has been linked to GCr phosphorylation in humans, it is likely that a same phenomenon occurs here, though it must be confirmed. Since, phosphorylation of the GCr reduces steroid-induced cytokine repression; this may explain the enhanced cytokine expression. These data show that phosphorylated forms of p38 and GCr play a role in GC resistance.

Support: NIH grants NIMH/RO1MH046801-16 and NIDCR/T32DE014320-6
Itk acts as a negative regulator in mast cell activation (91.19)
Authors: Evans John W , Falk Markus , Schattgen Stefan A , Kosaka Yoko , Berg Leslie J
Published: January 2023

Mast cells are key effectors of allergic and innate immune responses and have been implicated as 'immuno-modulators' in adaptive immunity. Mast cell activation through the immuno-receptor, FceR1, propagates a complex signaling cascade which culminates in a broad range of effector functions. Central to this signal transduction are various protein tyrosine kinase families including members of the Tec family.

The Tec family kinases, Itk and Btk, have been extensively studied in T cells and B cells, respectively, and have been shown to play positive signaling roles. Deficiency in either of these two kinases results in diminished signal transduction downstream of their respective immuno-receptors leading to defects in development as well as effector function.

In contrast to T cells and B cells, mast cells express both Itk and Btk at similar levels. Btk-deficient mast cells exhibit a decreased effector function consisting of reduced degranulation and cytokine production. Furthermore, this phenotype is accompanied by diminished signal transduction downstream of FceR1, as shown by decreased Ca2+ flux and PLCγ phosphorylation.

Here we show that Itk-deficient mast cells display almost the opposite phenotype. These mast cells show enhanced levels of Th2-associated cytokines as well as some pro-inflammatory factors. We show that signal transduction events downstream of FceR1, including Ca2+ flux and PLCγ phosphorylation, are enhanced in the absence of Itk. Additionally, we show that Itk interacts with and phosphorylates the phosphatase, SHIP-1, a negative regulator in mast cells. Finally, we show that Itk co-localizes with a negative signaling complex in wild-type mast cells. Taken together, these data indicate that Itk can impact mast cell activation by dampening the Ag-mediated response. This work suggests a novel role for Itk as a negative regulator in mast cell signaling.
Modulation of the Lipoxygenase Pathway in Bone-Marrow-derived Dendritic Cells by Peroxisome Proliferator Activated Receptor γ. (91.21)
Authors: Hontecillas Raquel , Mysiak Sara , Bassaganya-Riera Josep
Published: January 2023

Enzymes of the lipoxygenase (LOX) family generate lipid mediators that activate the nuclear receptor peroxisome proliferator activated receptor γ (PPAR γ) and regulate immune and inflammatory reactions. GMCSF upregulates the 5-LOX pathway whereas IL-4 suppresses 5-LOX and upregulates 15-LOX expression. The objective of this study was to evaluate whether, in addition of being a molecular target of the lipid mediators generated from arachidonate oxidation by LOX enzymes, PPAR γ regulates the expression of both 5- and 15-LOX. We show that during differentiation of BMDC in GMCSF and IL-4, the loss of PPAR γ resulted in upregulation of 15-LOX. Phenotypically, PPAR γ deficient BMDC differentiated in GMCSF and IL-4 expressed more CD40 and less CD36 than PPARγ-expressing cells. Also, PPAR γ-defficient cells differentiated in the presence of GMCSF alone had more 5-LOX mRNA than PPAR γ-expressing cells, which was paralleled by upregulation of PPAR δ. The loss of PPAR γ did not change the expression of 5-LOX activating protein. LPS stimulation of mature BMDC induced secretion of LTB4, a product of the 5-LOX pathway, in cells differentiated in GMCSF although the loss of PPAR γ did not have any effect on the amount of this lipid mediator released. Our results suggest that a negative feed-back loop is initiated following activation of PPAR γ that regulates further generation of more endogenous ligands.
Validation and application of a multiplex immunoassay for the detection of PGE2 induced inflammatory mediators of human decidual macrophages (92.15)
Authors: Casper John M , Senn Tennille , Cox Jennifer , Black Eric , Aronoff David M. , Mullenix Michael M.
Published: January 2023

A multiplex bead immunoassay was developed for the simultaneous quantitation of 8 inflammatory mediators including 6 cytokines (IL-1 β, IL-4, IL-6, IL-8, TNFα and IFNγ) and 2 eicosanoids (PGE2, and TXB2). The sensitivities of the cytokine assays are less than 5 pg/mL with the exception of IL-4 (19.9 pg/mL). The sensitivity of the PGE2 and TXB2 assays at 80% B/Bo are 64.2 and 65.9 pg/mL for PGE2 and TXB2 respectively. The antimicrobial actions of human decidual macrophages (DMs) isolated from the upper reproductive tract were characterized in the assay. Decidual macrophages are innate immune cells known to defend the upper reproductive tract from infection. PGE2 is a potent regulator of innate immunity in the reproductive tract during pregnancy and/or in response to infection. CD14+ DMs were incubated with bacteria, peptidoglycan or LPS and analyzed for the expression of the inflammatory mediators detected in the MultiBead(tm) multiplex immunoassay. The DMs produced PGE2 in response to bacteria, peptidoglycan or LPS with increased production of IL-8, TNFα, IL-6 and IL-1 β. The addition of 1 μM PGE2 decreased expression of TNFα and IL-1β and increased the expression of IL-6. The DMs produced PGE2 in response to inflammatory stimuli and modulated the expression of cytokines and eicosanoids in vitro. We conclude that PGE2 is involved in immunomodulation that may be relevant when PGE2 levels are increased during pregnancy and infection.
Bone marrow Ly6Chi Monocytes are recruited to injured kidney and differentiate into Ly6Clo profibrotic macrophages (92.7)
Authors: CASTANO ANA P , Lin Shuei Liong , Duffield Jeremy  S
Published: January 2023

Macrophages (M(s) play decisive roles in different models of organ fibrosis. Prevailing concepts support circulating monocyte heterogeneity, subpopulation discrete recruitment into injured tissues, differentiation into M(s, and possibly subpopulation discrete roles in injury and repair. Here we show discrete subpopulations of M(s in fibrotic kidney induced by unilateral ureteral obstruction (UUO) and their discrete functional roles in renal fibrosis. Fluorescence-activated cell sorting (FACS) of purified UUO kidney leukocytes reveals 3 populations of M(s separated by Ly6C: high, intermediate, and low. FACS of peripheral blood mononuclear cells (PBMC) showed a marked increase in Ly6chi-Mo in response to kidney injury. Depletion of M(s in CD11b-DTR mice during UUO fibrosis inhibits the progression of fibrosis by an indirect mechanism. However, Ly6clo kidney M(s were selectively depleted indicating their predominant role in fibrogenesis. To determine the origins of kidney M(s, we lineage-traced 3 different populations of monocytes (Mo) by adoptive transfer of CD45.1+ bone marrow (BM)-Ly6chi Mo or peripheral blood (PB) Ly6chi Mo or PB-Ly6clo-Mo into CD45.2 mice with UUO. Ly6Chi BM-Mo were avidly recruited to injured kidney, and differentiated into Ly6Chi, Ly6Cint, and Ly6Clo kidney M(s. PB-Mo also differentiated into 3 populations but 20-fold lower recruitment was observed. We characterized kidney-M(-subpopulation transcript levels by branched-chain-DNA-technology which demonstrated discrete differences in expression of genes including TNF(, IL1(, MIP2, MIP1(. We conclude that Ly6Chi BM-Mo are recruited to kidney by mobilization from BM, differentiate into 3 functionally discrete kidney M(s one of which is profibrotic, and resident M(s play a non-fibrogenic role in kidney injury.
CD34 is required for the infiltration of inflammatory cells into the colon in a mouse model of ulcerative colitis (93.2)
Authors: Maltby Steven J , Wohlfarth Carolin , McNagny Kelly , Hughes Michael R
Published: January 2023

The infiltration of eosinophils into gut tissue and release of granular mediators appears to play a critical role in the pathogenesis of inflammatory bowel diseases (IBD) such as ulcerative colitis. Using an allergic asthma model, we recently demonstrated that eosinophil migration into the lung requires surface expression of the sialomucin CD34 on mast cells and eosinophils. Based in part on these findings, we have investigated a similar role for CD34 in the migration of eosinophils and other inflammatory cells into the colon, and the effects of CD34 deletion on IBD development using a dextran sodium sulfate (DSS)-induced model of ulcerative colitis. We monitored weight loss, colon length, rectal bleeding and diarrhea and our findings demonstrate decreased disease severity in DSS-treated Cd34-/- mice. Post-mortem histology of DSS-treated mice also showed reduced inflammation of Cd34-/- colon compared to wild type controls. Interestingly, the percentage of peripheral blood eosinophils was reduced significantly more in wild type than Cd34-/- mice at the disease endpoint - corroborating a defect in the exit of eosinophils from circulation into the gut. These findings support the conclusion that CD34 plays a role in eosinophil migration into gut tissues and suggest that CD34 may serve as a viable target for pharmacological interventions across a range of inflammatory conditions.
Differential gene expression profiles in the spontaneously hypertensive rats administrated with anti-hypertensive drugs (93.20)
Authors: Park Min , Lee Kee-Mo , Kim Eun-Mi , Park Yong-Wook , Kee Seung-Jung , Kang Hyung-Sik
Published: January 2023

To identify and characterize the genes involved in the functional regulation of hypertension and arteriosclerosis-specific genes, changes in gene expression profiles were examined by DNA microarray from the spontaneously hypertensive rats (SHR) and normal the Wistar-Kyoto (WKY) rats after administration with anti-hypertension drugs, including angiotensin I-converting enzyme (ACE) inhibitor, enalapril and the calcium channel blocker, nifedifine. Among the genes that were distinct and differentially expressed in brain, heart and liver of SHR rats administrated with the drugs, 113 were specifically up-regulated, and 102 were down-regulated genes compared to those of WKY or SHR rats. The differential gene expression profiles in DNA microarray were corroborated by semi-quantitative RT-PCR. Of these genes, expression of TNFSF4, 6, 10, IL-6 receptor, IL-18 receptor accessory protein and CD24 genes was extensively exhibited in a hypertension-specific manner, and patterns for hypertension-associated gene expression was regulated by changes of their expression in angiotensin ¥± or H2O2-treated vascular smooth muscle cells.

These results will provide a fundamental basis to delineate the novel mechanisms, and a thorough investigation of the relationship between their expression and function will provide a better understanding in the development of new therapeutic drugs against cardiovascular diseases.

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2006-005-J03003)
NFkB and IkB expression are modulated by sex steroids during alcohol liver inflammation (93.24)
Authors: Lee Eric G , Mickle Bethany , Chastain Lerin , Gallucci Randle
Published: January 2023

Women are more susceptible to alcoholic liver disease (ALD) than men. The release of cytokines from Kupffer cells during inflammation is associated with the pathogenesis of ALD, and chronically ethanol fed female rats express cytokine mediators differently than male counterparts. Differences in inflammatory gene expression in females may have a role in gender related ALD pathology. This study examines the effects of sex steroids on the NFkB and IL-6 pathways in chronically ethanol fed rats. Ovarectomized, female, Wistar rats implanted with osmotic pumps releasing estrogen or testosterone were fed liquid diets with and without ethanol for two and four weeks. Estrogen receiving animals had lower mRNA expression of IL-6Rα, SOCS-3, PIAS-3, C/EBPβ, IkBα, and IkBβ at 2 weeks with little difference observed at 4 weeks. Ethanol consumption induced lower mRNA expression of IL-6Rα, PIAS-3, IkBα, and IkBβ in both testosterone and estrogen receiving animals with the lowest levels consistently observed in the estrogen group. Nuclear NFkB was greatest in the estrogen groups and lowest in the ethanol consuming testosterone group. IkBα protein expression was greatest in the estrogen receiving groups. The combined effects of estrogen and ethanol resulted in the lowest mRNA expression and highest NFkB and IkBα protein expression. Our data suggest that testosterone and estrogen distinctively affect NFkB, IkBα, and IL-6R expression and function and the effect of testosterone may be protective in ALD.

Funded by NIH/NIAAA grant R03AA015198
Presence of the functional Caspase-12 allele in Indian subpopulations (93.25)
Authors: Hermel Evan , Klapstein Kevin D , Yavari Mehdy
Published: January 2023

In rodents, caspase-12 (casp12) is a negative regulator of interleukin-1 production. Most humans lack a functional CASP12 gene, with a non-functional variant (CASP12p1), found in 100% of the Caucasian and East Asian population, and in approximately 80% of people of African descent. However, 20% of sub-Saharan Africans carry an intact allele of CASP12, which produces a full-length non-catalytic proenzyme and is associated with an increased risk of sepsis. Using PCR-based single nucleotide polymorphism analysis and DNA sequencing, we examined CASP12 allele distribution in individuals from Central and Southern Asia and found that CASP12 was significantly present in members of the Dravidian language group (the predominant ethnic and language group of southern India), particularly persons from Tamil Nadu. The CASP12 allele also occurs at a higher frequency in individuals with the Australoid morphotype that predominates in southern India, as compared to those with the Caucasoid morphotype prevalent in central and northern India. The data suggest that there is a north-to-south gradient of increasing prevalence for the CASP12 allele. As the CASP12p1 allele has been rigorously selected for in populations outside of Africa and southern Asia, we hypothesize that a pathogen that exploits the inflammatory immune response in Africa and southern India may be exerting positive selective pressure on CASP12.
The role of inflammation in diet-induced obesity (93.4)
Authors: Alexander Lindsey A , Slotterbeck Brandon D , Najjar Sonia M , McInerney Marcia F
Published: January 2023

Metabolic syndrome, affecting up to 25% of Americans, includes impaired glucose tolerance, central obesity, dyslipidemia, insulin resistance, and type 2 diabetes. Secondary complications include atherosclerosis, stroke, fatty liver disease, blindness, and others. Our current research investigates the role of inflammatory cells, adipocytes, Toll-like receptors (TLR), and cytokines in the metabolic syndrome. We have studied two mouse models of impaired hepatic insulin clearance leading to insulin resistance. In the L-SACC mouse model, CEACAM1, a molecule necessary for liver insulin receptor endocytosis, contains a point-mutation which abolishes an essential phosphorylation site (S503A). This dominant-negative CEACAM1 is targeted to the liver only. We have also examined CEACAM1 knock-out (Cc1 KO) mice, wherein CEACAM1 is absent from all tissues. We have induced obesity by feeding L-SACC, Cc1 KO, and wild-type mice either a high-fat or high-fat / high carbohydrate (Western) diet and compared the effects to regular diet-fed mice. We have observed dysregulated TLR, cellular, and cytokine responses by qPCR, serum analysis, and histological methods. We conclude that these mouse models of impaired insulin clearance will be valuable tools for investigating abnormal cellular responses in obesity and type 2 diabetes. This research is supported by the United States Department of Agriculture.
Nuclear factor-kappaB provides mechanistic link between inflammation and prostate cancer (93.8)
Authors: Vykhovanets Eugene , MacLennan Gregory T , Vykhovanets Olena , Gupta Sanjay
Published: January 2023

The mechanistic link between chronic inflammation and prostate cancer (PCa) is missing, despite accumulating evidence suggests a role for inflammation in the etiology of this disease. We hypothesized that nuclear factor-kappaB (NF-κβ) activation might result in the production of signals for mononuclear cell (MNC) accumulation into the prostate. We used transgenic NF-κB-Luc Tag mice that demonstrate the highest levels of NF-κB-dependent luminescence in vivo and ex vivo at 4 h after i/v IL-1β administration. In these mice, IL-1β-induced NF-κB activation correlated with MNC trafficking, expression of chemokines and IL-17 in the prostate. Next we determined the possibility of IL-17 producing cells to be targets of specific pro-inflammatory signals that initiate inflammation at sites of prostate inflammatory atrophy (PIA) in PCa patients. To immunophenotype the inflammatory cell infiltrates in formalin-fixed paraffin-embedded whole mount radical prostatectomy specimens two-color staining was employed. MNC were the predominant IL-17 producing cells in PIA lesions. The number of IL-17 positive cells in the acini of PIA lesions was 5-fold higher than in PCa areas; and 3-fold higher than around PCa or other pathologic patterns studied. Taken together, our findings support the hypothesis that NF-κB provides a mechanistic link between cell-mediated inflammation, PCa development and its subsequent progression.
Nitric Oxide and Carbon Monoxide mediate the anti-inflammatory effects of Remote Ischemic Preconditioning (RIPC) (94.12)
Authors: simao antonio  felipe , Souza-Filho Marcos  V P , Souto Fabricio Oliveira , Cunha Fernando Queiróz , Ribeiro Ronaldo Albuquerque
Published: January 2023

Remote ischemic preconditioning(RIPC) consists of short periods of ischemia followed by reperfusion, which induces protective anti-inflammatory effect on distance organs. Studies have shown that inflammatory parameters such as leukocyte migration are significantly reduced in pre-conditioned animals.However, the mechanism involved in this effect is not yet well established. This study evaluated if NO and CO is mediating the anti-inflammatory activity of RIPC.RIPC model is based on turnstile in right leg of a mouse for 10 minutes followed by 30 of reperfusion. Participation of NO and CO was investigated using inhibitors of iNOS (1400W; 3mg/kg or Aminoguanide; 50mg/kg) and HO-1 (ZnPPIX;10mg/kg) as pre-treatment 30 minutes before RIPC. Afterwards, inflammatory reaction was induced by intraperitoneal administration of Carragenin(500µg/cav).Four hours after the peritoneal cavities were washed with saline and number of migrated leukocytes was evaluated. Chemotaxis ability of neutrophils obtained from mice under RIPC treated or not with the inhibitors was analyzed in Boyden Chamber. Expression of CXCR2 and GRK2 in neutrophils was determined by flow cytometry and immunofluorescence, respectively.CO and NO inhibitors prevented the inhibitory effect of RIPC upon Cg-induced neutrophil migration into peritoneal cavity of animals, as well as, upon KC induced neutrophils chemotaxis in Boyden Chamber. The reduction of neutrophil chemotaxis by RICP correlated with CXCR2 internalization and increase of GRK2 expression in the neutrophils.CO and NO production prevented these effects of RICP.Thus,inhibitors of HO-1/CO and NOSi/NO pathways seem to mediate the anti-inflammatory effect of RICP.
A novel method to evaluate inhibitors of cell adhesion using physiological conditions generated using microfluidic flow cells (94.14)
Authors: Conant Carolyn G , Schwartz Michael , Ionescu-Zanetti Cristian
Published: January 2023

Development and assessment of cell adhesion inhibitors are fundamental to allergy, asthma, clotting, neurodegeneration, and cancer drug research and discovery. Here, we present a cell-based screen of adhesion inhibitors directed against VLA-4, a molecule found on the surface of most leukocytes and key to the inflammatory process. Reduction of VLA-4 mediated adhesion to the ligand, VCAM-1 , was evaluated using a micro-scale flow cell system designed to run automated shear flow protocols for cell biology experiments in high throughput. The screen was performed under physiologically-relevant shear flow conditions of 0.8 dyn/cm2. The flow was also exploited to streamline the experimental workflow and mitigate effects of inconsistent washing as is the case with such experiments performed in microplates using pipet based washing. The time to perform the entire experiment from coating the plate with recombinant VCAM-1 to manual data collection was reduced to under 3 hours compared to 10 hours for a standard microplate adhesion inhibition assay. Cell adhesion images were captured using a standard inverted microscopy setup and data were analyzed using proprietary software. Using three inhibitors against VLA-4, we showed a dose-dependent decrease in cell adhesion in two of the three compounds leading to a complete block of adhesion. IC50 values were determined to be 600pM and 1/32000 respectively. The third compound's IC50 was 10nM although complete inhibition was never observed; as well, homotypic cell aggregation was observed at a concentration of 1nM. Homotypic aggregation would not typically be observed in a microtiter plate adhesion assay screened for bulk fluorescence in a plate reader.
New Applications for Bispecific Antibodies in Wound Healing (94.16)
Authors: Cousens Leslie  P. , Terry Frances E , Falanga Vincent
Published: January 2023

Bispecific antibodies (BiAbs) have potential to confer injury-specific targeting to cells for enhancing wound repair. Studies are designed to test BiAbs for their ability to direct recruitment and trafficking of specific cell populations (i.e. stem cells or macrophages) to sites of injury. A new series of BiAbs for wound repair applications have been chemically heteroconjugated: monoclonal antibodies (mAbs) for specific cell populations (i.e. F4/80 on macrophages, or c-kit on stem cells) were linked to mAbs against injury-associated antigens (i.e. VCAM, fibrinopeptide A). Heteroconjugation efficiency was measured by SDS-PAGE/densitometry, demonstrating final product composition of 66.75%, 58.76%, and 24.80% active heteroconjugates of F4/80xFibA, F4/80xVCAM, and cKitxVCAM, respectively. Specific BiAb binding to macrophage or stem cell populations was observed by flow cytometry. Functional BiAb binding was quantified in a novel biological assay. With these three distinct BiAbs successfully heteroconjugated and validated in vitro, studies are underway to test trafficking of BiAb-armed cells to injured tissue in murine models of wound repair: full thickness skin wounds and external muscle crush injury.
CalDAG-GEF-I is required for E-selectin-dependent slow leukocyte rolling (94.2)
Authors: Zarbock Alexander , Mueller Helena , Bergmeier Wolfgang , Lowell Clifford A. , Van Aken Hugo , Wagner Denisa D. , Ley Klaus
Published: January 2023

During leukocyte recruitment, leukocytes are exposed to different adhesion molecules and chemokines. E-selectin binding to PSGL-1 and chemoattractant binding to Gαi-coupled receptors can modulate the rolling velocity by activating β2-integrins. The guanine nucleotide exchange factor Ca2+ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEF-I) catalyzes GDP for GTP exchange in Rap1 and 2 and thereby controls the activation of β-integrins. CalDAG-GEF-I-/- neutrophils have an adhesion and migration defect in response to chemokine activation. To test the role of CalDAG-GEFI in E-selectin-mediated slow rolling, we investigated calDAG-GEF-I-/- mice by intravital microscopy of the cremaster muscle and in autoperfused flow chambers. In the flow chamber, rolling velocity of neutrophils from WT mice on E-selectin was 2.2 µm/s and adding ICAM-1 to E-selectin reduced rolling velocity to 1.0 µm/s. This velocity reduction was partially abrogated in calDAG-GEF-I-/- mice, suggesting that this molecule is involved in E-mediated slow rolling. Mixed chimeric mice confirmed that calDAG-GEF-I-/- leukocytes had an elevated rolling velocity in TNF-α-induced inflammation in vivo. The physiologic importance of our findings is shown by near complete inhibition of neutrophil recruitment into the inflamed peritoneal cavity of calDAG-GEF-1-/- leukocytes treated with pertussis toxin to block Gαi signaling. These data show that calDAG-GEF-1 partially mediates slow rolling following E-selectin engagement.