bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasoactive intestinal peptide 10-28 enhances natural killer cell cytotoxicity (134.84)
Authors: Suck Garnet , Veljkovic Veljko , Chu Sixian , Niam Madelaine , Tan Suet Mien , Branch Donald R. , Koh Mickey B. C.
Published: January 2023

Objective: Vasoactive intestinal peptide (VIP) and its synthetic analog VIP14-28 enhance NK cytotoxicity. This prompted us to study interaction of the naturally occurring peptide and orphan ligand VIP 10-28 with NK.

Results: VIP10-28 stimulation (10-8-10-10 M, 30 min.) increased NK-92 cytotoxicity against K562 max. from 52% +/-2.6% SEM (control) to 68% +/-2.6% SEM (E:T 1:1), similar to long-term IL-2 stimulated NK from different donors (4/6), max. from 38% +/-4.2 SEM (control) to 51% +/-0.9% SEM (E:T 1:1). VIP10-28 did not seem to induce degranulation, since granzyme B levels remained unchanged in NK-92. However, stimulation with 10-8-10-10 M VIP10-28 for 5- 60 min. significantly induced tyrosine phosphorylation with ERK identified as one of the stimulated kinases. We used Informational Spectrum Method (ISM) to predict VIP10-28 NK receptor interactions. ISM results suggested potential binding of VIP10-28 to LFA-1α (F = 0.03, peptide residues 16-24) and with lower affinity to CD44 or MAC-1, but unlikely to NKG2D, -C, -A, NKp30, -44, -46, CD56, CD94, KIR2DL4, NKR-P1, CD45 or VIP receptors VPAC-1/ VPAC-2.

Conclusion: The neuroendocrine peptide VIP10-28 induces increased NK cytotoxicity potentially through LFA-1 signaling pathways involving tyrosine kinase activation of ERK kinases.
Enhanced immunity following ingestion of plant derived polysaccharides (134.87)
Authors: Hedges Jodi F. , Rask Kerri M. , Jutila Mark A.
Published: January 2023

Considering the recent increased interest in dietary supplements by the public, characterization of their mechanisms and safety are warranted. Yamoa(tm), bark from the Funtumia elastica tree, has anecdotal positive effects in asthma patients. We have determined that polysaccharide components of Yamoa(tm) and other dietary supplements stimulate innate immunity similarly to LPS, in part by affecting γδ T cells. We hypothesized that orally administered Yamoa(tm) would effectively target this mucosal T cell subset and result in changes in peripheral and tissue-associated immune cells, and provide benefit in mucosal disease settings. Increases in an inflammatory subset of γδ T cells, and B cells were detected in peripheral blood in bovine calves fed Yamoa(tm). Cannulation of the efferent lymphatic ducts of the gut in calves was used to analyze the responses of tissue-derived cells after ingestion of Yamoa(tm). Therapeutic treatment of mice by i.p. injection of Yamoa(tm) and its derived polysaccharides was effective in reduction of fecal CFUs in a mouse model of Salmonella-induced enterocolitis. Interestingly, whereas long term ingestion of low doses of polysaccharides had neutral or negative effects on CFUs in this model, a single prophylactic oral dose of Yamoa(tm) resulted in decreased CFUs. These data suggest that the practical and relevant route of ingestion of plant-derived polysaccharides can affect innate immunity.
How is the diacylated lipopeptide FSL-1 processed after recognition by TLR2 ? (135.1)
Authors: Haque Mohammad Shamsul , Hasebe Akira , Iyori Mitsuhiro , Kiura Kazuto , Ohtani Makoto , Shibata Ken-ichiro
Published: January 2023

The diacylated lipopeptide FSL-1 is recognized by TLR2/TLR6, but it still remains unclear how FSL-1 ligands are processed after recognition. Nystatin had no effect on the FSL-1 uptake by macrophages, whereas both chlorpromazine and methyl-β-cyclodextrin inhibited the FSL-1uptake. Intenalization of FSL-1 was downregulated by knockdown of clathrin mRNA. These results suggested that FSL-1 was found to be internalized into the cells by a clathrin-dependent endocytosis. In addition, it was found that FSL-1 was not colocalized with TLR2 in the cytosol, and there were no differences in the level of FSL-1 uptake by peritoneal macrophages from TLR2+/+ and TLR2-/- mice. FSL-1 stimulation induced downregulation of the surface expression level of TLR2, suggesting that FSL-1 stimulation is required for the internalization of TLR2. Thus, this study demonstrated that FSL-1 was internalized into macrophages in a clathrin-dependent manner and TLR2 was not required for the internalization, although FSL-1 stimulation was required for internalization of TLR2. In addition, HEK 293 cells stably expressing CD14 or CD36, but not HEK293 cells, were involved in the uptake of FSL-1 by macrophages, suggesting that they function as an endocytic receptor for FSL-1 as well as a coreceptor of TLR2.
Activation of MyD88-mediated Signaling by MHC Class II Ligands Stimulates Inflammation (135.22)
Authors: Fernandez Stefan , Kissner Teri L , Ruthel Gordon T , Ulrich Robert G , Kamal Saikh U
Published: January 2023

Agonists binding to Toll-like receptor (TLR) or interleukin 1 receptor (IL-1R) activate MyD88-mediated signaling, which leads to pro-inflammatory responses. Here we report that MyD88 also mediates a component of intracellular signaling initiated by MHC class II, receptor-ligand interactions. Treating human primary monocytes with staphylococcal enterotoxin A (SEA), SEB, or anti-MHC class II monoclonal antibodies up-regulated MyD88 expression, increased expression of IL-1R1 accessory protein, induced activation of NF-kB and the subsequent release of pro-inflammatory cytokines. Similar to MHC class II activation of monocytes in isolation, (i.e., in the absence of T cells) MyD88-mediated pro-inflammatory cytokine expression was observed in primary monocytes selected after stimulation of peripheral blood mononuclear cells with SEA or SEB. Consistent with these results, increased secretion of pro-inflammatory cytokines was also observed in MHC class II + murine spleen cells. This effect was drastically reduced in MHC class II+, MyD88-deficient spleen cells. Importantly, stimulation of primary human monocytes or monocytic cell lines with SEB or lipopolysaccharide induced translocation to the membrane, de novo synthesis, and tyrosine phosphorylation of MyD88. These results demonstrate a MyD88-mediated signaling mechanism for MHC class II, which induces expression of genes encoding pro-inflammatory cytokines.
Regulation of Macrophage Activation by a Novel Splice Variant of the Voltage-gated Sodium Channel NaV1.5 (135.27)
Authors: Carrithers Michael , Carrithers Lisette
Published: January 2023

Novel splice variants of the voltage-gated sodium channels, NaV1.5 and NaV1.6, are expressed in human monocyte-derived macrophages and THP-1 cells, a human monocytic cell line. Our prior work demonstrated that macrophage NaV1.5 localizes to the late endosome in primed human macrophages and differentiated THP-1 cells and regulates endosomal acidification and phagocytosis. Here we show that macrophage NaV1.5 regulates intracellular signaling associated with cell priming. THP-1 cells that express NaV1.5 (wild type and control shRNA cells) and those that do not (NaV1.5 shRNA-mediated knockdown) were differentiated with phorbol ester for 3 days and then primed sequentially with interferon-gamma and LPS. Analysis of global mRNA expression using Affymetrix oligonucleotide arrays demonstrated decreased expression of several inflammatory response genes and increased expression of a pro-survival growth factor in NaV1.5 knockdown cells. These results were confirmed by real time PCR analysis. In addition, shRNA-mediated knockdown of macrophage NaV1.5 inhibited LPS-mediated activation of the transcription factor NFkappaB and stimulation-induced apoptosis. Preliminary experiments suggest that knockdown of NaV1.5 prevents a decrease in cytosolic calcium that is necessary for optimal activation of NFkappaB. These results suggest that a variant of human NaV1.5 may regulate intracellular signaling during macrophage priming.

This work is support by an award from the National Multiple Sclerosis Society (RG-3888) and the National Institutes of Health (K02-NS047457-01).
Mycobacteria induce the inactive form of cyclooxygenase-2 (COX-2) without inducing endoplasmic reticulum (ER) stress responses in local macrophages (Mνll]) (135.29)
Authors: Pantuso Traci , Kogiso Mari , Shinohara Tsutomu , Henriksen Ruth Ann , Dorey C. Kathleen , Shibata Yoshimi
Published: January 2023

In response to pathogens, activated MØ frequently express COX-2, which mediates increased release of prostaglandin E2 (PGE2), regulating host defense and inflammatory responses. We previously demonstrated, however, that i.p. administration of HK- Mycobacterium bovis BCG or M. tuberculosis resulted within 24h in increased COX-2 formation, which was subcellularly dissociated from the nuclear envelope (NE), enzymatically inactive and mediated no PGE2 release in peritoneal MØ. In contrast, when normal peritoneal MØ isolated from mice or RAW264.7 MØ were activated for 24h in vitro by BCG, they expressed increased COX-2, which was subcellularly localized in the NE and mediated increased release of PGE2. To assess whether production of inactive COX-2 is associated with ER stress-dependent signaling, we measured selected ER stress protein levels in peritoneal MØ from C57Bl/6 mice and RAW264.7 MØ that were treated with BCG in vitro or in vivo. Literature indicates that in vivo administration of endotoxin has been shown to up-regulate ER stress responses. We found that ER stress proteins growth arrest and DNA-damage-inducible gene 153, glucose regulated protein 78, and the phosphorylation of eukaryotic initiation factor two were up-regulated in RAW264.7 MØ when stimulated with endotoxin, tunicamycin or thapsigargin. However, no detectable changes in ER stress proteins were found in BCG-treated RAW264.7 MØ, in vitro BCG-treated peritoneal MØ or in vivo BCG-treated peritoneal MØ. Our results indicate that mycobacterial activation of MØ COX-2 expression in vivo or in vitro does not involve ER stress/unfolded protein responses. (Supported by NIH HL71711, DOD DAMD 17-03-1-0004, BCCRP 06BS-04-9615)
TLT2 expression and function are conserved between mouse and human (135.37)
Authors: Thomas Kimberly A , King R Glenn , Justement Louis B
Published: January 2023

Trem-like transcript 2 (TLT2) is an innate immune receptor belonging to the TREM family, but unlike other TREM receptors, it is also expressed on cells of the lymphoid lineage. Murine TLT2 (mTLT2) is expressed on neutrophils, macrophages, and B cells, but not on monocytes or T cells. Within the B cell compartment, mTLT2 expression is highest on MZ and B1 cells, followed by T1/T2 cells, and then follicular B cells. Expression of mTLT2 is upregulated on neutrophils and macrophages, but not B cells, in response to inflammatory stimuli. Ligation of mTLT2 on B cells results in enhanced migration towards various chemokines in vitro. Human TLT2 (hTLT2) and mTLT2 exhibit 60% homology, but little is known regarding hTLT2 expression and function. Therefore, studies were performed to characterize its expression profile and function. Neutrophils, macrophages, and B cells express hTLT2, but not T cells. In contrast to mouse, hTLT2 is expressed on human monocytes at levels comparable to macrophages. A hierarchy of hTLT2 expression is observed in the B cell compartment in which hTLT2 expression is highest on mature, naïve B cells, and decreases upon activation and participation in germinal center reactions. Human B cells and neutrophils exhibit enhanced migration towards chemokines upon hTLT2 ligation in vitro, similar to that observed in mouse. Thus, TLT2 expression patterns and function are largely conserved between human and mouse.
TLR4/MD-2 and RP105/MD-1 differentially regulate LPS responsiveness in B cells (135.4)
Authors: Nagai Yoshinori , Ohta Shoichiro , Miyake Kensuke , Takatsu Kiyoshi
Published: January 2023

[Aims] TLR4/MD-2 is an essential complex for LPS recognition. RP105/MD-1 is a homologue of TLR4/MD-2 and is expressed in mainly B cells. RP105 or MD-1-deficient B cells show impaired LPS responsiveness, suggesting that B cells require both TLR4/MD-2 and RP105/MD-1 signals for LPS responses. However, precise mechanisms that functionally couple with the two types of complex remain unclear. Furthermore, as LPS stimulation activates B cells through both receptors, it is difficult to analyze the signals via TLR4/MD-2 and RP105/MD-1 differentially. To resolve this, we used agonistic mAbs to TLR4 and RP105 to explore differential roles for TLR4/MD-2 and RP105/MD-1 in B cell responses to LPS. [Methods] Mouse splenic B cells were stimulated with αTLR4 mAb, αRP105 mAb, or LPS in combination with IL-4. Expression of CD86, BAFF receptors (TACI, BAFF-R, BMCA), and CD138 was analyzed by FACS. For proliferation analysis, the B cells were stimulated with vehicles for 3 days and pulsed with 0.5 μCi [3H]-TdR. The μ to γ1 class switch recombination (CSR) was examined by analyzing the proportion of surface IgG1+ cells by FACS. [Results and Discussion] The αTLR4 mAb and αRP105 mAb induced massive B cell proliferation like as LPS. Expression of CD86 and BAFF receptors was induced by low doses of αRP105 (10 ng/ml), whereas those expression was very low by αTLR4 stimulation even at high doses (1 μg/ml). Combination of αTLR4 + IL-4 as well as LPS + IL-4 forced B cells to induce sIgG1+ cells. In contrast, αRP105 + IL-4 never showed this activity. CD138 expression was induced on B cells stimulated with αTLR4 but not with αRP105. These findings suggest that TLR4 and RP105 differentially regulate B cell response to LPS.
IFN-γ and IL-4 differentially regulate TLR-triggered induction of antimicrobial peptides by altering vitamin D metabolism (135.49)
Authors: Edfeldt Kristina , Liu Philip T. , Chun Rene , Krutzik Stephan R. , Adams John S. , Hewison Martin , Modlin Robert L.
Published: January 2023

Innate immunity is important for host defense against microbial infections and vitamin D has been shown to play a role in this response. Vitamin D is required for Toll-like receptor (TLR)-triggered induction of the antimicrobial peptides cathelicidin and DEFB4, but how acquired immunity influences this system is not known. The objective of this study was to assess the role of acquired immune cytokines on TLR-triggered vitamin D-dependent induction of antimicrobial peptides.

We showed that acquired immune cytokine responses influence antimicrobial pathways in innate immunity by regulating vitamin D metabolism. The Th1 cytokine IFN-γ induced bioconversion of 25-hydroxyvitamin D3 (25D3) to its active metabolite 1,25-dihydroxyvitamin D3 and resulted in a subsequent increase in cathelicidin mRNA in cells treated with a TLR2/1 ligand. In contrast, the Th2 cytokine IL-4 lead to induction of 25D3 catabolism by inducing 24-hydroxylase enzyme activity and consequently inhibited TLR2/1-triggered induction of the antimicrobial peptides cathelicidin and DEFB4.

In conclusion our results support a role for acquired immune cytokines in regulating vitamin D-dependent antimicrobial pathways in innate immunity.

KE is a Wenner-Gren Fellow
Long-term biphasic effects of estrogen exposure on murine bone-marrow derived macrophage TLR4 expression (135.52)
Authors: Rettew Jennifer A , Huet Yvette M , Marriott Ian
Published: January 2023

Numerous studies have shown that estrogens augment immune responses and have been demonstrated to increase resistance to bacterial infections. In addition, it has been shown that production of inflammatory cytokines following administration of LPS is attenuated in ovariectomized animals, which is reversed following exogenous estrogen administration. We have recently demonstrated that chronic in vivo exposure to estrogen, but not acute in vitro exposure, augments Toll-like receptor 4 (TLR4) expression on peritoneal macrophages although the mechanisms that underlie this disparity are unclear. In this study we have investigated the effect of in vivo removal of endogenous estrogen and high dose estrogen replacement on TLR4 expression on bone-marrow derived macrophages and/or their progenitor cells. We report that these cells isolated from ovariectomized animals that received high dose estrogen treatment have significantly less (~20%) cell surface TLR4 expression than those derived from sham treated mice or untreated gonadectomized animals following 7 days in culture. Importantly, this effect was not reflected by changes in total cell TLR4 content suggesting that estrogen does not affect TLR4 production. However, longer-term (14-16 day) culture of these cells resulted in an apparent reversal of this pattern with restoration of TLR4 expression on bone-marrow derived macrophages isolated from estrogen treated animals and reduction in the expression of this pattern recognition receptor on cells from untreated gonadectomized mice in a manner that is consistent with our previous observations in acutely isolated peritoneal macrophages. Interestingly, in vitro treatment of bone-marrow derived cells with varying doses of estrogen failed to mimic these effects suggesting that this reproductive hormone exerts its effects prior to macrophage maturation.
TLR agonists synergize with CD40L/IFN-γ to promote human dendritic cell synthesis of IL-12 (135.65)
Authors: Huang Alex , Carreno Beatriz M. , Linette Gerald P.
Published: January 2023

CD40L provides a critical signal to license DC for the effective generation of T cell memory. To investigate additional signals that might synergize with CD40L, we tested TLR agonists to promote IL-12 production by DC matured with the combination of CD40L/IFN-γ. Human monocyte-derived immature DC established by standard 6 day culture in GM-CSF/IL-4 were activated with human CD40L/IFN-γ plus optimal concentrations of individual TLR agonists for an additional 48 h and the supernatants were assayed for IL-12 p70 by ELISA. TLR 1/2, 4, 5, 6, and 8 agonists each resulted in significant increases in IL-12 production as compared to CD40L/IFN-γ alone with mean fold increases of 2.3, 4.0, 2.1, 3.5, and 8.8 respectively. TLR agonists 3, 7, and 9 had no effect. To further assess the potential synergy of TLR agonists with CD40L/IFN-γ mDC, we studied the capacity of CMV pp65 NLV peptide-pulsed mDC to stimulate antigen-specific CD8+ T cells from normal CMV sero-positive donors. The antigen-specific CD8 response as measured by tetramer-fold increase was significantly higher at day 10 using TLR/CD40L/IFN-γ activated mDC compared to conventional CD40L/IFN-γ mDC. We conclude that multiple maturation signals are required to license DC for the optimal generation of T cell memory.
Granulysin induces recruitment and activation of antigen presenting cells and acts as an immune adjuvant and alarmin (135.69)
Authors: Tewary Poonam , Yang De , de la Rosa Gonzalo , Li Yana , Krensky Alan , Clayberger Carol , Oppenheim Joost J
Published: January 2023

Granulysin (GNLY) is a 9-KD antimicrobial protein present in the granules of CTLs and NK cells. It kills a variety of pathogens, possesses antitumor activity and is expressed in diverse disease settings. Recently it was shown GNLY chemoattracts and activates monocytes. Here we demonstrate that GNLY (1) induces directional migration of monocyte derived DCs, (2) activates human dendritic cells (DCs) leading to the upregulation of phenotypic markers and production of proinflammatory cytokines and (3) induces functional maturation of DCs with the capacity to stimulate a mixed lymphocyte reaction (MLR). Intraperitoneal injection of human GNLY in mice causes a marked recruitment of inflammatory leucocytes and subsequent immunization with GNLY and OVA promotes antigen-specific immune responses, indicating that GNLY can activate both innate and adaptive immune responses. Thus, based on its capacity to recruit and activate antigen presenting cells and promote an in vivo antigen-specific immune response, we consider GNLY an immune alarmin.
NLRC4 is an innate immune detector of the bacterial type III secretion apparatus (135.72)
Authors: Miao Edward A , Mao Dat , Yudkovsky Natalya , Bonnea Richard , Aderem Alan
Published: January 2023

The mammalian innate immune system uses Toll-like receptors (TLRs) and Nod-LRRs (NLRs) to detect microbial components during infection. Often these molecules work in concert; for example, the TLRs can stimulate the production of the pro-forms of the cytokines IL-1β and IL-18, whereas certain NLRs trigger their subsequent proteolytic processing via caspase 1. Gram negative bacteria utilize type III secretion systems (T3SS) to deliver virulence factors to the cytosol of host cells, where they modulate cell physiology to favour the pathogen. We show here that NLRC4/Ipaf detects the basal body rod component of the T3SS apparatus (rod protein) from S. typhimurium (PrgJ), Escherichia coli (EprJ and EscI), Burkholderia pseudomallei (BsaK), and Shigella flexneri (MxiI). These rod proteins share a sequence motif that is essential for detection by NLRC4; a similar motif is found in flagellin which is also detected by NLRC4. S. typhimurium has two T3SS: Salmonella pathogenicity island-1 (SPI-1), which encodes the rod protein PrgJ, and SPI-2, which encodes the rod protein SsaI. While PrgJ is detected by NLRC4, SsaI is not, and this evasion is required for virulence in mice. Our results indicate that macrophages respond to the activity of T3SS, not by sensing the specific translocated virulence factors, but rather by detecting the translocation apparatus that is conserved between pathogens. This reduces the number of host detectors required, a survival strategy that is divergent from that used by plants, where translocated effectors are detected on a gene for gene basis. Furthermore, the specific detection of the virulence machinery permits the discrimination between pathogenic and non pathogenic bacteria.
Regulation of IL-1beta and IL-23 production by beta-glucan in human dendritic cells (135.76)
Authors: Lyakh Lyudmila A , Cardone Marco , Riboldi Elena , Trinchieri Giorgio
Published: January 2023

We have previously demonstrated in human monocyte-derived dendritic cells (DCs), interleukin-23 (IL-23) is efficiently induced by the Dectin-1 ligands alone or in combination with Toll receptor 2/6 (TLR2/6) ligand Pam2C but not by the TLR4 ligand LPS alone. We found that the ability of various stimuli to induce Th17 cells depends not only on their induction of IL-23, IL-6, and TGF-β production in DCs, but also on their ability to induce bioactive IL-1β.

We considered whether the Dectin-1 receptor ligands were able directly or indirectly to activate the inflammasome. We found that only particulate but not soluble forms of β-glucan induced secretion of IL-1β in human DCs, and caspase-1 inhibitors blocked its secretion. LPS did not induce IL-1β secretion unless ATP was added to activate the inflammasome. Moreover, treatment with oxidized ATP or apyrase impaired the secretion of IL-1β by β-glucan-treated DCs.

We also observed that caspase-1 inhibitors or an IL-1 receptor antagonist significantly decreased the production of IL-23 by DCs stimulated with β-glucan. Our data indicate that endogenous IL-1β is involved in regulation of the IL-23 production. Thus, these results strongly suggest that activation of the inflammasome is necessary both for the production of IL-23 and for the development of Th17 responses. We are currently analyzing the molecular mechanisms of the inflammasome activation by dectin-1 ligands alone or in combination with TLR ligands.

This work was supported by the intramural program of the National Cancer Institute, NIH.
NLRs in mouse hepatocytes respond to specific ligands to activate NFκB (135.78)
Authors: Scott Melanie J , Liao Hong , Billiar Timothy R
Published: January 2023

Hepatocytes are microbial-responsive cells that express and activate Toll like receptors (TLRs). NACHT-LRR receptors (NLRs) are a family of more recently identified intracellular pattern recognition receptors. NOD1 and NOD2 are NLRs that respond to specific microbial ligands and activate NFκB through activation of RIP2/RICK. We hypothesized that hepatocytes express and activate NOD1/NOD2 signaling pathways as part of their function to initiate and regulate the innate immune system. High levels of NOD1, NOD2, and RIP2/RICK mRNA and protein expression were detected by quantitative PCR or Western blot in untreated primary isolated C57BL/6 hepatocytes. Hepatocytes were then treated with LPS (100ng/mL), iE-DAP (100ng/mL; NOD1-ligand), or MDP (10μg/mL; NOD2-ligand) for up to 60min. NOD-ligands were endotoxin free. NFκB activation by EMSA in hepatocytes treated with iE-DAP was similar or higher than after LPS-stimulation. However there was minimal NFκB activation after NOD2-ligand (MDP). NOD1/2, RIP2 mRNA expression increased significantly by 1h after stimulation with LPS, MDP or iE-DAP and was maximal by 4h, remaining elevated at 24h of stimulation. Together these data show hepatocytes respond to microbial ligands specific for NOD1 or NOD2 and increase their expression suggesting a role for these proteins in hepatic responses to infection.

Work supported in part by funding from NIH grant R01-GM-50441
Structure of NALP1/NLRP1 leucine-rich repeats and potential ligand binding (135.79)
Authors: Jin Tengchuan , jiang Jiansheng , Perry Andrew , Xiao Tsan
Published: January 2023

NALP1/NLRP1 is a prototype NLR family member and is involved in inflammasome formation and activation of caspase-1 in response to bacteria infections. The primary stimuli of NLRP1 appears to be the bacterial cell wall components, though the molecular mechanisms of recognition has remained elusive. The structure of a fragment of NLRP1 containing its leucine-rich repeats has been determined. The structure illustrates an unusual tetramerization of the leucine-rich repeats. There is a potential ligand binding site that appears to have affinity for sugar molecules.
Differentially Expressed MicroRNAs in Allergic Asthma Target Genes underlying Airway Hyper-Responsiveness and Goblet Cell Hyperplasia (136.22)
Authors: polikepahad Sumanth , Creighton Chad J , Zhu Huifeng , Harris Alan , Coarfa Cristian , Berel Dror , Milosavljevic Aleksandar , Corry David B , Gunaratne Preethi H
Published: January 2023

Asthma is an allergic disease that results in the obstruction of airways as a result of goblet cell hyperplasia and airway hyper-reactivity (AHR). Broad-spectrum P2-receptor antagonists and glucocorticoids are commonly used therapeutic agents for this disease. Niflumic acid (NA), an inhibitor of calcium-activated chloride channels is used to overcome corticosteroid-resistant asthma. Using Next Generation Sequencing, we dissected the asthma-related small RNAome in the lung before and after (FC) allergen challenge. We find a significant induction of microRNAs in FC from 40% to 74% suggesting an important role for miRNAs in asthma. Also, we uncovered 460 miRNA-up:mRNA-down and 17 miRNA-down:mRNA-up pairs that are significantly correlated in naïve and FC lung. The latter category contains a number of key genes induced during asthma and include miR-106b:P2X, miR-205:P2Y, miR-145:CLCA3 and miR-133a:GM-CSF. We also uncovered two novel miRNAs Asth-miR-1 and Asth-miR-2. Asth-miR-1 is predicted to target Toll-like receptor (TLR) adaptor TIRAP and IRAK which associates to activate NF-Kb, AP-1 and IRFs, and under some conditions, induce allergic lung disease. Asth-miR-2 is predicted to target ryanodine receptor 2 (RyR) that mediates Ca2+ release that induces airway smooth muscle contraction and bronchoconstriction. The integration of miR-106b, 205, 145 a, 133a and Asth-miR-1 and Asth-miR-1 2 with current therapies can potentially significantly enhance the efficacy and specificity of drugs used to combat asthma.
Epigenetic modifications control the phenotype of alternatively activated macrophages (136.4)
Authors: Ishii Makoto , Wen Haitao , Corsa Callie A S , Hogaboam Cory M , Dou Yali , Kunkel Steven L
Published: January 2023

Although alternatively activated (M2) macrophages play critical roles in chronic diseases, little is known about the acquisition and maintenance of their phenotype. We investigated the mechanisms whereby chromatin remodeling can influence the expression of genes that define M2 macrophages. Bone marrow-derived macrophages were incubated with IL-4 (10 ng/ml) and the expression of M2 marker genes (Ym1 and found in inflammatory zone-1 (FIZZ1)) and the histone H3 lysine-27 demethylase Jmjd3 was measured by PCR. ChIP assays were performed to determine levels of histone H3 lysine-4 trimethylation (H3K4me3), histone H3 lysine-27 trimethylation (H3K27me3), and Jmjd3 associated with the promoter regions of Ym1 and FIZZ1. Macrophages treated with IL-4 were found to increase the expression of mRNA levels of Ym1 and FIZZ1. Concomitant with this augmented expression pattern were an increase in H3K4me3 levels and a decrease in H3K27me3 levels; a pattern consistent with enhanced gene expression. Furthermore, Jmjd3 mRNA and Jmjd3 recruitment to Ym1 and FIZZ1 genes were increased in macrophages treated with IL-4, suggesting that Jmjd3 participates in erasing H3K27me3 marks on the promoter regions of Ym1 and FIZZ1. We conclude that the M2-macrophage phenotype is epigenetically regulated, in part, via induction of the H3K27 demethylase Jmjd3. (NIH grants P01-HL031963, HL092845, HL89216 and HL031237)
Regulatory T Cells Suppress Expression of IL-2 by Responder T Cells via a Transcriptional Mechanism (136.40)
Authors: Luther Rita J , Randolph David , Deshane Jessy , Maynard Craig , Janowski Karen , Oliver Jimmy , Weaver Casey T
Published: January 2023

Interleukin-2 (IL-2) is a type I cytokine that undergoes transcriptional activation downstream of T-cell receptor engagement and CD28 co-stimulation. IL-2 has been shown to have a role in self-tolerance as the presence of this cytokine, shown in several mouse models, necessitates the presence of a sufficient population of T regulatory cells (T reg). Tregs have a critical role in limiting the development of autoreactive effector T cells from naïve precursors that have escaped negative selection within the thymus. Ongoing studies indicate several mechanisms by which Tregs suppress induction of effector T cell responses, one of which is the inhibition of IL-2 gene expression. While several studies have indicated an inhibition of IL-2 mRNA and protein expression by responder CD4+ T cells co-cultured with T regs, the mechanism (s) remains unclear, although effects on both IL-2 gene transcription and mRNA stability have been implicated. In order to examine transcriptional and post-transcriptional effects on IL-2 expression, we have generated an IL-2 BAC-in transgenic reporter mouse model, 2BiT, in which a Thy1.l reporter is expressed under control of the extended Il2 locus. Herein we show that the Thy1.1 IL-2 reporter is expressed with fidelity to the endogenous IL-2 genes. Co-culture assays indicate that transcriptional activation of the reporter in responder T cells is inhibited in the presence of T regs, despite modest effects on IL-2 mRNA half-life (t1/2). These findings indicate that T regulatory cells suppress IL-2 expression primarily through inhibition of transcription rather than modulation of IL-2 mRNA stability.
Acetylcholine Receptor-alpha Subunit Over-Expression in Experimental Myasthenia Gravis: A Possible Role for the Autoantigen in Pathogenesis (137.1)
Authors: Sheng JianRong , Prabhakar Bellur S , Meriggioli Matthew N
Published: January 2023

Previous studies have shown an increased expression of AChR alpha (AChR-α) subunit transcripts in experimental autoimmune myasthenia gravis (EAMG). We examined the mRNA and protein expression of AChR-α, as well as the pattern of α-bungarotoxin labeling in muscle tissue from EAMG mice. AChR-α expression was profoundly increased in endplates from mice with severe EAMG, but this expression was distinct and greatly in excess of α-bungarotoxin labeling. These results suggest that loss of functional AChR increases transcription of AChR-α mRNA, but the expressed protein is "functionally inert," failing to compensate for loss of AChR, and very likely enhancing immunogenicity.
Title: CD8+CD62LhiCD25- cells induce skin inflammation in a psoriasis-like disease model and exhibit a Th1 phenotype (137.19)
Authors: Napierata Lee , Stedman Nancy , Benoit Stephen , Collins Mary , Nickerson-Nutter Cheryl , Young Deborah , Ma Hak-Ling
Published: January 2023

Previously we have shown that adoptive transfer of CD4+CD45RBhiCD25- T cells (naïve CD4 cells), into scid/scid mice can induce psoriasis-like disease. The affected mice develop scaly and raised skin plaques and immune features in this model mimic those of human psoriasis. Data from others suggests that skin infiltrating CD8+ T cells contribute to disease exacerbations in human psoriasis. To understand the role of CD8 cells in the mouse model of psoriasis, we adoptively transferred either naïve CD4 T cells, or CD8+ CD62LhiCD25- cells (naïve CD8 T cells) or combined naïve CD4 and CD8 cells into scid/scid mice and monitored disease progression. We found that adoptive transfer of naïve CD8+ T cells alone resulted in slightly less severe skin lesions when compared to either naïve CD4 T cell transfer or combined CD4 and CD8 T cell transfer. We also found that mice receiving CD8+ T cells experienced slightly reduced epidermal hyperplasia and inflammation than mice given other combinations of T cell transfer. Quantitative RT-PCR analysis of the mouse ear RNA and intracellular cytokine staining of the draining lymph node cells from these mice revealed that in comparison to mice given naïve CD4 cells, mice receiving CD8 cells expressed mainly a Th1 cytokine profile. Results here suggest that CD8 cells may also play an important role in perpetuating the development of skin lesions and exhibit mainly a Th1 phenotype.
Two populations of alternatively activated M2 macrophages (TLR4+ vs. Tim-3+) regulate inflammation and fibrosis in the heart during coxsackievirus-induced myocarditis (137.20)
Authors: Fairweather DeLisa , Frisancho-Kiss Sylvia , Coronado Michael J. , Frisancho J. Augusto , Cihakova Daniela , Rose Noel R.
Published: January 2023

Previously we showed that Toll-like receptor 4 (TLR4) and T cell Ig mucin-3 (Tim-3) cross-regulate each others expression on CD11b+ cells, with TLR4 increasing and Tim-3 reducing cardiac inflammation. We show here that TLR4 and Tim-3 are expressed on GR1+F4/80+IL-4R+ alternatively activated M2 macrophages from the heart during acute myocarditis, but are not expressed on GR1-F4/80+IL-4R- classically activated M1 macrophages. GR1+ cells also express the mannose receptor (CD206), caspase-1, IL-1β and IL-10. In gonadectomized male mice, M2 cells express significantly more Tim-3 and significantly less IL-1β with significantly reduced inflammation. In contrast, complement receptor 1/2 (CR1/2) deficient mice have a significantly increased TLR4+casp-1+IL-1β+ M2 population. Elevated expression of IL-1β by M2 macrophages in the heart leads to significantly increased myocarditis, fibrosis and heart failure in CR1/2 deficient mice. These data indicate that different M2 populations have contrasting roles on the development of heart disease: TLR4+casp-1+IL-1β+ M2 are associated with increased inflammation and fibrosis, while Tim-3+ M2 with decreased inflammation. Deciphering how M2 macrophage populations interact with autoreactive cells and antibodies will be critical in understanding the pathogenesis of autoimmune disease.

Supported by NIH grants R01 HL087033 (DF) and R01 HL67290 (NRR).
T cell receptor V beta usage in HLA-DQ6αβ8αβ mice displaying spontaneous polyarthritis (137.31)
Authors: Sidhu Maninder , Bradley David S.
Published: January 2023

Spontaneous polyarthritis (SP), an autoimmune disease that develops spontaneously in HLA-DQ6 αβ8aαβ transgenic (tg) mice, is characterized by lymphocyte infiltration in joints leading to chronic polyarthritis in the extremities similar to human rheumatoid arthritis. We examine here the CD4 T cell receptor (TCR) expression in the DQ6/8 tg mice during SP. CD4 T cells were isolated from the lymph nodes of arthritic and non-arthritic DQ6/8 tg mice at various time points, phenotyped via flow cytometry with V beta usage characterized in the CD4+CD25+ T cell population. Preferential usage of V β8.2 was increased from 9% in 2 month naïve mice to 15% in 4 month old and 20% by 8mo old arthritic mice. V β17 expressing T cells also increased from 0.02% in 2 month naïve mice to 2.8% in 4 month old arthritic mice. This is consistent with other studies that have indicated an association of Vβ8.2 TCR in the Experimental Autoimmune Encephalomyelitis and collagen-induced arthritis models. These results support our hypothesis that activated CD4 T cells expressing Vβ8.2 TCR are the predominantly pathogenic; autoreactive T cells involved in SP. Selective depletion of these T cells using anti-Vβ8.2 antibodies will confirm their involvement. Application of such strategies of immune intervention can be helpful in humans to control or reduce the disease progression in the stages of disease initiation. The study was supported by NIH-NIAMS AR45657
Synergistic role of diacylglycerol kinases α and ζ in T cell development and self-tolerance (137.37)
Authors: Zhong Xiaoping , Wan Chi-Keung , Guo Rishu
Published: January 2023

Signal for the T cell receptor (TCR) plays critical roles in T cell development and function. TCR signaling can induce positive or negative selection of thymocytes, induces activation or anergy of mature T cells, and induce conventional T cell activation or regulatory T cells to suppress immune function. The mechanisms that modulate TCR signaling to direct these distinct outcomes have been poorly understood. Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA) through phosphorylation. In mammals, ten DGK isoforms have been identified. Using both a gain of function and a loss of function approaches, we have found that DGK ( and ( are important regulators for T cells. Enhanced DGK activity inhibits TCR signaling and T cell maturation. Deficiency of either DGK( or ( causes T cells hyperresponsive to TCR stimulation and resistant to anergy induction. Loss of both DGK( and ( in mice results in impairment of positive selection, spontaneous T cell activation, impairment of regulatory T cell function, and autoimmunity. We further demonstrate that DGKs function both as signal terminator by inhibiting DAG-mediated signaling and as signaling initiator by generating PA.
Innate immune CD11b+Gr-1+ cells, myeloid- derived suppressor cells, influence the adaptive immune response during demyelinating disease (137.6)
Authors: Olson Julie K , Bowen Jenna L
Published: January 2023

Innate immune CD11b+Gr-1+ cells are a heterogeneous population of cells which include neutrophils and myeloid- derived suppressor cells (MDSC). CD11b+Gr-1+ cells that express Ly6C are MDSC which can directly suppress T cell responses. Theiler's murine encephalomyelitis virus (TMEV) is a mouse model of multiple sclerosis in which mice develop a chronic progressive demyelinating disease associated with a myelin- specific CD4+ T cell response. We have previously shown that the innate immune response directly affects the initiation and progression of demyelinating disease. Thus, we wanted to determine the role of CD11b+Gr-1+ cells in the innate immune response during TMEV- induced demyelinating disease. TMEV- infected mice had a large number of infiltrating CD11b+Gr-1+ cells composed of Ly6Chigh cells in the central nervous system (CNS) during the innate immune response. Depletion of Gr-1+ cells in TMEV-infected mice resulted in increased expression of IFNγ, IL-12, and IL-17 in the CNS during the innate immune response and altered development of demyelinating disease. Most importantly, CD11b+Gr-1+ cells suppressed the activation of CD4+ and CD8+ T cells in TMEV- infected mice. These results suggest that innate immune CD11b+Gr-1+ cells, specifically the MDSC, influence the adaptive immune response and modulate the development and progression of demyelinating disease.

This work was supported by the National Multiple Sclerosis Society Grant RG3625.
NK cell development from a novel progenitor found in the murine lung (138.10)
Authors: Halim Timotheus You Fu , Takei Fumio
Published: January 2023

NK cells in the lung are phenotypically different from those in the spleen or bone marrow (BM). We have identified NK cell progenitors (NKPs) in normal mouse lung. Lung NKPs are lineage- NK1.1- CD122+ B220+/- and are phenotypically similar to conventional BM NKPs. Cells with the same phenotype are also found in peripheral blood. Lung NKPs cultured on OP9 stromal cells with cytokines expand over 2×104 fold and mature into NK1.1+CD3- NK cells. The frequencies of NKPs among the lineage-NK1.1-CD122+B220+ population in the lung and lineage-NK1.1-CD122+B220- cells in the BM are 0.16 and 0.14, respectively. However, NK cells generated in vitro from lung NKPs and BM NKPs significantly differ in the expression of the Ly49 family of NK cell receptors. Most NK cells develop in vitro from lung NKPs express multiple Ly49s whereas most of those develop from BM NKPs express CD94/NKG2 but not Ly49. Lung NKPs develop into CD3-NK1.1+ NK cells upon adoptive transplant into NOD/SCID IL2Rγ-/- hosts. Thus, we have identified NKPs in the lung. They likely develop in the BM and migrate to the lung through peripheral blood. This supports the emerging hypothesis that NK cell development includes final maturation stages outside of the BM, where the local microenvironment influences development. The contribution of lung NKPs to the lung NK cell population in vivo is currently being investigated.
KPNA1 is a putative substrate of the RAG1 ubiquitin ligase (138.11)
Authors: Jones Jessica M , Simkus Carrie , Bhattacharyya Anamika
Published: January 2023

The RAG1 V(D)J recombinase encompasses DNA binding/cleavage and ubiquitin ligase activities. The nuclear transport protein karyopherin alpha 1 (KPNA1) binds to RAG1 upstream of its ubiquitin ligase domain, but this interaction is not required for nuclear localization of RAG1. We found that the isolated ubiquitin ligase domain of RAG1 (amino acids 218-389) promoted ubiquitylation of purified KPNA1 in a reaction supported by the ubiquitin conjugating (E2) enzyme UbcH2/Rad6 or UbcH5a. KPNA1 is the first putative substrate identified for the RAG1 ubiquitin ligase. Ubiquitylation of KPNA1 required the lysine/arginine-rich region spanning RAG1 amino acids 218-263 upstream of the RAG1 ubiquitin ligase domain, but RAG1 was still able to undergo auto-ubiquitylation in this region even in the presence of KPNA1. RAG1 did not promote rapid ubiquitin chain extension following mono-ubiquitylation of substrate, regardless of the E2 used. Substitutions of amino acids surrounding the third, non-canonical Zn coordination site of the RAG1 RING domain abrogated functional interaction with E2 enzymes, and this was significantly correlated with reduction in the ability of full length RAG1 to support recombination of extra-chromosomal substrates. These data suggest that RAG1-dependent mono-ubiquitylation of a substrate, possibly KPNA1, is required for optimal levels of V(D)J recombination.
Polychromatic immunophenotyping and probability binning algorithm reveal distinct disease clusters in patients with Common Variable Immunodeficiency (138.12)
Authors: Kalina Tomas , Stuchly Jan , Janda Ales , Ruzickova Sarka , Litzman Jiri , Sediva Anna , Vlkova Marcela
Published: January 2023

Common Variable Immunodeficiency (CVID) is a disease where only small fraction of cases has known molecular defect. Classification of CVID patients is based on B-cell phenotype and count.

Using polychromatic flow cytometry we set out to analyze B-cell phenotype in a single six-color tube in a cohort of 49 patients and 50 controls. The aim of the study was to find an expert free approach for phenotype analysis that would cover all differences in the 6-color space and form groups of patients with similar phenotype. We used "probability binning" algorithm for a creation of 1024 bins (eg. six-color gates) that were optimally covering the cells' distribution in the whole B-cell compartment. We have created a matrix file with numerical representation of all cells in all bins. Next, we analyzed this matrix in MeV software, which was developed originally for the microarray data analysis. "Self-Organizing Tree Analysis" created a tree of related individuals. First branching split the whole cohort into control-like phenotype branch (28% of CVID patients, 5 clusters) and CVID branch (94% of CVID patients in 6 clusters). Independent sample from the same patient 3-13 month later was clustered to either the same (11 pts), neighbor (4 pts) or further away (2 pts), thus confirming the reproducibility of this approach.

The "signature" bins for each cluster were characterized.

Grant support MSM0021620813 and IGA NR/9198-3
Role of Ikaros in mast cell development and differentiation (138.21)
Authors: Rao Kavitha N , Gregory Gregory D , Winandy Susan , Brown Melissa A
Published: January 2023

The transcription factor Ikaros has been implicated in the regulation of hematopoiesis at the level of T, B, NK and dendritic cells. In this study we examine the role of Ikaros in the development and differentiation of mast cells, using mice lacking Ikaros (Iknull). In vitro derived bone marrow mast cells (BMMCs) from Iknull mice expressed decreased levels of mRNA encoding transcription factors required for optimal mast cell development (MITF, GATA1, GATA2 and STAT5a), and mast cell-specific proteases (chymase, tryptase and carboxypeptidase). Upon activation through FcεRI crosslinking, Iknull mast cells revealed a hyper-activation phenotype as assessed by increased degranulation, cytokine secretion and expression of co-stimulatory molecules including OX40 ligand. Furthermore, Iknull BMMCs expressed reduced levels of several cell surface adhesion molecules and integrins including CD49d (integrin alpha 4), which is required for homing of mast cell precursors to the intestine. Consistent with this observation, in vivo examination of Iknull mice for presence of mast cells in various tissues revealed a striking deficiency of mast cells in the peritoneal cavity, and limiting dilution analysis demonstrated severely reduced number of mast cell precursors in the intestine. Taken together, our data suggest that Ikaros plays an important role in mast cell development, differentiation and activation.

This research has been supported by JDRF-3-2007-617 and NIH RO1 CA047992.
The transcription factor E47 controls the cell cycle quiescence and development of multipotent hematopoietic progenitors (138.3)
Authors: Yang Qi , Kardava Lela , Leger Anthony St , Martincic Kathleen , Varnum-Finney Barbara , Bernstein Irwin D , Milcarek Christine , Borghesi Lisa
Published: January 2023

It is of great interest to understand the mechanisms controlling the development and maintenance of multi-potential bone marrow progenitors, the key hematopoietic compartment that seeds the entire immune system. Using E47 knockout (KO) mice, we found that the transcription factor E47 plays a critical role in the cell cycle regulation and development of the multipotent hematopoietic subsets. Multipotent LSKs (Lineage-Sca-1+c-Kit+) from E47 KO mice had significantly increased BrdU incorporation, decreased proportion of progenitors in G0, and hypersensitivity to a mitotoxin 5-Fluorouracil, suggesting loss of cell cycle quiescence. Total LSKs contain both self-renewing hematopoietic stem cells (HSCs) and non-renewing multipotential progenitors (MPPs). Using three independent phenotypic definitions, we found that E47 KO mice have numerically unperturbed HSCs but a 2-4 fold reduction in the MPPs. Moreover, the residual MPPs in E47 KO mice fail to fully up-regulate flk2 and lack V(D)J rearrangement, suggesting compromised lymphoid differentiation potential. Finally, both loss-of-function and gain-of-function experiments identified a key stem cell regulator, p21, as E47 target in the multipotent LSKs. Taken together, our data define an important role of the transcription factor E47 in controlling the development, differentiation and cell cycle regulation of multipotent hematopoietic progenitors.