bluuebunny/crossref_metadata_2025 huggingface dataset:
Coactivator Associated Arginine Methyltransferase 1 (CARM1) is reciprocally regulated by phosphorylation and O‐GlcNAcylation
Authors: Sakabe Kaoru , Hart Gerald W
Published: June 2021

Enzymatic modification of serine and threonine residues on nucleocytoplasmic proteins with single N‐acetylglucosamine moiety through a β‐glycosidic linkage (O‐GlcNAc) is a dynamic post‐translational modification. Much like phosphorylation, protein O‐GlcNAcylation is responsive to cell stimuli and serves to regulate protein function. However unlike phosphorylation, which is mediated by more than 500 kinases, GlcNAcylation is regulated by transient targeting of a single catalytic subunit, O‐GlcNAc Transferase (OGT), to a multitude of substrates by interactor proteins. Recently, through a yeast two‐hybrid approach, we identified Coactivator Associated Arginine Methyltransferase (CARM1) as a putative binding partner with OGT. CARM1 methylates Arg residues of proteins, such as p300 and histone H3. Here, we demonstrate that not only does CARM1 associate with OGT, it also is O‐GlcNAcylated. Additionally, we show that it is reciprocally regulated by phosphorylation and O‐GlcNAcylation. When CARM1 becomes hyper‐phosphorylated during M phase, O‐GlcNAc on CARM1 decreases. This reciprocal relationship regulates substrate specificity, since methylation on Arg 17 of histone H3, a CARM1 specific substrate, increases dramatically during M phase. Overexpression of OGT alters not only the modification status of CARM1, but also the methylation of histone H3 by decreasing mitotic phosphorylation of CARM1 and concomitantly decreasing histone H3 methylation. Aberrant methylation of histone H3 caused by OGT overexpression could explain the gross defects associated with altering O‐GlcNAc levels during cell cycle. Supported by NIH HD13563. Dr. Hart receives royalty received by the university on the sales of the 110.6 antibody. Terms of the arrangement are managed by JHU.
G12 signaling through JNK promotes breast cancer cell invasion
Authors: Juneja Juhi , Kelly Patrick , Casey Patrick J
Published: June 2021

The G12 family of heterotrimeric G proteins has been implicated in such cellular processes as cytoskeletal rearrangements, cell‐cell adhesion and oncogenic transformation. Recently, work done in our laboratory has shown that G12 signaling via Rho induces a striking increase in breast cancer cell invasion in vitro and in vivo. In a continuation of this study, we further characterized G12 signaling leading to invasion of breast cancer cells. We show that c‐Jun N‐terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively active Gα12 leads to increased JNK and c‐Jun phosphorylation in the MDA‐MB‐231 breast cancer cell line. Chemical inhibition of JNK or knockdown of JNK by siRNA significantly decreases G12‐induced JNK activation as well as the ability of cells to invade Matrigel in in vitro transwell invasion assays. Furthermore, treatment of these cells with the Rho kinase (ROCK) inhibitor reduces G12‐induced JNK and c‐Jun activation and also G12‐induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. This suggests that a Rho‐ROCK‐JNK signaling axis is stimulated by G12 to promote breast cancer cell invasion. We are currently studying which G protein coupled receptor(s) G12 couples to in order to engage this pathway. This work was supported by a Human Frontier Science Program Fellowship (to J.J.).
Purification and characterization of the ternary complex between phosducin‐like protein, the G protein β subunit and the cytosolic chaperonin ‐ an important intermediate in the assembly of the G protein βγ dimer
Authors: Lai Chun Wan J , Ludtke Paul J. , Stowell Caleb J. , Mabey Alicia A. , Willardson Barry M.
Published: June 2021

Phosducin‐like protein 1 (PhLP1) has been recently shown to be an essential co‐chaperone, along with the cytosolic chaperonin containing the tailless‐complex polypeptide 1 (CCT), in the folding and assembly of the nascent G protein βγ subunit dimer (Gβγ). It has been proposed that a ternary PhLP1‐Gβ‐CCT complex forms during the folding process with nascent Gβ in the CCT folding cavity and PhLP above the cavity contacting Gβ and the apical domains of CCT. If PhLP1 is phosphorylated in a cluster of serine residues near its N‐terminus (S18–20) by the protein kinase CK2, it will release Gβ from CCT in a PhLP1‐Gβ complex that is then able to interact with Gγ and form the Gβγ dimer. To further test the proposed mechanism of Gβγ assembly, we have isolated the PhLP1‐Gβ‐CCT ternary complex in both states of PhLP1 phosphorylation from insect cells over‐expressing both PhLP and Gβ using a tandem affinity purification method. In addition, we have isolated the Gβ‐CCT complex in the absence of PhLP1. These complexes have been subjected to structural determination by cryo‐electron microscopy in an effort to determine the mechanism by which PhLP1 phosphorylation results in the release of PhLP1‐Gβ from CCT.
RGS2 gene product from candidate hypertension allele shows decreased plasma membrane association and inhibition of Gq
Authors: Gu Steven , Tirgari Sam , Heximer Scott
Published: June 2021

Hypertension is a leading cause of mortality and morbidity in North America. Recent work has identified the Regulator of G‐protein Signaling 2(RGS2) as a candidate gene in the development of hypertension. Specifically, a single nucleotide polymorphism resulting in a missense mutation (R44H) has been correlated with hypertensive patients in a subset of the Japanese population. In our work, we identify the functional characteristics of the R44H mutation and the mechanisms by which they occur. Single cell calcium imaging shows that the mutant protein is less able to inhibit Gq signaling. Previously, we have shown that plasma membrane (PM) localization via RGS‐lipid interactions dependent on the amphipathic alpha helix is important for efficient Gq inhibition. Confocal microscopy of the R44H mutant shows greatly reduced tonic association with the PM. Finally, tryptophan spectroscopy of the helix domain shows an inability to associate with liposomes. In conclusion, we have shown that RGS2 R44H is a poor inhibitor of the M1‐muscarinic receptor and that this reduced function is a result of deficient RGS2‐lipid interactions leading to the inability to associate with the PM.
The C type natriuretic peptide receptor associates with AHNAK in human aortic vascular smooth muscle cells
Authors: Alli Abdel A , Gower William R
Published: June 2021

We recently identified AHNAK as a binding partner for the C type natriuretic peptide receptor (NPR‐C) in normal rat gastric mucosa (RGM1) cells. To investigate the role of AHNAK in NPR‐C function in different cell types and species we examined the expression and interaction of both proteins in multiple cell lines. Antibodies to NPR subtypes and AHNAK domains were used to examine expression by immunoblotting, colocalization by immunohistochemistry, and interaction by immunoprecipitation in multiple mammalian cell lines. Mass spectrometry was used to confirm the identity of the proteins from the immunoprecipitation studies. GST fusion proteins of the N‐terminal, internal repeating, and C‐terminal domains of AHNAK were purified and used to perform GST pulldown assays to verify the association of the two proteins and determine specificity. Coding sequences of human NPR‐C and AHNAK were subcloned into mammalian two hybrid expression vectors to ascertain the interaction in situ. NPR‐C was found to be the most abundantly expressed NPR subtype and AHNAK was found to also be expressed in human aortic vascular smooth muscle cells (AoVSMCs). NPR‐C and AHNAK colocalized to the plasma membrane of AoVSMCs. NPR‐C bound to AHNAK isoform 1 in vitro. The interaction was attributed to a single AHNAK domain. This research was supported by an American Heart Association Predoctoral fellowship and a Veterans Affairs Merit grant.
Differential regulation of caveolin‐2 phosphorylation at serine 23 and 36 by caveolin‐1 and cell cycle in human endothelial cells.
Authors: Sowa Grzegorz , Xie Leike
Published: June 2021

In the present study, we are focusing on regulation of serine phosphorylation of caveolin‐2 in endothelial cells. We show that phosphorylation of caveolin‐2 at serines 23 and 36 can be differentially regulated by caveolin‐1 and in endothelial cells synchronized in mitosis. To directly address the role of caveolin‐1 in regulating phosphorylation of endogenous caveolin‐2, we have used siRNA approach. The specific knockdown of caveolin‐1 in endothelial cells decreases caveolin‐2 phosphorylation at serine 23 but not serine 36. Furthermore, immunofluorescence microscopy studies determined that serine 23‐phosphorylated caveolin‐2 mostly localizes to plasma membrane caveolae. Thus, upregulation of serine 23 phosphorylation of caveolin‐2 depends on caveolin‐1‐driven targeting to plasma membrane caveolae. Interestingly, although, serine 36 phosphorylation does not seem to be regulated by caveolin‐1, it can be selectively upregulated in endothelial cells synchronized in mitosis. The latter data suggests a possible involvement of serine 36‐phosphorylated caveolin‐2 in modulating endothelial cell cycle, in particular mitosis.
Activity dependent cleavage and protein phosphorylation regulate human glutamate decarboxylase GAD65 activity
Authors: Buddhala Chandana , Wei Jianning , Wu Jang Yen
Published: June 2021

Brain L‐glutamate decarboxylase (GAD) is responsible for the synthesis of neurotransmitter GABA. There are two GAD isoforms, namely GAD65 and GAD67. The two are regulated differentially by protein phosphorylation. GAD65 is activated by phosphorylation mediated by PKC‐ε whereas GAD67 is inhibited by phosphorylation mediated by PKA. The phosphorylation site of GAD67 was identified as Threonine 91 based on MALDI‐TOF and site‐directed mutation analysis. (Biochemistry 43:6182–6189, 2004). Here we further report several lines of evidence on the regulation of GAD activity during neuronal activity with special focus on GAD65. Under stimulation condition, GAD65 activity is increased due to increase in phosphorylation through translocation of PKC‐ε to become membrane‐anchored. Several potential phosphorylation sites have been implicated in the regulation of GAD65 activity. In addition, cleavage of GAD65 by calpain also increased GAD65 activity. A model depicting activity‐dependent regulation of GAD65 by phosphorylation and proteolytic cleavage is proposed.
Roles for STYX in MAPK signaling and nuclear transport during germ cell maturation
Authors: Karnak David , Carter Balin , Torgersen Knut Martin , Wishart Matthew J
Published: June 2021

Phospho‐serine, ‐threonine, or ‐tyrosine binding protein (STYX) is the archetype of a unique class of proteins termed, dead‐phosphatases, which structurally mimic protein tyrosine phosphatases (PTPs). In contrast to PTPs, STYX contains a glycine in place of the essential catalytic cysteine. We have shown through knock‐out of STYX function in mice that the protein plays an essential role in the differentiation of germ cells. Although STYX lacks phosphatase activity it is thought to function as a protein‐protein adaptor, which recognizes the phosphorylated cellular substrates of PTPs. The goal of this project is to determine how STYX interaction with MAPKs and nuclear transport factors affects cellular differentiation. Here, we show that STYX expression negatively regulates growth factor mediated activation of specific MAPKs in mammalian cell culture. Also, we identify a karyopherin family member in immune precipitations with STYX from whole testis, thereby raising the possibility that their interaction regulates nucleo‐cytoplasmic transport of STYX and MAPK. Our ongoing work focuses on the cellular mechanisms by which STYX modulates MAPK signaling to control germ cell differentiation. This work was supported, in part, through the BIRCWH‐NIH career development program (MJW).
Regulation of Dual specificity protein phosphatase‐2 (DUSP2) by hypoxia
Authors: Lin Shih‐Chieh , Tsai Shaw‐Jenq
Published: June 2021

Constitutive activation of MAP kinases was found in many cancers, and it's still an enigma that needs to be revealed. DUSP2 is a member of dual specificity protein phosphatase that regulates the activity of MAP kinases. Until now, the regulation of DUSP2 expression has not yet investigated. Hypoxia occurs in many solid tumors and has been shown to be a factor of increasing the MAP kinase activity. Therefore, we hypothesize that hypoxia‐induced MAP kinase activity may be mediated via deregulation of DUSP2 expression. We predicted a putative hypoxia response element (HRE) in the DUSP2 promoter region by using bioinformatic tool. Expression of DUSP2 was decreased by hypoxia treatment in HeLa cancer cells and normal endometrial stroma cells. Knockdown of HIF‐1α or HIF‐2αby siRNA reversed hypoxia‐mediated inhibition of DUSP2 expression. Promoter activity assay and site‐direct mutagenesis showed that predicted putative HRE was essential for downregulation of DUSP2 expression under hypoxic condition. Chromatin immunoprecipitation (ChIP) assay further demonstrated the binding of HIF‐1α to the HRE of DUSP2 promoter. ERK1/2, the downstream target of DUSP2, was activated by hypoxia and was inversely correlated with the expression of DUSP2. Taken together, we demonstrate that hypoxia downregulates the expression of DUSP2 thus enhances the activity of ERK1/2, which may be an important factor for cells to survive in hypoxic environment.
SMIT2 mediates all myo‐inositol uptake in apical membranes of rat small intestine
Authors: Aouameur Rym , Cal Sandra Da , Bissonnette Pierre , Coady Michael J. , Lapointe Jean‐Yves
Published: June 2021

This study presents the characterization of MI uptake in rat intestine using purified membrane preparations (BBMv). The two Na+‐coupled myo‐inositol (MI) cotransporters identified (SMIT1 and SMIT2) can be differentiated through inhibition studies using the selective substrates D‐chiro‐inositol (DCI, specific for SMIT2) and L‐fucose (specific for SMIT1). Results show that SMIT2 is exclusively responsible for apical MI transport in rat intestine. Other sugar transport systems present in apical membranes (SGLT1 and GLUT5) lacked any significant contribution to MI uptake. Functional analysis of rat SMIT2 activity determined using electrophysiological studies in Xenopus oocytes, demonstrated similarities to the activities of SMIT2 from species (rabbit and human), displaying high affinities for MI (0.150 ± 0.040 mM), DCI (0.31 ± 0.06 mM) and Pz (0.016 ± 0.007 mM), low affinity for glucose (36 ± 7 mM) and no affinity for L‐fucose. Electrophysiological studies essentially confirmed those found in rat intestinal BBMv with exception of glucose affinity which was about 40‐fold higher in vesicles (Ki = 0.94 ± 0.35 mM) when compared to oocytes. Finally, GLUT2 expressed in oocytes did not mediate any significant radiolabelled MI uptake, indicating that this transport system does not participate in the basolateral exit of MI from small intestine.
Regulation of thiol enzyme activity by (−)‐epigallocatechin gallate through covalent binding to cysteine
Authors: Mori Taiki , Ishii Takeshi , Tanaka Tomoko , Mizuno Daisuke , Kumazawa Shigenori , Miyoshi Noriyuki , Akagawa Mitsugu , Nakayama Tsutomu
Published: June 2021

(−)‐Epigallocatechin gallate (EGCg), which has an antitumor activity, is a major component of polyphenol in green tea. Some mechanisms on the anticancer effect of EGCg have been proposed, and EGCg‐protein interaction is thought to be a significant step. However, the mechanisms on the regulation of protein function through binding are still unclear. To obtain the proof of functional modulation by EGCg, we used thiol enzymes, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and papain, as model proteins. Incubation of GAPDH with EGCg resulted in a dose‐dependent loss of the enzyme activity, which was accompanied by the loss of free thiol groups. Furthermore, by mass spectrometry (MS) and Western blotting with redox cycling staining, it is clarified that EGCg covalently binds to the enzyme. In addition, the results on the modification of papain were similar to that of GAPDH. To identify the binding site of GAPDH by EGCg, both the native and EGCg‐treated GAPDH were purified with catechol affinity resin and then digested with trypsin, and the resulting peptides were subjected to MS. We identified the EGCg‐binding peptides containing cysteine residues. These results suggest that modulation of GAPDH activity by EGCg is due to the selective modification of the cysteine residues in the GAPDH molecule. These findings may contribute to the understanding of anticancer mechanisms based on binding of tea catechins to proteins.
Critical role for nucleostemin in protein synthesis and muscle cell differentiation
Authors: Romanova Liudmila , Hirai Hiroyuki , Asakura Atsushi , Kikyo Nobuaki
Published: June 2021

Nucleostemin (NS) is a nucleolar protein that was shown to be primarily expressed in proliferating cells and essential for proliferation but the precise functions of NS remained unclear. Here, we reveal a significant role for NS as an integrated component of ribosome biogenesis, particularly pre‐rRNA processing. The supporting data demonstrated physical interactions of NS with other processing‐related proteins, nucleolar retention of these proteins by NS, and inhibition of pre‐rRNA processing and protein synthesis in NS‐depleted cells. We also showed that unlike in other cell lineages, NS was not downregulated in differentiated skeletal muscle cells, which maintain highly active protein synthesis after exit from the cell cycle. Moreover, progress of muscle differentiation could be modulated by NS in a dose‐dependent manner. These results underscore a fundamental involvement of NS in cell proliferation and differentiation through its contribution to ribosome biogenesis and protein synthesis.

This work was supported by Muscular Dystrophy Association, Korea Institute of Science and Technology, Nash Foundation and the NIH.
Beneficial effects of the unsaponifiable matter from shark liver oil on oxidative stress of hepatocytes by tert‐butyl hydroperoxide in vitro and in vivo
Authors: Lee Kwang‐Won , Yang Hyok
Published: June 2021

The objective of this study was to evaluate the protective effects of the unsaponifiable matter (UM) of shark liver oils on the tert‐butyl hydroperoxide (t‐BHP)‐induced oxidative stress injury observed in HepG2 cells and rat liver. After saponification, the final UM was obtained, and contained squalene, fatty acid and alcoxyglycerol based on by GC‐MS analysis. The treatment of the hepatocytes with the shark liver oil UM significantly reversed the t‐BHP‐induced cell cytotoxicity. Also, UM exhibited in vitro ferric‐reducing antioxidant power and 2,2‐diphenyl‐1‐picrylhydroazyl free radical‐scavenging activities. The in vivo study showed that pretreatment with UM (1000 or 3000 mg/kg) by gavage for 5 d before a single dose of t‐BHP (0.5 mmol/kg i.p.) and reduced the lipid oxidation. Histopathologic examination of the rat livers showed that UM reduced the incidence of liver lesions induced by t‐BHP. Based on the results described above, we speculate that UM has the potential to play a role in the hepatic prevention of oxidative damage in living systems.
T cell engagement downregulates vasoactive intestinal peptide receptor‐1 by a JNK MAPK dependent mechanism in primary, murine CD4 T cells
Authors: Vomhof‐DeKrey Emilie Erin , Hermann Rebecca , Benton Keith , Dorsam Sheri , Dorsam Glenn
Published: June 2021

This study investigates the signaling pathways that regulate mRNA expression of vasoactive intestinal peptide receptor‐1 (VPACR‐1) in murine, primary CD4 T cells. VPACR‐1 is an anti‐proliferative, G‐protein coupled receptor that is highly expressed on naïve T cells, but is downregulated upon activation. To date, the signaling molecules involved in mediating VPACR‐1 downregulation have yet to be identified. Splenic CD4 T cells isolated from C57BL/6 mice by magnetic beads (Miltenyi) were pretreated with several pharmacological inhibitors and activated with plate bound anti‐CD3 for 24 hrs. Total RNA was isolated, and VPACR‐1 mRNA levels were measured by qPCR. Our results show that the JNK MAPK pathway downstream from src kinases Fyn/Lck is involved in TCR‐induced VPACR‐1 downregulation. Preliminarily, the adaptor molecule, Zap70, and the JNK upstream G‐protein signaling molecule, Rac1, also appear to downregulate VPACR‐1. Surprisingly, we did not collect evidence to support other MAPK pathways, such as Ras→PKC→ERK and p38, as having roles in downregulating VPACR‐1. Furthermore, two other common T cell signaling molecules, calcineurin and PI3‐K, were not statistically involved in VPACR‐1 regulation. By studying the signaling pathways that regulate VPACR‐1, we can gain greater insight into the role of this receptor in infectious disease, autoimmunity and T cell biology. 1KO1 DK064828, COBRE‐ 2P20RR015566
IMMUNOREGULATION OF EXPERIMENTAL MYASTHENIA GRAVIS
Authors: Sheng JianRong , Prabhakar Bellur S , Meriggioli Matthew N
Published: June 2021

Recently, dendritic cells (DCs) and regulatory T‐cells (Tregs) have been shown to possess potent capabilities to tolerize T‐cells in an antigen‐specific manner. Strategies mobilizing specific subsets of DCs and Tregs have shown promise in experimental models of T‐cell mediated autoimmune disease. We have observed that the selective activation of particular subsets of DCs utilizing granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) had profound effects on the induction of the antibody mediated autoimmune disease, experimental autoimmune myasthenia gravis (EAMG). We now show that treatment with GM‐CSF significantly ameliorates well‐established, chronic EAMG and effectively down‐modulates anti‐AChR T cell and pathogenic antibody responses. Furthermore, we show that GM‐CSF treatment shifts the cytokine response to a Th2 pattern and that CD4+CD25+ cells are critically involved in the disease suppressive effect.
Interaction of programmed cell death‐1(PD‐1) with its ligand PD‐ligand 1(PD‐L1) has a co‐inhibitory function in NKT cells
Authors: Chang Woo‐Sung , Kim Ji‐Yeon , Kim Dong‐Hyeon , Kang Chang‐Yuil
Published: June 2021

Natural killer T cells (NKT cells) are a distinct subset of T lymphocytes that have invariant T cell receptor (TCR) restricted to CD1d, the MHC class I like‐protein. It is well known that the response of the NKT cells to glycolipids presented by CD1d critically affects innate and adaptive immune response. In the NKT cell response to antigens, co‐stimulatory pathway related to co‐stimulatory molecules such as CD28, ICOS, GITR has been established. However, expression of co‐inhibitory molecules on NKT cells or involvement of co‐inhibitory pathway in the NKT cell response is poorly investigated. We show here that programmed cell death‐1 (PD‐1) and its ligand PD‐L1, known as co‐inhibitory molecules in the interaction of conventional T cells with antigen presenting cells (APCs), were expressed on NKT cells and their interaction could modulate activation of NKT cells as a co‐inhibitors. We found that PD‐1 and PD‐L1 was constitutively expressed on naïve NKT cells and that expression of PD‐L1 was increased at early time points in activation of NKT cell with alpha‐galactosylceramide (aGC). In addition, the blockade of the interaction of PD‐1 and PD‐L1 by blocking monoclonal antibodies led to increase levels of IFN‐γ in sera from aGC‐treated mice. Our findings suggest that the interaction of PD‐1 with PD‐L1 can be defined as a novel co‐inhibitory pathway to regulate activation of NKT cells by antigens.
Leukocyte‐associated immunoglobulin‐like receptor (LAIR)‐2 is a soluble competitor of the collagen/LAIR‐1 inhibitory immune interaction
Authors: Meyaard Linde , van den Berg Maaike , de Ruiter Talitha , Raynal Nicholas , van Roon Joel , Lenting Peter , Jin Boquan , Lebbink Robert Jan
Published: June 2021

Leukocyte‐associated Ig‐like receptor (LAIR)‐1 is a collagen‐receptor that inhibits immune cell function upon collagen binding (Lebbink et al, J Exp Med, 2006). Next to LAIR‐1, the human genome encodes LAIR‐2, a putative soluble homologue. We now show that the LAIR‐2 gene is broadly transcribed in human peripheral blood mononuclear cells, mirroring the expression profile of LAIR‐1. LAIR‐2 protein is expressed as a soluble receptor exhibiting high affinity for various collagen molecules to which it binds in a hydroxyproline‐dependent manner. In vitro stimulation of peripheral blood mononuclear cells induces secretion of LAIR‐2. Interestingly, high amounts of LAIR‐2 are detected in urine of pregnant women, indicating that the soluble receptor is indeed produced in vivo and can be cleared from the body via urine. Furthermore, LAIR‐2 levels are increased in synovial fluid of patients with rheumatoid arthritis as compared to osteoarthritis patients. We hypothesize that soluble LAIR‐2 may function as a natural competitor for LAIR‐1, thereby regulating its inhibitory potential. Indeed, LAIR‐2 prevents binding of human LAIR‐1 to collagens and LAIR‐1 crosslinking in vitro, revealing a novel mechanism of immune regulation by a soluble LAIR receptor regulating the inhibitory potential of the membrane‐bound LAIR‐1 via competition for ligands.

Supported by the Netherlands Organization for Scientific Research (NWO)
Immunostimulatory activities of CMB, the water extract of Cordyceps militaris fruit bodies, in primary macrophages
Authors: Kim Kyungjae , Shin Seulmee , Kwon Jeunghak , Lee Sungwon , Kong Hyunseok , Lee Chongkil
Published: June 2021

Cordyceps militaris has been used in traditional folk medicine to treat numerous diseases and reported to have antitumor and immunomodulatory activities in vivo and in vitro. The pharmacological and biochemical mechanisms of CMB on macrophages have not been clearly elucidated. We examined whether CMB induces the production of pro‐inflammatory cytokines and the expression of co‐stimulatory molecules. CMB increased the production of nitric oxide (NO) and pro‐inflammatory cytokines such as Interferon‐1 beta (IL‐1β), Interferon‐6 (IL‐6), tumor necrosis factor‐alpha (TNF‐α) and prostaglandin E2 (PGE2) dose‐dependently. The change of gene expression level by CMB treatment was analyzed by using RT‐PCR, and the associated mRNA expression was increased same as cytokine production. Western blot analysis showed the augmentation of cytokine related protein on CMB treatment, and the expression of co‐stimulatory molecules such as ICAM‐1, B7‐1, B7‐2 also was enhanced with the increment of CMB concentration.

These results provide the clues for understanding the macrophage activation mechanism by CMB in innate and adaptive immunity, and suggest that CMB could be useful immunomodulatory agent for treating immunological diseases.
Engagement of the A2B adenosine receptor modifies murine DC maturation
Authors: Wilson Jeffrey Michael , Ross William , Linden Joel , Ernst Peter B
Published: June 2021

Background: Adenosine exerts anti‐inflammatory effects on most immune and inflammatory cells, including the cytokine response of dendritic cells (DC). It is not clear which adenosine receptors mediate this response or whether adenosine affects other aspects of DC maturation. Methods: Cytokine production and co‐stimulatory marker expression were assayed in murine bone marrow‐derived DCs (BMDC) primed with LPS in the presence or absence of the non‐selective adenosine agonist NECA. Responses were compared in BMDCs from WT or adenosine receptor knock‐out mice, or with adenosine receptor subtype selective antagonists. Results: NECA inhibited TNF‐α and IL‐12 protein in a concentration dependent manner, whereas IL‐10 production was increased. NECA significantly impaired CD86 expression compared to vehicle control as measured by mean fluorescence (p <0.05). The A2A adenosine receptor (A2AAR) and A2BAR were found to be the predominant adenosine receptors expressed in BMDCs by real‐time RT‐PCR assessment of mRNA transcripts. Using adenosine receptor subtype selective antagonists and BMDCs derived from A2AAR−/−and A2BAR−/−mice, it was shown that NECA modulation of TNF‐α, IL‐12, IL‐10, and CD86 responses require A2BAR. Conclusions: These data indicate that engagement of A2BAR impairs murine BMDC maturation, and suggest that adenosine may favor tolerance through actions on DCs.
Vitamin D receptor expression is required for the normal development and function of iNKT cells
Authors: Yu Sanhong , Cantorna Margherita T.
Published: June 2021

CD1d‐reactive natural killer T (NKT) cells with an invariant T cell receptor Vα14 rearrangement are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. Vitamin D is a potent regulator of conventional T cell development and function but there is no information on whether vitamin D regulates NKT cells. Expression of the vitamin D receptor (VDR) is required for normal development and function of iNKT cells. The iNKT cells from VDR KO mice are intrinsically defective and lack T‐bet expression that result in a population of immature NK1.1− iNKT cells that express normal levels of CD122. Bone marrow transplantation studies showed that non‐hematopoetic cell expression of the VDR was essential for intact iNKT cell responses. Extrinsic factors that impact iNKT cell development and function in the VDR KO mice included a failure of the liver to support homeostatic proliferation and reduced thymic expression of CD1d and other factors important for optimal antigen presentation in the thymus. VDR deficiency resulted in reduced thymic CD1d expression that impaired the ability of thymocytes but not peripheral DC to act as antigen presenting cells. The defects in VDR KO iNKT cells stem from both intrinsic and extrinsic requirement for VDR expression.
TRAF3 inhibits signaling by Toll‐like receptors in B lymphocytes
Authors: Xie Ping , Brown Lindsey , Stunz Laura L. , Bishop Gail A.
Published: June 2021

TRAF3, a cytoplasmic adaptor protein, binds directly to a number of receptors of the tumor necrosis factor receptor (TNF‐R) superfamily. To elucidate the physiological functions of TRAF3 in B lymphocytes, we recently generated B cell‐specific TRAF3‐deficient mice, and reported that TRAF3 is a critical regulator of peripheral B cell homeostasis. In light of the evidence that TRAF3 is required for type I interferon production induced by Toll‐like receptors (TLRs) in myeloid cells, the present study further investigated whether and how TRAF3 regulates signaling by TLRs in B lymphocytes. Interestingly, we found that B cell responses to agonists for TLR3, 4, 7/8 and 9, including surface molecule upregulation, cytokine production, and Ig isotype switching, were markedly enhanced by TRAF3 deficiency. Our findings indicate that in contrast to myeloid cells, TRAF3 inhibits TLR signaling in B lymphocytes, and suggest that TRAF3 may play important roles in modulating humoral immunity in response to pathogen infections.

This study is supported by a grant from the American Heart Association (PX), NIH grants AI28847, AI49993, CA099997, and a VA Career Award (GAB).
MR1 uses an endocytic pathway to activate mucosal‐associated invariant T cells
Authors: Huang Shouxiong , Gilfillan Susan , Kim Sojung , Thompson Bruce , Wang Xiaoli , Fremont Daved , Lantz Olivier , Hansen Ted
Published: June 2021

Mucosal Associated Invariant T (MAIT) cells are an “innate” T cell population expressing an invariant TCRα chain, mVαl9 in mice and hVα7.2 in humans. Strikingly similar to CD 1d‐restricted iNKT cells, MAIT cells have a memory phenotype and rapidly release IFN‐γ, IL‐4, 5, and 10 upon TCR ligation. Interestingly, MAIT cells are also restricted by an MHC class I like molecule, MR1. But, unlike iNKT cells, MAIT cells require B cells and gut commensal flora for development and/or expansion. Although the ligand presented by MR1 is unknown, evidence strongly suggests that MAIT cell activation is ligand‐dependent. Here we demonstrate that MR1 antigen presentation is not affected by either the proteasome or the class I chaperones. However, the class II chaperone invariant chain, Ii, physically associates with MR1 and promotes its endosomal trafficking. Functionally, MAIT cell activation is enhanced by Ii overexpression but strikingly diminished by silencing endogenous Ii. Furthermore, confocal microscopy confirms localization of MR1 in late endosomes and inhibiting the acidification of these compartments reduces MR1 surface expression and ablates MAIT cell activation. These data indicate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to potentially sample both endocytosed and endogenous antigens.
Loss of invariant chain expression with vaccinia virus infection of APC
Authors: Wang Nan , Li Ping , Blum Janice S.
Published: June 2021

Vaccinia virus (VV) has been used as a vaccine to eradicate smallpox and tested as a vaccine vector for HIV and tumor immunotherapy. The immunoevasive properties of VV, however have raised concerns with respect to the use of this virus in immune deficient patients, the elderly and children. VV infection can disrupt components of both host innate and adaptive immunity. VV infection of antigen presentation cells (APC) perturbs MHC class II‐mediated presentation of peptides, endogenous and exogenous antigens. In monitoring VV infection of human B cell lines, virus infection was observed to dramatically reduce expression of the class II chaperone invariant chain (Ii) in a time dependent manner. By contrast, the surface expression of MHC class II molecules was maintained along with cell viability up to 24h after exposure to virus. A reduction in Ii and class II mRNA levels was detected upon VV infection of APC. Analysis of Ii degradation in VV treated cells, revealed Ii proteolysis contributed to the reduced steady state of Ii levels in these APC. Yet, VV infection of APC altered the expression of lysosomal proteases likely influencing Ii degradation. These results demonstrate that at later stages of VV infection, reduced cellular Ii levels may contribute to defects in class II presentation.
Differential gene expression with co‐engagement of FcεRI and CC chemokine receptor 1
Authors: Aye Cho Cho , Morohoshi Kei , Fukuda Ken , Fifadara Nimita , Toda Masako , Ono Santa Jeremy
Published: June 2021

The classical pathway for mast cell degranulation is mediated through the high affinity IgE receptor FcεRI but the optimal mast cell degranulation in the conjunctiva also requires CC chemokine ligand 3 (CCL3). To examine the transcriptional changes underlying the rat basophilic leukemia mast cell line's response to costimulation of FceRI and CCL3 receptor CC chemokine receptor 1 (CCR1), we screened more than 31,000 probe sets using Affymetrix Rat Expression Array 230 2.0. Genes whose expression changed only with activation of both receptors were designated as synergy‐related genes. Among the 212 synergy‐related genes, 93 genes are functionally annotated, fourteen genes are involved in signaling pathways, 112 genes are up‐regulated while 100 genes are down‐regulated. The expression changes of the most up‐regulated synergy‐related genes including Epithelial Membrane Protein 1 (Emp1), Regulator of G protein Signaling 1 (Rgs1), and RT1 class Ib, locus S3 (RT1‐S3) were validated at both mRNA and protein levels. Their function was also analyzed by siRNA inhibition followed by degranulation and chemotaxis assays. This project is funded by MCCID EU 504926 EST program, European Commission, Brussels, Belgium.
Immunoreceptor tyrosine‐based activation motif (ITAM)‐containing adapters DAP12 and FcRγ required for E‐selectin mediated slow rolling
Authors: Zarbock Alexander , Lowell Clifford A. , Ley Klaus
Published: June 2021

E‐selectin binding to PSGL‐1 can induce slow rolling by activating the β2‐integrin LFA‐1 through a spleen tyrosine kinase (Syk)‐dependent pathway. Immunoreceptor tyrosine‐base activation motifs (ITAMs) are required for Syk activation downstream from B‐cell and Fc receptors. Neutrophils express two ITAM‐containing adaptor molecules, FcRγ and DAP12. To test their role in E‐selectin‐mediated slow rolling, we investigated Fcrg−/− and Tyrobp−/− as well as Fcrg−/− Tyrobp−/− mice by intravital microscopy of the cremaster muscle and in autoperfused flow chambers. In wild‐type mice, rolling velocity on E‐selectin was 2.2 μm/s. Adding ICAM‐1 to the substrate reduced rolling velocity to 1.0 μm/s. This velocity reduction was partially blunted in Fcrg−/− or Tyrobp−/− and completely eliminated in Fcrg−/− Tyrobp−/− mice, suggesting that the ITAM domains of DAP12 and FcRγ are partially redundant for E‐mediated slow rolling. Lethally irradiated WT mice reconstituted with bone marrow from Fcrg−/− Tyrobp−/−mice and WT mice revealed that leukocytes from Fcrg−/− Tyrobp−/− mice showed markedly elevated rolling velocities in TNF‐α‐induced inflammation in vivo. Phosphorylation of Syk and p38 MAPK following E‐selectin engagement was completely abolished in Fcrg−/− Tyrobp−/− mice. The physiologic importance of our findings is shown by near complete inhibition of neutrophil recruitment into the inflamed peritoneal cavity of Fcrg−/− Tyrobp−/− treated with pertussis toxin to block Gαi signaling. These data show that the DAP12 and FcRγ provide the ITAM domains necessary for downstream signaling and slow rolling following E‐selectin engagement.
Severity of murine arthritis induced by Freund's complete adjuvant is reduced by prior infection with the tapeworm parasite Hymenolepis diminuta
Authors: Beaurepaire Cecile , Wang Arthur , Andruski Ben , McDougall Jason , Sharkey Keith , McKay Derek
Published: June 2021

Infection with helminth parasites can prevent or reduce the severity of colitis and airway inflammation in murine models of human inflammatory bowel disease and asthma. We postulated that infection with H. diminuta, which is spontaneously expelled from mice, would ameliorate the inflammation evoked by direct injection of Freund's complete adjuvant (FCA) into the knee joint, as a model of inflammatory joint disease. Male mice were orally gavaged with 5 viable H. diminuta cysticercoids in 100μl PBS and 8 days later received FCA (50 μl) into the right knee joint. Controls: a) PBS gavage + PBS knee injection, b) H. diminuta gavage + PBS injection, c) PBS gavage + FCA injection. Knee diameter was measured over a 7 day period and on sacrifice, blood samples were collected, spleen cells stimulated for cytokine production, knee joints extracted for myeloperoxidase (MPO) activity and RT‐PCR performed for cytokine mRNA expression. Mice infected with H. diminuta were significantly protected from FCA‐induced arthritis as shown by reduced knee swelling, fewer circulating neutrophils, lower MPO levels in the joint and reduced splenocyte TNFα production. Thus, our results show that prior infection with the helminth parasite, H. diminuta, significantly protects mice from the arthritic effects of FCA. Funded by CIHR.
T lymphocytes specific for vascular smooth muscle cells in a murine model of vasculitis
Authors: Baiu Dana Carina , Hart Michael N.
Published: June 2021

We developed an inducible murine model of vasculitis. In this model, transfer of T cells to syngeneic recipients, after in vitro sensitization by co‐culture with syngeneic vascular cells, results in lymphocytic and granulomatous‐like systemic vasculitis. T cells are pathogenic in the absence of antibodies, because the vasculitis model has been successfully replicated in mice deficient in B cells (JgH‐J). We applied a limited proteomic approach combining iterative subcellular fractionation and antigen presentation to identify potential antigenic targets in vascular tissue that may contribute to T cell‐mediated inflammation. Activation of pathogenic T cells was evaluated by flow cytometry detection of early and late‐activation surface markers, intracellular cytokines and cell proliferation. Proteins from fractions enriched in endoplasmic reticulum / Golgi vesicles induced activation of CD4 T cells previously sensitized by live vascular smooth muscle cells. The data suggest that, in this mouse model, T cells specific for vascular smooth muscle autoantigens, possibly located in the endoplasmic reticulum / Golgi membrane fractions, are pathogenic for vasculitis.

NIH grant R01HL48658
Th17 Differentiation by Dendritic Cells is Dependent on IL‐13
Authors: Baldeviano Geral Christian , Barin Jobert G. , Kimura Miho , Rose Noel R. , Cihakova Daniela
Published: June 2021

Interleukin‐17 (IL‐17) drives the pathogenesis of a number of autoimmune disease models, including experimental autoimmune myocarditis (EAM). We have previously found that IL‐13 deficient (IL‐13 KO) mice developed severe EAM. To assess whether IL‐17 modulates the pathogenic response in heartss of IL‐13 KO mice, we determined the levels of IL‐17. Interestingly, IL‐17 was significantly decreased in spleen, heart (R&D Systems ELISA), and draining lymph nodes (by RT‐PCR) of IL‐13 KO mice at day 21 of EAM. Naïve IL‐13 deficient CD4+ T cells differentiated into Th‐17 cells and CD4+ FOXP 3+ T regulatory cells similar to naïve WT BALB/c T cells. However, bone marrow derived dendritic cells (BMDCs) from IL‐13KO mice were deficient in driving OVA323–339‐specific Th‐17 differentiation from naïve DO11.10 CD4+ T cells. This defect was partially restored when the BMDCs were cultured in the presence of recombinant IL‐13. IL‐13 deficient BMDCs expressed less MHCII, CD80, CD86 and CD40 upon maturation with LPS, b‐glucan curdlan, or antiCD40 mAb. Furthermore, IL‐13 deficient BMDCs produced less proinflammatory cytokines IL‐6 and TNFalpha after maturation with LPS. Thus, IL‐13 is required for DC maturation and cytokine production, which limits its ability to induce Th17. In summary, a decrease of IL‐17 in hearts of IL‐13 KO mice during EAM represents a general defect of IL‐13 KO DCs in inducing Th‐17.

Supported by 2R01 HL67290
Expansion of CD4+ regulatory T cells by BMDC in the absence of exogenous IL‐2 and TGFβ is determined by the strength of the TCR signal
Authors: Turner Michael Stuart , Morel Penelope A
Published: June 2021

Our group has previously reported that onset of Type 1 diabetes could be delayed in NOD mice by administration of BMDC grown with GM‐CSF & IL‐4 (G4DC) and enriched for high‐exfpression of MHC II and co‐stimulatory molecules, whereas DC grown with only GM‐CSF (GMDC) were not therapeutically effective. By studying the interaction of tolerogenic G4DC with CD4+ T cells from BDC2.5 TCR Tg mice, we have found that G4DC can induce proliferation of both Foxp3+ (Treg) and Foxp3− (Th) CD4+ T cells, in the absence of exogenous IL‐2 or TGFβ.

In these experiments CFSE‐labeled BDC2.5 CD4+ T cells were cultured with G4DC, pulsed with increasing doses of high‐ and low‐affinity peptide mimetopes. The presence of Treg in proliferating in and non‐proliferating T cells was assessed after 7 days by ICC for FoxP3. We observed a significant expansion of FoxP3+ Treg when low doses of specific peptides were used, whereas high peptide doses induced expansion of effector T cells. This phenomenon is not unique to self‐reactive T cells since similar results were obtained in the OVA‐specific DO11.10 TCR Tg system.

Interestingly, G4DC also induced expansion of Tregs from nontransgenic NOD CD4+ T cells, whether or not the DCs were peptide‐pulsed, suggesting that G4DC from NOD mice may present low levels of endogenous self‐antigen.

These data suggest that therapeutic G4DC function, in part, by inducing Treg expansion and that, in the absence of exogenous TGF‐β, the strength of the TCR signal is critical for Treg expansion/induction.

Funded by the American Diabetes Association (PM).
17a‐ethynyl‐5‐androsten‐3ß, 7ß, 17ß‐triol (HE3286): a novel synthetic steroid with broad based anti‐inflammatory activity
Authors: Auci Dominick Leo , Nicoletti Ferdinando , Pieters Raymond , Reading Christopher , Frincke James , Boyle David , Offner Halina
Published: June 2021

HE3286 is a novel synthetic steroid that limits acute and chronic inflammation in rodents. HE3286 limited carrageenan‐induced pleurisy, LPS‐induced NFkB activation, and TNFa and IL‐6 levels. Oral HE3286 treated established disease in models of adjuvant and collagen‐induced arthritis, experimental autoimmune encephalitis, ulcerative colitis, and collagen antibody‐induced arthritis. Dose dependent reductions were seen in disease score and levels of pro‐inflammatory cytokines including IL‐6. Unlike corticosteroids, HE3286 was not immune suppressive in classical models, including human MLR and mitogen induced lymphocyte proliferation, murine DTH, OVA immunization, and reporter antigen popliteal lymph node assays. HE3286 is currently in Phase I/II trials in patients with chronic inflammatory conditions. By possibly mimicking a key component of the body's own natural hormonal system, HE3286 may reverse acute inflammation and prevent the transition to chronic disease. This approach may be a breakthrough in treating inflammatory diseases.
Optimizing anti‐tumor immunity: Mechanisms and reversion of systemic iNKT defects in murine prostate cancer
Authors: Nowak Michael , Ilyinski Petr , Arredouani M. Simo , Sanda Martin , Balk Stephen P , Exley Mark A
Published: June 2021

Breaking tolerance to self‐antigens represents a promising treatment approach, especially for cancers of noncritical organs. Invariant natural killer T cells(iNKT) recognize glycolipids presented on CD1 molecules by dendritic and related cells and regulate immune reactions.

In numerous model studies, α‐GalCer administration activates iNKT to inhibit pre‐existing and spontaneous tumors as well as metastases.

Importantly, in multiples types of model and human cancers numerical iNKT defects have been found. Moreover, decreased iNKT numbers were accompanied by reversible functional iNKT defects such as decreased IFN‐γ production in advanced prostate cancer and progressive multiple myeloma, resulting in a deleterious Th2 cytokine response of iNKT in such patients.

We analyzed numerical and functional NKT cell defects in the murine TRAMP prostate cancer (PCa) model. In this model, probasin‐directed expression of SV40 T antigen drives the sequential development of dysplasia (prostatic intraepithelial dysplasia, PIN), locally invasive tumors, and metastatic disease resembling features of human PCa.

Tumor‐bearing 20 wks. old TRAMP mice revealed systemic decreases in iNKT numbers. Furthermore, iNKT from TRAMP mice exhibited upon α‐GalCer stimulation a clear Th2 bias both in vitro and in vivo compared to WT littermates. Since we have shown that the latter iNKT defects are reversible in vitro, we are investigating reversing these defects in vivo.

We are analyzing the mechanisms which contribute to the observed Th2 bias to optimize iNKT anti‐tumor effector function towards Th1 type in tumor‐bearing mice. Focus is examining impact on tumor progression in TRAMP mice upon adoptive transfere of in vitro expanded iNKT, as in our concurrent iNKT clinical trial for cancer.