bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective human cysteine protease inhibition mediates Ixodes scapularis blood feeding success
Authors: Kotsyfakis Michail , Karim Shahid , Anderson Jennifer , Andersen John , Valenzuela Jesus , Mather Thomas , Ribeiro Jose
Published: June 2021

Ixodes scapularis is the main vector of Lyme disease in the eastern United States. These hematophagous arthropods have developed unique strategies to circumvent vertebrate defense mechanisms and among them, saliva secretion in the sites of infestation has been shown as important for both blood meal completion and pathogen transmission. Here we report a duplication event of cystatin genes in tick genome ‐ named as sialostatins L and L2 ‐ that results in a transcription regulated boost of saliva inhibitory activity against a relatively limited number of vertebrate papain like cysteine proteases during blood meal. Tick treatment with sialostatin L2 dsRNA resulted immediate rejection and subsequent death of 40% of the silenced ticks, when attached on a rabbit. The rest 60% of the silenced ticks that managed to attach to the rabbit they weighed 3 times lesser than the control group after completion of their meal. Given the role of the targeted enzymes in vertebrate immunity, we uncover that host immunomodulation is implicated in the deleterious phenotype of silenced ticks making I. scapularis cystatins attractive targets for the development of anti tick vaccines. Indeed vaccination of guinea pigs with sialostatin L2 protein leads to their protection against tick blood feeding, while on‐going experiments aim to show whether such a vaccination affects Lyme disease transmission as well.
KLF5 enhances SREBP‐1 action in androgen‐dependent induction of FASN in prostate cancer cell line
Authors: Lee Min‐Young , Kim Hee Eun , Park Byeong‐Woo , Kim Kyung‐Sup
Published: June 2021

The Kruppel like factor 5 (KLF5) is a zinc‐finger transcription factor that plays important roles in regulating all aspects of cellular signaling involved in cell proliferation and oncogenesis. In the present study, KLF5 interacts with sterol regulatory element‐binding protein‐1 (SREBP‐1) in regulating the expression of lipogenic protein, fatty acid synthase (FASN), which plays important roles in androgen‐dependent prostate cancer cells. Genes involved in lipid metabolism, including FASN, are reported to be up‐regulated by androgens in prostate cancer cells through a coordinated cascade controlled by the activation of SREBP‐1. The promoter and intron 1 region of FASN gene contains multiple GC‐boxes that might show response to KLF5. Deletion and mutation analyses indicated that KLF5 binds to the FASN gene and strongly induces its activity. Furthermore, KLF5 protein is able to bind to SREBP‐1 and enhance the SREBP‐1 regulated activation of FASN. Transfection of KLF5 into prostate cancer cells enhanced FASN promoter activity, and conversely, silencing of KLF5 by siRNA down‐regulated FASN expression. The multiple GC motifs are scattered throughout the upstream sequence and first intron of FASN gene whereas these are scarce in the promoters of other lipogenic and cholesterogenic genes such as acetyl‐CoA carboxylase, ATP‐citrate lyase, LDL receptor, HMG‐CoA synthase and HMG‐CoA reductase, which makes FASN gene very unique among them. Taken together, FASN gene could be activated by the synergistic action of KLF5 and SREBP‐1 which is increased by androgen treatment.
Network‐based identification of critical transcription regulators in the metabolic syndrome in mice
Authors: Wu Sulin , Tran Linh , Liao James C , Lusis Aldons Jake , Drake Thomas A
Published: June 2021

Systematic approaches that integrate genetic and gene expression data have been employed to elucidate gene interactions underlying traits. In this study, 53 transcription factors and 460 of their know target genes are analyzed by employing network component analysis (NCA) method to infer transcription factor activities (TFAs). Linkage analysis was applied with the TFA data to map the genetic determinates that regulate the activity of individual transcription factors. Chromosome regions controlling the level of activity of a particular transcription factor are mapped as TFA quantitative trait loci (tQTLs). In this study, we report the sexually dimorphic TFAs in somatic tissues of mice. These transcription factors exhibited highly tissue‐specific patterns of activity and most of them have varying activity levels that are significantly correlated with clinical traits involved in the metabolic syndrome. Genetic analysis provided evidence of the reuglators of sexually dimorphic transcription factors, as the activities of certain transcription factors were controlled by tQTL hotspots that exhibited tissue‐/sex‐specific pattern. Several transcription factors (Myb, Bcl6, Ets2, Fli1, Cebpa, Rarb and Sfpi1) are found to be associated with metabolic traits and investigation of their biological roles in metabolism may reveal molecular mechanism involved in metabolic syndrome in mouse.
Excessive phosphorylation of STATs leads to reduced chondrocyte proliferation and shortened bones in mucopolysaccharidosis VII mice
Authors: Metcalf Jason Andrew , Hilton Matthew , Zhang Yanming , Tittiger Mindy , Ma Xiucui , Haskins Mark , Long Fanxin , Ponder Katherine Parker
Published: June 2021

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease in which a deficiency in β‐glucuronidase activity results in glycosaminoglycan (GAG) accumulation in the growth plate, causing shortened long bones in various species through unclear mechanisms. Immunohistochemistry and RNA in‐situ hybridization showed that MPS VII mouse growth plates accumulate massive amounts of the GAG chondroitin‐4‐sulfate (C4S), have 53% of normal chondrocyte proliferation, express reduced levels of hypertrophic chondrocyte genes, and exhibit excessive phosphorylation of STAT proteins. In contrast, MPS I mice have less GAG storage and their bones and growth plates are nearly normal. Phosphorylated STATs are thought to mediate bone shortening in achondroplasia, a disease in which STATs are excessively phosphorylated by overactivation of fibroblast growth factor receptor 3 (FGFR3). Therefore, it was postulated that accumulation of C4S overstimulates FGFR3 in MPS VII. However, MPS VII mice that are FGFR3 deficient had shortened bones, suggesting that FGFR3 is not required for the bone defect. We propose that MPS VII bone disease is caused by a chondrocyte proliferation defect that may be mediated by STATs phosphorylated by an alternative pathway. Funding provided by Howard Hughes Medical Institute, Ryan Foundation, Mucopolysaccharidosis Society, and NIH (DK066448 and DK54481).
Naphthalene assimilation in Pseudomonas fluorescens: plasmid localization of the gene for OmpW
Authors: Neher Tracy Marie , Lueking Donald R.
Published: June 2021

Naphthalene, and other polycyclic aromatic hydrocarbons (PAHs), are listed as pollutants by the EPA. Naphthalene has been shown to be catabolized by a plasmid‐encoded pathway, however, the precise mechanism utilized for assimilation remains unclear. Previous studies have suggested that Pseudomonas fluorescens utilizes an active transport system for naphthalene assimilation. In this regard, examination of a differentially expressed cDNA library from naphthalene grown cells revealed the presence of a number of transport related proteins. One of these proteins, OmpW, has been proposed to function as a porin for hydrophobic molecules, although its specific function is unknown. The gene for this 22 kD protein has been cloned, sequenced and its cellular localization evaluated. In addition, the large catabolic plasmid possessed by P. fluorescens has been isolated and a cured strain of P. fluorescens has been generated by incompatibility curing. By using oligonucleotide primers specific for OmpW, the gene encoding OmpW has been shown to be specifically localized to the naphthalene catabolic plasmid possessed by P. fluorescens. Cured strains of P. fluorescens do not possess their large catabolic plasmid, do not grow on naphthalene and do not possess the gene encoding OmpW. These results provide circumstantial evidence for the involvement of OmpW in naphthalene metabolism. Support: MTU Incentive Account E35597
The high affinity choline transporter: topology and choline binding site as studied by site‐directed mutagenesis
Authors: Urashima Kousuke , Kubo Yuji , Nishikawa Shizue , Nakamura Toru , Hayashi Kengo , Yamada Haruhiko , Osawa Chieko , Okuda Takashi , Haga Tatsuya
Published: June 2021

The high affinity choline transporter (CHT1) is expressed in cholinergic neuron. CHT1 takes up choline and provides it for acetylcholine synthesis. CHT1 has two putative glycosylation sites (N69, N301). We found that N69Q mutant was glycosylated as well as wild type CHT1, but N301Q mutant was not glycosylated and not expressed on the cell surface. We have constructed a number of mutants substituted with cysteine and examined the accessibility of impermeable SH reagent to each cysteine residue in these mutants expressed in HEK293 cells. Obtained results indicated that CHT1 has the extracellular amino terminus and 13 transmembrane segments. Several acidic or basic amino acid residues in the putative third and 12th transmembrane segments were indicated to be involved in the choline uptake activity. In particular, the E451D mutant had approximately five times higher Km for choline uptake and five times lower Kd for HC3 binding as compared with wild type hCHT1, indicating that the E451 residue is involved in recognition of choline and HC3.
A New Link in the Biosynthesis and Transport of Lipid A ‐ Interaction of MsbA and LpxK
Authors: Six David A. , Ma Bing , Raetz Christian R.H.
Published: June 2021

Gram‐negative bacteria depend on the biosynthesis and transport of lipopolysaccharide (LPS) for viability, and thus, both pathways contain important targets for antibiotic development. Tantalizing evidence suggests that certain enzymes involved in the biosynthesis and transport of lipid A, the hydrophobic anchor of LPS, may interact. The genes that encode the lipid A biosynthetic 4'‐kinase, LpxK, and the inner membrane transporter, MsbA, overlap in many species, including Escherichia coli. Interestingly, predicted sequences from marine organisms contain fused msbA and lpxK genes. Several of these marine fusion proteins do not heterologously express in E. coli. However, a fusion of E. coli MsbA and E. coli LpxK, joined by a protein linker sequence, expresses well in E. coli and possesses both MsbA and LpxK activities. Moreover, this fusion protein is able to complement an E. coli null mutant of both msbA and lpxK. The functional N‐terminally‐His‐tagged fusion protein was confirmed to be present as a single 104‐kDa fusion protein. These findings suggest that MsbA may directly interact with LpxK. This hypothesis is supported by immunoprecipitation experiments confirming that Flag‐tagged LpxK pulls down His‐tagged MsbA. Further studies will investigate whether the MsbA‐LpxK interaction contributes to the optimal function of LPS biosynthesis and transport and whether MsbA and LpxK are part of a larger biosynthetic complex localized to the inner membrane. This research was supported by National Institutes of Health Grant GM‐51310 to C. R. H. Raetz.
Effect of CI‐976 on ferritin secretion and viability in Aedes aegypti
Authors: Geiser Dawn L. , Shen Meng‐Chieh , Mayo Jonathan J. , Winzerling Joy J.
Published: June 2021

In mammals, iron taken into somatic cells is stored in ferritin or incorporated into iron containing proteins. Very little ferritin is found circulating in mammalian blood; most is retained in the cytoplasm. Ferritin is a multimer of 24 subunits of heavy and light chains. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored to prevent oxidative damage. We are studying how mosquitoes and mosquito cells deal with this iron load. Iron treatment of A. aegypti larval CCL‐125 cells induces ferritin co‐localization with the Golgi apparatus and discrete vesicles within the cytoplasm. When these cells are exposed to high levels of iron, iron accumulates in the membranes. In contrast to mammalian cells, mosquito cells secrete ferritin into the culture medium in response to iron treatment. In addition, a 59Fe pulse‐chase experiment showed that both membrane and secreted ferritin were loaded with iron. To evaluate the effects of preventing ferritin secretion in the presence of iron load, we tested several known secretory inhibitors. Treatment of CCL‐125 cells with CI‐976 and iron disrupts ferritin secretion and decreases cell viability. Studies are underway to determine the effects of CI‐976 and RNAi on mosquito viability and fecundity following the iron load of a blood meal. This work was supported by funds from the National Institutes of Health (GM056812), the Agricultural Experiment Station, the College of Agriculture and Life Sciences and the BIO5 Institute for Collaborative Bioresearch at The University of Arizona.
Characterization of cholesterol metabolism and apolipoprotein E glycosylation in the liver of a Niemann‐Pick Type C1 animal model
Authors: LIM Mei‐Li , CHUA Ching‐Ching , HO Jacqueline Zyet‐Ee , WONG Boon‐Seng
Published: June 2021

Niemann‐Pick type C1 (NPC1) disease is a cholesterol‐storage disorder characterized by liver dysfunction. NPC1 is highly expressed in the liver and the protein regulates the transport of cholesterol from late endosomes/lysosomes to other cellular compartments involved in intracellular cholesterol homeostasis. Another major protein involved in cholesterol metabolism is apolipoprotein E (ApoE). ApoE is also highly expressed in the liver and function as a major component of lipoprotein in cholesterol homeostasis. Altered ApoE expression is associated with defective low‐density lipoprotein receptor (LDL‐R) and may cause hypercholesterolemia. Earlier studies have shown that ApoE is differentially sialylated. However, the functional significance of this chemical alteration on ApoE and its association with cholesterol homeostasis is unknown. In this study, we will present the correlation between the glycan profile on hepatic ApoE and cholesterol metabolism at early and late stages of pathology in a NPC1 animal model. In addition, we will show the connection between changes in cholesterol metabolism with the biochemical profile of high‐density lipoprotein (HDL), LDL and triglycerides at different stages of disease development in the liver.
Expression and regulation of ERp57 in hepatocellular carcinoma
Authors: Grindel Brian J. , Farach‐Carson Mary Cindy , Bennett Joseph , Snapp Erik
Published: June 2021

ERp57/PDIA3, a 1,25D3 binding protein with thiol‐oxidoreductase activity, resides in the endoplasmic reticulum (ER) owing to a C‐terminal QEDL. ERp57 partitions among cell surface, cytoplasm and nucleus with functions including 1,25D3 binding, ion transport, and redox modulation of transcription. A tissue array immunoscreen of normal and diseased liver sections showed dynamic changes in expression in both cytoplasm and nucleus. To understand expression of ERp57 in hepatocellular carcinoma (HCC), its steady state distribution was assessed in HepG2 cells using: 1) GFP (green fluorescent protein) tagged ERp57 and, 2) subcellular fractionation. We also measured the response to various treatments including PMA, 1,25D3, or TNF‐α. High resolution imaging demonstrates localization of ERp57‐GFP in the ER along with additional peri‐nuclear staining. A small (<5%) fraction of ERp57 also exists in the cytosol. Co‐treatment with PMA, alone and in combination with 1,25D3 for times from 15′ to 1 h did not reorganize EPp57‐GFP. In contrast, TNF‐α treatment of HepG2 cells moved cytoplasmic ERp57‐GFP to the nucleus in 15 min. Ongoing studies will determine if ERp57 binds to NFκB in HepG2 cells or in normal hepatocytes after TNF‐α treatment. These studies will shed light on redox regulation of transcription by nuclear ERp57 during TNF‐α signaling in normal and transformed liver cells. Supported by HHMI and the CTCR‐UD.
Activation of NFkB with Serine Phosphorylation by Otitis Media Pathogens in Epithelial Cells
Authors: Moon Sung K. , Woo Jeong‐Im , Pan Huiqi , Lee Haa‐Yung , Gellibolian Robert , Lim David J.
Published: June 2021

Rel/NFkB proteins are required for regulating genes related to inflammation. Recently, we showed that activation of p65 is involved in nontypeable Haemophilus influenzae (NTHi)‐induced MCP‐1 up‐regulation in the spiral ligament fibrocytes (Moon et al, 2007). We aim to elucidate the specific subunits of NFkB activated upon exposure to otitis media (OM) pathogens such as NTHi and S. pneumoniae (SP) and to identify the phosphorylated serine residue of the NFkB subunits involved. Luciferase assay was performed with vectors containing multiple copies of the NFkB consensus sequence, demonstrating that NFkB is activated upon exposure to OM pathognes. Immunolabeling showed that p65, p105/p50 and Rel‐B were translocated to the nucleus upon exposure to NTHi, SP or TNF. In contrast, p100/p52 and c‐Rel were not activated upon exposure to OM pathogens. Immunoblotting showed that Ser536 of p65 and Ser933 of p105 are phosphorylated upon exposure to NTHi, SP or TNF. Interestingly, Ser468 and Ser276 of p65 were phosphorylated by SP or TNF, but not by NTHi. TNF showed a faster kinetics in activating NFkB than NTHi or SP. Gene Arrays demonstrated that SP up‐regulates IkBa and RhoA compared to NTHi. It is suggested that a variety of signaling pathways are involved in OM pathogen‐induced NFkB activation. Identifying the pathogen‐activated NFkB with signaling pathways will enables us to better understand host‐pathogen interactions.
The first crystal structure of cyclic GMP‐dependent protein kinase Iβ dimerization domain reveals the molecular features of an extended leucine/isoleucine zipper
Authors: Kim Choel , Smith‐Nguyen Eric V. , Casteel Darren E. , Sankaran Banumathi , Spraggon Glen , Pilz Renate B. , Taylor Susan S.
Published: June 2021

Subcellular targeting of cyclic GMP‐dependent protein kinase (PKG) provides an important mechanism for achieving substrate specificity and is mediated by the N‐terminal dimerization (ND) domain, which binds to defined sets of protein substrates in an isotype dependent manner. Although ND of PKG Iβ has previously been shown to provide a unique docking surface for TFII‐I and IRAG, the structural detail of this protein‐protein recognition surface was unknown. In order to understand the subcellular targeting mechanism of PKG Iβ and the molecular detail of the protein docking motif, we solved a crystal structure of the ND of PKG Iβ at 2.1 angstrom. The structure reveals two N‐terminal helices warping around each other into a left‐handed helix and forming an extended leu/ile zipper with 10 pairs of leu/ile packing in a “knobs‐into‐holes” manner. The interaction interface consists of negatively charged residues surrounding a pronounced hydrophobic patch. Surprisingly, the structure also reveals two positively charged residues at the d positions (rather than at the canonical g positions) forming interhelical electrostatic interactions with two negatively charged residues at the e positions in a mirror symmetry fashion. The electrostatic pairs flanking the interaction interface may aid in stabilizing the docking surface crucial for specifically binding IRAG and TFII‐I.
Analysis of transcription and protein changes in response to human RET activation in SKN‐SH and transfected COS cells.
Authors: Torres Martha Angelina , Poku Maryann , Vega Quinn
Published: June 2021

RET is a mammalian receptor tyrosine kinase normally activated after association with a protein co‐receptor GFR‐a (1–4) and its corresponding ligand (GDNF, Neurturin, Artemin or Persephin). RET is required for nervous system development and RET mutations are associated with both cancer (MEN2A, MEN2B and FMTC) and developmental abnormalities (Hirschsprung's Disease). In order to analyze how the cell responds to RET activation, isoform expression, internalization and downstream transcription were measured in RET expressing cells. QPCR was used to measure the production of the two major isoforms of RET‐RET9 and RET51. In these experiments, RET 9 levels increased ∼ 4‐fold in comparison to RET 51. With respect to internalization, the loss of RET was identified over time with a loss of the RET protein by 6 hours. With respect to signaling, downstream luciferase promoter assays demonstrated that wild type RET increased transcription over phosphotyrosine mutants and kinase inactive RET. Future experiments will confirm these changes and study the effect of both specific tyrosines and naturally occurring mutations on RET signaling.

(Supported by NIH Grant R15‐NS048043)
Gene Expression Patterns and Pathway Functions of SEB Induced human PBMC
Authors: Campbell Katherine , Zahrte Darin , Jett Marti , Mendis Chanaka
Published: June 2021

Staphylococcal Enterotoxin B (SEB) is one of several exotoxins produced by staphylococcus aureus that may have the ability to cause lethal shock in humans. We have successfully tracked and located signal transduction events induced by SEB by further investigating specific gene expression profile induced by SEB in human Peripheral Blood Mononuclear Cells (PBMCs). Prior work done in our lab has identified p38 as a key signal transduction component of multiple signal transduction pathways induced by SEB in human PBMCs. Our strategy was to compare the gene expression profile of SEB to the p‐38 inhibitor (SB203580) by using Reverse Transcription Polymerase Chain Reactions (RT‐PCR). Comparison of the two gene profiles (with and without the inhibitor) together with the protein profiles allowed us to validate the effectiveness of the p38 inhibitor as a potential target to block SEB induced signaling activities in human PBMCs.
Interplay between TRPC3 and SNAREs regulate Neurosecretion
Authors: Bollimuntha sunitha , Watt John A , Singh Brij B
Published: June 2021

Ca2+ is undoubtedly the lead player when it comes to vesicle fusion and exocytosis, but the source and the signals for Ca2+ influx may vary. The C3 Transient Receptor Potential Canonical (TRPC3) channel is a store operated Ca2+ channel which was shown previously to interact with synaptobrevin II ‐ a key protein in vesicle exocytosis. The objective of our study was to investigate the role of TRPC3 in vesicle fusion and neurosecretion in Vasopressin (VP) producing neurons of the hypothalamic Supraoptic nucleus. The VP laden neurosecretory vesicles are present all over the neuronal axis and the axonal endings terminating in the neural lobe (NL) of pituitary gland. Immunohistochemistry as well as electron microscopy demonstrate the localization of TRPC3 to the VP neurosecretory vesicles and plasma membrane. Co‐Immunoprecipitation studies with NL and PC12 cells reveal an activation dependant interaction of TRPC3 with synaptobrevin II, Syntaxin1A, and SNAP25. Interaction of TRPC3 with SNARE components is dynamic and is abolished in the absence of Ca2+ or by blocking TRPC3 with SKF96365. The physiological significance of these interactions in NL and PC12 cells respectively is underscored by an increase VP and human growth hormone release following TRPC3 activation. These results put forward a plausible role of TRPC3 in a neurosecretory paradigm.
Phosphodiesterase (PDE) activity measured by micro‐isothermal calorimetry; a study of cyclic nucleotide analogues as substrates and inhibitors of PDEs1a, 1b, 1c, 2, 4, 5, 6, 8, 9, and 10
Authors: Hinds Thomas Richard , Poppe Heiko , Tang Xiao‐Bo , Rybalkin Sergei D , Butt Elke , Beavo Joseph A
Published: June 2021

Identification of cellular signal transduction pathways that are hypothesized to be regulated by cyclic nucleotides often have been probed using various cyclic nucleotide analogues. However, the effect that these probes have on different PDEs has in general not been studied. Since radioactive analogs are not available, we employed micro isothermal calorimetry to measure the heat of hydrolysis of the analogs. Analogues that did not serve as substrates were tested to determine if they could be inhibitors. We show that this assay method gives similar results to the standard radioactive assay using tritiated cyclic nucleotides. Somewhat unexpectedly we find that many of the 11 most commonly used cyclic nucleotide analogues can act as substrates for multiple PDEs and most of the others are inhibitors. We also find that these analogues can inhibit different PDEs by various mechanisms including competitive, non‐competitive, and linear mixed kinetics. The kinetics obtained suggests that the active sites of dimeric PDEs must interact with one another in order to display non‐competitive and linear mixed kinetics. In addition, we provide evidence that inhibition of PDEs with so‐called selective analogs can indirectly increase both PKA and PKG mediated phosphoylation. Since these analogs can inhibit PDEs and thereby increase cAMP or cGMP, it is clear that care must be taken in the interpretation of results obtained when they are used for studying cellular regulatory pathways.
Genesis of tear duct‐associated lymphoid tissue is independent of Id2, RORγt but requires Cbfβ2 transcriptional regulator
Authors: Nagatake Takahiro , Fukuyama Satoshi , Tachibana Masashi , Taniuchi Ichiro , Kim Dong‐Young , Takamura Kaoru , Sato Shintaro , Kunisawa Jun , Kiyono Hiroshi
Published: June 2021

We have previously reported the presence of tear duct‐associated lymphoid tissue (TALT) in the murine lacrimal sac and demonstrated that the organogenesis program of TALT was distinctively different from other secondary lymphoid organs including Peyer's patch (PP) and nasopharynx‐associated lymphoid tissue (NALT). Indeed, developmental program of TALT did not require a common tissue genesis program family of Id2, RORγt and LTα1 β2/LTβR‐mediated signals. However, essential molecule for the TALT genesis remained to be identified. Transcriptional factor complexes composed of Cbfβ2 and Runx1 were recently shown to play an important role for development of PP and peripheral lymph nodes (pLNs) by regulating early differentiation of lymphoid tissue inducer (LTi) lineage cells in fetal liver. Here we demonstrate that mice deficient for Cbfβ2 lack both TALT and PP, while mice deficient for P1‐Runx1 isoform exhibited normal TALT development with decreased numbers of PP. These findings suggest conserved functions of Cbfβ2 in the common developmental program of secondary lymphoid tissues, and an involvement of other Runx protein(s) as a partner of Cbfβ2 in initiating TALT development.
Identification of age‐related changes in both hematopoietic progenitors and thymic stromal components contributing to thymopoietic capacity
Authors: Thoman Marilyn Louise
Published: June 2021

Thymopoiesis dramatically decreases with age. Hematopoietic progenitors as well as the thymic structural cells undergo functional alterations with age. To dissect the contribution of each of these thymic components to the overall process of involution, a series of bone marrow‐irradiation chimeras were constructed in which either young or aged host mice were reconstituted with bone marrow progenitors derived from young or aged donor mice. Both donor type progenitors are equal in reconstituting young host thymuses as measured by total cellularity. In contrast, the aged thymic environment was less well reconstituted by either type donor. Further the aged bone marrow was less effective than the young‐derived progenitors in this environment. Specifically the thymus in an aged host reconstituted with young bone marrow progenitors was 4 fold larger than that reconstituted with aged bone marrow. This size differential was found from 4–24 weeks post transplant. Subset analysis further reveals that the aged progenitors within the aged thymic microenvironment are deficient in the production of mature CD4 and CD8+ T cells. In competitive repopulation assays, aged progenitors are clearly deficient. Together the results indicate that interaction with young thymic stromal elements overcomes at least some age‐associated functional deficiencies in T cell progenitors.

Support NIH RO1AG09948 and RO1AG028077 to MLT
Inhibition of Ectopic Adipocyte Accretion within Aging Thymic Environment Stimulates Thymopoiesis
Authors: Dixit Vishwa Deep , Youm Yun‐Hee , Rim Jong‐Seop , Sun Yuxiang , Smith Roy , Smith Steven , Nakata Chiaki
Published: June 2021

With advancing age, the thymic lymphoid and stromal cell microenvironment is progressively replaced with adipocytes, leading to reduction in output of naive T cells in a process recognized as thymic involution. Using magnetic resonance imaging we demonstrate that compared to 25 year old healthy adults, by 45 years of age greater than 75% of human thymus is replaced with adipose tissue. Herein, we report an age‐related increase in epithelial to mesenchymal transition (EMT) in thymus and further differentiation of thymic fibroblasts to adipocytes by activation of PPARγ, and aP2 pro‐adipogenic pathways. The stromal cells undergoing adipogenic transformation were unable to support the T cell development of lymphoid progenitors. The thymus specific inhibition of PPARγ, aP2 by means of pro‐longevity intervention, caloric restriction reversed adipogenic progression. Reduction of EMT genes in aging thymus by CR led to regeneration of cortical epithelium and thymic vessels at cortico‐medullary junction with increased early thymocyte progenitors naïve T cell output and improvement of peripheral T cell receptor diversity. Up regulation of EMT genes and specific gain of function of PPARγ and aP2 pathway in thymic stromal cell in absence of ghrelin signaling increased thymic involution. Collectively, these data suggest that inhibiting the pro‐adipogenic pathways within thymus could offer a new mechanism to forestall thymic aging and promote renewal.
Identification of two early differentiated populations within CD4+ and CD8+ TEMRA T‐cells
Authors: Derhovanessian Evelyna , Koch Sven , Haehnel Karin , Pawelec Graham
Published: June 2021

Antigen‐experienced T‐cells can be divided into functionally distinct populations, according to the expression of different cell surface markers, such as CD45RA, CCR7, CD27 and CD28. Effector T‐cells re‐expressing CD45RA and lacking the chemokine receptor CCR7 (TEMRA cells), are considered to be late differentiated cells, with limited proliferative but high functional capacity. Using 10‐color flow cytometry, we subdivided circulating CD4+ and CD8+ effector memory (EM) (CCR7−, CD45RA−) and TEMRA (CCR7−, CD45RA+) cells of healthy individuals and cancer patients according to their surface expression of CD27 and CD28, as well as CD57, a marker up‐regulated on very late‐differentiated T‐cells. In both cohorts, we identified two subsets of early differentiated CD27+CD28+ and CD27+CD28‐lymphocytes, with very low CD57 expression in both the CD4+ and CD8+ TEMRA population, whereas most of the CD28−CD27− TEMRA cells were strongly positive for this putative marker of T cell senescence. Further functional analysis, combined with cell surface phenotyping showed that these two CD27+ TEMRA populations represent early‐differentiated T‐cells with higher proliferative capacity and lower IFN‐γ secretion compared to the late differentiated CD28−CD27− TEMRA cells. This work was supported by EC‐LSHG‐CT‐2007–036894 “LifeSpan” and DFG‐SFB685‐B4.
CD28 enhances in vivo clonal expansion by CD4+ T cells without increasing sensitivity to antigen
Authors: Pagán Antonio José , Moon James J. , Chu H. Hamlet , Pew Marion Pepper , Jenkins Marc K.
Published: June 2021

Costimulatory signals are required for optimal CD4+ T cell immunity. In vitro studies and experiments with monoclonal T cells have shown that ligation of CD28 by B7 enhances CD4+ T cell proliferation, effector function, and survival upon priming. One model posits that CD28 acts by amplifying signals transduced through the T cell receptor (TCR). A prediction of this model is that increasing TCR signal strength would compensate for the defective T cell proliferation observed in the absence of CD28. We tested this prediction by using peptide:major histocompatibility complex II tetramers and magnetic bead enrichment to measure the clonal expansion of polyclonal normal or CD28‐deficient T cells in response to subcutaneous immunization with a wide range of antigen doses emulsified in Freund's Adjuvant. CD28‐deficient and normal T cells accumulated to a peak level at a similar antigen dose. However, the number of CD28‐deficient T cells present at the peak dose was 5‐fold lower than the number of normal T cells. Furthermore, intravenous or subcutaneous immunization with a high dose of soluble antigen plus lipopolysaccharide did not increase the numbers of antigen‐specific CD28‐deficient T cells above that of naïve mice. These data suggest that CD28 is essential for T cells to respond at all to soluble antigen and enhances T cell responsiveness to emulsified antigen by a mechanism that does not involve TCR signal amplification.
Regulation of the differentiation of IL‐17‐secreting CD8 T cells
Authors: Yeh Norman , Kaplan Mark
Published: June 2021

Inflammatory immune responses are regulated by T cell subsets that secrete specific panels of cytokines. Similar to CD4+ T cells, CD8+ T cells can differentiate into Tc1 and Tc2 subsets that secrete IFNã and IL‐4. Recently IL‐17‐secreting CD8+ T cells have been described, though the relationship of these cells to other CD8+ T cell subsets or Th17 cells is unclear. We have found that like Th17 cells, IL‐17 secretion from Tc17 cells is dependent on Stat3. Compared to IL‐12 induced Tc1 cells, Tc17 cells have significantly lower expression of Eomesodermin and T‐bet and Tc17 programming is inhibited by Stat1 and T‐bet. Expression of perforin, Granzyme B, and Fas ligand, genes responsible for cytotoxicity, is lower in Tc17 cells than in Tc1 cells and remains low in the absence of Stat3, Stat1, and T‐bet suggesting that these factors do not negatively regulate cytotoxicity genes in Tc17 cultures. These data suggest that Tc17 cells share requirements for transcription factor regulation with Th17 cells and may differ significantly in their function from Tc1 cells.
CD4 T Cells That Promote Extrafollicular B Cell Responses
Authors: Poholek Amanda , Odegard Jared , Craft Joseph
Published: June 2021

The role of follicular helper T cells (TFH) in development and maintenance of the germinal center is well known; however the role of T cells in the extrafollicular B cell response is less understood. We have described a novel subset of activated IL‐21 producing CD4 T cells that are located at extrafollicular sites in murine lupus. These cells are CXCR5lo and are marked by downregulation of P selectin glycoprotein ligand 1 (PSGL1). To determine if the IL‐21+ CXCR5lo PSGL1lo population exists in a normal immune response, we used adoptive transfer of ovalbumin (ova) specific TCR transgenic CD4 T cells to track their development following ova immunization. We find that PSGL1lo cells arise following ova immunization, with significant expansion when the antigen is administered chronically. Like TFH cells, this population requires ICOS for its development; however, unlike the former, B cells do not appear to be required. In the absence of the PSGL1lo population, isotype switched antibody production to ova is lost. These cells make IL‐21; however, only a subset makes IL‐4 and is CXCR5hi. By microscopy, PSGL1lo cells are found in the B cell follicle and at extrafollicular sites. Based on this data, we hypothesize that IL‐21+ CXCR5lo PSGL1lo cells are important for extrafollicular responses. We have termed this novel population T helper extrafollicular cells or THEF cells.
Tristetraprolin down‐regulates E47 mRNA stability in old splenic murine B cells
Authors: Blomberg Bonnie B , Landin Ana Marie , Alvarez Juan P , Riley Richard L , Frasca Daniela
Published: June 2021

The E2A‐encoded transcription factor E47, which regulates class switch recombination (CSR) in splenic B cells, is down‐regulated in aged murine B cells, due to increased E47 mRNA decay. We have previously shown that part of the decreasedstability of E47 mRNA seen in aged B cells is mediated by tristetraprolin, TTP, which binds AU‐rich elements in the 3′‐untranslated region (3′‐UTR) of mRNAs leading to their rapid degradation. Levels of TTP are higher and phosphorylation of TTP is lower in old B cells than in young B cells, generating more mRNA degradation. The pro‐B cell line 107.2 transfected with the TTP‐siRNA suppressed TTP and led to enhancedaccumulation of E47 mRNA. These results altogether suggest that TTP plays a relevant role in the degradation of E47 mRNA and in the control of CSR and somatic hypermutation of Ig, critical for high affinity Ig and optimal response to pathogens. We have also introduced the coding region of E47 (lacking the 3′‐UTR) by retroviral transduction of young and old splenic activated B cells. This over‐expression of E47 up‐regulated activation‐induced cytidine deaminase and CSR in both young and old, restoring the old levels to that of normal young levels.

This work is supported by NIH AG‐17618, AG‐23717 (BBB) and AG025256 and AI064591 (RLR)
Aging B cells show decreased proliferative capacity but are able to express normal levels of activation markers
Authors: Blaeser Anthony Ryan , Vogel Laura
Published: June 2021

Aging is associated with a dysfunction in the humoral immune response. This dysfunction may be due to intrinsic B cell defects or defects in the microenvironment. Previous in vivo studies by our lab found decreased antigen‐induced expansion by aged B cells but could not rule out the possibility of decreased migration. In the current study we examine early activation events of B cells from middle aged (12 month) and older (16 month) mice in response to LPS and anti‐CD40 stimulation in vitro. There was no significant difference in the ability of middle aged or older B cells to up‐regulate CD86 and CD38 and to down‐regulate CD69 when compared to their young (2–3 mo) counterparts. Middle aged B cells show a slight decrease, however, in the formation of blast cells at both 24 and 48 hours while old B cells appear to produce higher numbers of blast cells at 24 hours and become dysfunctional at 48 hr. There also was a slight decrease in proliferative response in both middle aged and older B cells when compared to young. Thus, middle aged and older B cells show comparative abilities to up and down‐regulate surface markers but show some defects in their response to stimulation. Funding provided by NIH grant.
Increase in CD4+ / CD25+ / Fox P3+ Regulatory T Cells following chemical sympathectomy with 6‐Hydroxydopamine
Authors: Bhowmick Sourojit , Singh Anurag , Thrall Roger S , O’Rourke james , Cone Robert E
Published: June 2021

Ablation of the Sympathetic Nervous System (SNS) by systemic 6‐Hydroxydopamine (6‐OHDA) enhances the production of IgM antibodies, depresses hypersensitivity and the production of CD 8+ Regulatory T Cells. Here we investigated the effect of chemical sympathectomy induced by 6‐OHDA on CD4+ / CD25+ / Fox P3+ Regulatory T Cells (T Regs) in C57BL/6 (B6) mice. Two days after i.p injection of 6‐OHDA there was a significant increase (40–60%) in the number of T‐Regs in the spleen and inguinal lymph nodes as seen by flowcytometry. This increase in T‐Reg number in 6‐OHDA‐ treated mice did not occur in mice that received desipramine, which prevents the uptake of 6‐OHDA by the nerve terminals. However contact sensitivity to trinitrophenol (TNP) could not be induced in 6‐OHDA treated mice. The suppression of anti CD3‐induced in vitro proliferation of T Effector Cells by T Regs from 6‐OHDA‐treated donors was quantitatively equivalent to that from donors receiving PBS. These results suggest that ablation of the SNS can induce or expand systemic T Regs.
Antigen specificity acquisition of CD4+ regulatory T cells via acquired pMHC I complexes
Authors: Xiang Jim
Published: June 2021

Antigen‐specific CD4+ regulatory T (Tr) cells have important roles in preventing autoimmune diseases, but they also suppress effective anti‐tumor immunity. However, the molecular mechanisms regulating their acquisition of antigen specificity, particularly with respect to CD8+ T cells, are as yet unclear. In this study, we generated CD4+ Tr cells by stimulating naïve T cells derived from ovalbumin (OVA)‐specific T cell receptor transgenic OT II mice with OVA‐pulsed dendritic cells (DCOVA) that had been transduced with an IL‐10‐expressing adenoviral vector (DCOVA/IL‐10). These DCOVA/IL‐10 secreting IL‐10 induced CD4+ Tr cells that secreted IFN‐γ ‐and IL‐10, but not IL‐4. We showed that these CD4+ Tr cells acquired OVA peptide/major histocompatibility complex class I (pMHC I) following either in vitro or in vivo stimulation with DCOVA/IL‐10. We also demonstrated that IL‐10 secretion by these pMHC I‐carrying CD4+ Tr cells was critical to their suppression of OVA‐specific CD8+ T cell responses and antitumor immunity. However, the pMHC I complexes acquired by the CD4+Tr cells also importantly affected for their activity, enhancing their abilities to suppress OVA‐specific anti‐tumor cytotoxic T lymphocyte responses in vivo by ≈ 700% relative to analogous CD4+Tr cells incapable of expressing pMHC I complexes (i.e., Kb−/−Tr). The above conclusion has also been confirmed by the evidence that the non‐specific CD4+25+ Tr cells become antigen specific and contribute enhanced suppressive effect via acquired pMHC I by uptake of DCOVA‐released exosomes carrying pMHC I. Taken together, our data suggest that CD4+Tr cells that have acquired pMHCI complexes much more efficiently target antigen‐specific CD8+ T cells in vivo, and that this may be an important means of augmenting the antigen‐specificity of CD4+Tr cells in general.
Salmonella enterica serovar Typhimurium Induces Rapid Migration of Dendritic Cells into the Gut Lumen
Authors: Bertelli Eugenio , Regoli Marì , Clare Simon , Hautefort Isabelle , Hinton Jay , Ivory Kamal , Nicoletti Claudio , Arques Juan
Published: June 2021

Dendritic cells (DC)s are key elements for the immune responses and can operate as antigen sampling cells. We report now that they can also migrate into the gut lumen upon bacterial challenge. Isolated intestinal loops in BALB/c mice, C57/BL6 and C57/BL6 MyD88 knock out (KO) mice were infected with non invasive GFP‐labelled S. Typhimurium strain SL1344 (?SPI1, rpsM‐gfp), a derivative of the same strain lacking SPI1 and both fliC and fljB flagellin encoding genes, E. coli DH5a (/pZEP16, rpsM‐gfp), or PBS. After three hours, luminal content was harvested, washed and DCs were separated. FACS was carried out to characterize and enumerate the recovered cells. Confocal microscopy (CM) and TEM were also performed.

Approximately 7 × 104 cells were present in loops treated with flagellated S. typhimurium SL1344, but not in loops belonging to the C57/BL6 MyD88 KO mice. Phenotypic analysis showed the presence of CD11c+, CX3CR1+, MHCII+, CD8a− and CD11b− cells associated with Salmonella. CM confirmed the increase of CD11c+ DCs in the gut lamina propria. TEM and CM showed bacteria associated with luminal DCs. These data show that Salmonella induces flagellin‐ and Toll‐like receptor‐dependent DC migration into the gut lumen.
Decoding Immunodominance of an Antiviral CD8+ T Cell Response
Authors: Kotturi Maya , Scott Iain , Wolfe Tom , Peters Bjoern , Sidney John , von Herrath Matthias G. , Buchmeier Michael J. , Grey Howard , Sette Alessandro
Published: June 2021

The primary CD8+ T cell response against lymphocytic choriomeningitis virus (LCMV) infection of C57BL/6J mice is directed against 28 H‐2b‐restricted epitopes derived from the glycoprotein, nucleoprotein and RNA polymerase L protein. Within these 28 epitopes a clear immunodominance hierarchy is observed. Some major epitopes dominate the response, while some epitopes are intermediate and others are relatively minor contributors to the overall response. To probe the mechanisms regulating CD8+ T cell immunodominance, we manipulated the number of epitopes seen during acute LCMV infection. Deletion of epitope‐specific responses, either by using a LCMV viral variant lacking 4 major epitopes, or C57BL/6J mice altogether lacking expression of H‐2Kb, resulted in an overall decrease of the response, minor compensatory effects, and no detectable responses against new epitopes. Increasing the number of epitopes available for recognition by use of H‐2b F1 hybrid mice, or delivery of LCMV antigens by recombinant vaccinia virus also did not dramatically alter the immunodominance pattern. These data suggest that immunodominance hierarchy might be intrinsic to the epitope and its antigen of origin. We are currently investigating these issues by directly measuring the hierarchies in MHC binding affinities, the yield of epitope production as a result of cellular processing, and by quantifying the existing naïve T cell repertoire by the use of a sensitive tetramer‐based enrichment method.
Differential induction of CD4 and CD8 T cell responses to coxsackievirus B3
Authors: Kemball Christopher , Harkins Stephanie , Whitton J Lindsay
Published: June 2021

Coxsackievirus B (CVB) infections can cause considerable morbidity and mortality in the human population, and are the most common infectious source of myocarditis. Previous studies suggest that CVB3 induces minimal CD8 T cell responses, and it is unclear whether CVB3 infection drives a CD4 T cell response. In this study, we generated a recombinant CVB3 (rCVB3.6) that encodes both a CD8 and CD4 T cell epitope derived from lymphocytic choriomeningitis virus (LCMV), which we used to investigate CVB3‐specific CD4 T cell induction, and to compare the efficiency of MHC Class I and II antigen presentation. Highly sensitive in vivo and ex vivo assays did not detect endogenous CVB3‐specific CD8 and CD4 T cell responses during acute infection. However, LCMV‐specific memory CD8 and CD4 T cells expanded in number following secondary challenge with rCVB3.6, suggesting that both epitopes were presented during CVB3 infection. Using a transgenic T cell adoptive transfer model, we found that CVB3‐specific CD4 transgenic T cells proliferated in rCVB3.6‐infected mice, whereas CVB3‐specific transgenic CD8 T cells did not divide. In summary, these data show that there is a substantial difference in the efficiency of MHC Class I versus Class II antigen presentation during CVB3 infection, and in the generation of virus‐specific CD8 versus CD4 T cell responses. This work was supported by NIH R01 AI‐42314 (J.L.W.) and T32 NS41219‐06 (C.C.K.).
CD62Lhigh IL7Rhigh CD4+ T cells with memory potential are primed early following infection with Leishmania major
Authors: Colpitts Sara Lynn , Dalton Nicole , Rodriguez Carlos , Scott Phillip
Published: June 2021

The development of vaccines against diseases requiring cell‐mediated immunity has been hampered by our lack of understanding of the memory T cells required to maintain immunity. Our lab has shown that a population of CD62Lhigh central memory T (TCM) cells mediates long‐term immunity to the intracellular protozoan Leishmania major. Our current studies are aimed at how TCM cells develop following infection. Using both polyclonal and transgenic donor T cells, we have identified two populations of Leishmania‐specific CD4+ T cells that emerge early following infection. One population has undergone greater than six rounds of division, expresses low levels of CD62L, and produces IFNγ in accordance with an effector phenotype (TEFF). We believe the second population to be themselves, or the precursors of, TCM cells based on their expression of high levels of CD62L and CCR7, which mediate lymph node (LN)‐homing, the IL‐7R, which promotes memory T cell survival, and the production of IL‐2. Sorting the two populations and restimulating them in vivo with antigen revealed only the CD62Lhigh cells could survive, consistent with their potential to become memory T cells. We have also found that while both cell types are primed in the first few days following infection, TEFF cells emerge from cells resident in the draining LN at the time of infection, and cells that are recruited into the LN at later times contribute to the TCM population.