bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐precision mapping of the protein interaction network for the human transcription machinery reveals a novel class of cellular regulatory factors
Authors: Coulombe Benoit , Cloutier Philippe , Jeronimo Célia , Al‐Khoury Racha , Lavallée‐Adam Mathieu , Blanchette Mathieu , Forget Diane
Published: June 2021

We performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Many identified interaction partners were targeted in reciprocal tagging experiments in order to confirm some interactions and to enrich the dataset. High‐confidence interactions were selected computationally using an algorithm that we developed and trained using machine learning to minimize the rate of both false‐positives and false‐negatives. The data produced with 100 affinity tagged proteins was used to (1) build a high‐definition map of interactions that connect components of the transcription and RNA processing machineries in human cells; (2) show that transcription and RNA processing factors from the soluble cellular fraction are associated with proteins that specifically regulate the formation (e.g. assembly, localization and/or stability) of protein complexes; and, (3) assign a putative function to a number of previously‐uncharacterized proteins on a ‘‘guilt by association’’ basis. A number of previously‐uncharacterized proteins that we further characterized functionally and biochemically define a novel class of regulatory factors that target RNA polymerase II and other transcription factors prior and/or after the transcription reaction on chromatin DNA.
A systems biology approach for the validation of eQTL in obesity
Authors: Estrada‐Smith Daria , Munoz Gloria , Wang Shilang , Sorensen Peter , Dobrin Radu , Rawls John , Pomp Daniel
Published: June 2021

M16 mice are polygenic obese models created by 27 generations of selection for weight gain from an ICR background. Recent transcriptional analysis of adipose and hypothalamus from ~300 M16 × ICR F2 animals identified expression variants (eQTL) and cross‐tissue co‐expression networks of coordinately expressed genes that regulate many processes including satiety, circadian oscillations and stress responses.

The purpose of this project is to experimentally validate effects of eQTL and genes in co‐expression networks on obesity phenotypes. Approaches include a Zebrafish screen of morhpholinos, over‐expression and expression knock down by RNAi in adipocyte differentiation, and sequence analysis of regulatory regions. Preliminary results suggest a significant effect of several candidate genes on fat utilization and storage in Zebrafish morpholinos. Sequence analysis of regulatory intervals show variations that alter transcription factor binding sites. These differences may underlie changes in the regulation of gene expression in obese mice. Studies on changes in adipocyte differentiation as a consequence of altered expression levels are just being initiated. Given the biological complexity of fat accretion and energy regulation, a systems biology approach utilizing cumulative statistical and experimental methods may provide needed insight into the molecular basis of obesity.
Effect of denervation on the generation of glycerol‐3‐phosphate (G3P) in brown adipose tissue (BAT) from rats adapted to a cafeteria diet
Authors: Chaves Valéria Ernestânia , Frasson Danúbia , Santos Maria Emília Soares Martins , Garófalo Maria Antonieta Rissato , Kettelhut Isis do Carmo , Migliorini Renato Hélios
Published: June 2021

Hydrolysis of stored triacylglycerol (TAG) to produce fatty acids is an obligatory step in the process of activation of heat production in BAT in both nonshivering and diet‐induced thermogenesis. There are three pathways for generation of the G3P needed for TAG synthesis and storage: glycolysis, glyceroneogenesis and glycerokinase (GyK) activity. Here we investigated the effect of BAT denervation on G3P synthesis in rats adapted to a cafeteria diet. Experiments were performed 7 days after surgical hemidenervation of BAT from rats previously fed for 3 weeks a cafeteria diet. BAT denervation blocked the increase in GyK activity (6.8±0.9 vs 14.5±1.7 nmol.mg protein−1. min−1), but did not effect the increase in glucose uptake (3.74±0.68 vs 3.84±0.47 ng.mg tissue−1. min−1) and the decrease in glyceroneogenesis, evaluated by PEPCK activity (5.8±0.7 vs 5.1±0.8 nmol.mg protein−1. min−1) and by the rate of incorporation of 14C‐pyruvate into TAG‐glycerol (2.2±0.1 vs 2.1±0.2 nmol.g BAT−1. 2h−1), induced by cafeteria diet. The data suggest that in BAT from cafeteria diet‐fed rats the generation of G3P from glucose (via glucose) and glyceroneogenesis, in contrast to GyK, are not under control of the sympathetic nervous system. Financial Support FAPESP, CNPq.
BD™ Heparin Binding Plate: An Introduction to its Applications
Authors: Wiles Michelle , Whittle Jason David , Novak Christopher
Published: June 2021

BD™ Heparin Binding Plate: An Introduction to its Applications

Until now researchers have been constrained by the inability to immobilise heparin and heparan sulphate onto solid supports without having to first modify the molecule in some way – e.g. biotinylation or conjugation to a carrier protein. The BD™ Heparin Binding Plate (formerly EpranEx), allows researchers to simply and efficiently immobilise these molecules without modification, retaining their functional ability to interact with other biomolecules. Numerous heparin fractions and a range of heparan sulphates have been successfully bound to the plate. Heparin‐binding molecules (IL8 and LINK_TSG6) were used to demonstrate the functional activity of several heparin fractions with reproducible results (n=24). Binding capacity of the plate was determined to be 200 ng/well using 35S heparin. The BD™ Heparin Binding Plate facilitates the development of novel assays to study the wide range of biological functions that involve the participation of heparin or heparan sulphate.
Anti‐angiogenic Potential of Tunicamycin is NOT Reversible by Vascular Endothelial Growth Factor VEGF165
Authors: Banerjee A , Baksi K. , Burgos M. , Banerjee D. K.
Published: June 2021

Angiogenesis is essential for tumor progression. Current approaches inhibiting angiogeneis and eliminating breast tumor growth had limited success. Our laboratory has been focusing on glycotherapeutics and studying the dynamic relationship between the lipid‐linked oligosaccharide biosynthesis and angiogenesis. We have previously observed that tunicamycin, an N‐acetylglucosaminyl 1‐phopshate transferase inhibitor arrested capillary endothelial cells in G1, developed unfolded protein response and induced apoptosis. Reduced expression of Bcl‐2, cyclin D1 and PCNA has confirmed it. To evaluate if the anti‐angiogenic potential of tunicamycin could sustain the tumor–derived cytokine(s), we have used VEGF. Cells cultured with VEGF165 (10–100 ng/ml) supported proliferation but failed to overcome the tunicamycin inhibition. Total VEGF receptor‐1 (Flt‐1) expression has remained unchanged in G1 but maximally activated after 3 hours of VEGF treatment. Total and the activated VEGF receptor were however reduced after 3 and 12 hours of tunicamycin treatment. Thus, we conclude that VEGF is unable to neutralize the anti‐angiogenic potential of tunicamycin. Supported by NIH U54‐CA096297 and Susan G. Komen Breast Cancer Foundation BCTR5820 (DKB), and NIH/NCRR/RCMI grant G12‐RR03035 (KB).
Expression and characterization of two novel transporter orthologs, BOCT1 and BOCT2
Authors: Bennett Katie M. , Stoll James
Published: June 2021

The objective of this study is to characterize two novel members of the organic cation transporter (OCT) family: BOCT1 and BOCT2. Primary aims include the expression of recombinant BOCT1 and BOCT2 protein in cells, functional characterization, and generation of monoclonal antibodies. The novel genes and two technical control transporters, OCT1 and OCTN2, were expressed as fusion proteins, stably transfected into HEK‐293 cells, and assayed for organic cation uptake. OCT1 caused a significant increase in choline (1.5‐fold), tetraethylammonium (2‐fold), and 1‐methyl–4‐phenylpyridinium (40‐fold) uptake, and OCTN2 caused a significant increase in carnitine (12‐fold) uptake. However, BOCT1 and BOCT2 did not show any effect, suggesting that they do not function in organic cation transport despite homology to the OCT family. In preparation for making monoclonal antibodies, a DNA fragment encoding ∼60 amino acids of both BOCT1 and BOCT2 was amplified via PCR and each cloned into the pRSET plasmid vector containing a 6‐His peptide tag sequence. Upon induction in E.coli, peptides accumulated in the insoluble inclusion body fractions, which were solubilized in 8M urea. The inclusion bodies were further purified by metal affinity chromatography, and the ∼12kD peptides were analyzed by SDS‐PAGE. Refolding conditions were optimized, and the peptides were prepared for immunization into mice. Funded by NIH R15 GM070578.
Structural basis of Vta1 function in the multi‐vesicular body sorting pathway
Authors: Xiao Junyu , Xia Hengchuan , Zhou Jiahai , Azmi Ishara , Davies Brian A , Katzmann David J , Xu Zhaohui
Published: June 2021

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity but its mechanism of action was poorly understood. We report the high‐resolution crystal structures of the Did2‐ and Vps60‐binding N‐terminal domain and the Vps4‐binding C‐terminal domain of S. cerevisiae Vta1. The C‐terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C‐terminal domain stabilizes the ATP‐dependent double ring assembly of Vps4. In addition, the MIT motif containing N‐terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT‐III proteins. This provides an additional level of regulation and coordination for ESCRT‐III assembly and disassembly.
The role of calcium/calmodulin‐dependent protein phosphatase (PP2B) isoforms in exocytosis in Paramecium tetraurelia
Authors: Gaines Ray , Fraga Dean
Published: June 2021

Paramecium tetraurelia are ciliated protozoa that contain membrane‐docked vesicles called trichocysts. Trichocysts fuse with the membrane in a calcium‐dependent manner and release proteins that form long crystalline spines, which help Paramecium escape predators. A bacteria‐mediated RNA interference (RNAi) protocol was used to determine if members of the calcium/calmodulin‐dependent protein phosphatase family (PP2B) present in Paramecium were involved in trichocyst discharge as suggested by previous work. After treatment, Paramecium cells were selected and exocytosis triggered by the addition of a saturated picric acid solution. After normalization, the extent of exocytosis was compared and indicated that RNAi treatment with one of the isoforms of Paramecium PP2B resulted in a reduced efficiency of exocytosis. The number of cells exhibiting full exocytosis was reduced from 76% in control cells (n=129) to 25% in PP2B treated cells (n=190). Cells exhibiting dramatically reduced exocytosis (<50% full discharge) increased from 2% (control treatment) to 41% (PP2B treatment). Based upon these results, we conclude that PP2B has a role in the regulation of calcium‐dependent exocytosis in Paramecium tetraurelia. Funding for this project was provided by the Wilson Research Awards and the Howard Hughes Medical Institute.
The Anti‐Proliferative Effect of Resveratrol in T47D Cancer Cell Line is Enhanced by VACM‐1/cul‐5
Authors: Lubbers Justin , Harper Emily , Hledin Michael P , Marquez Gabe , Burnatowska‐Hledin Maria A
Published: June 2021

Vasopressin‐activated calcium‐mobilizing (VACM‐1) receptor, a cul‐5 gene product, has been shown to inhibit cellular growth in the T47D breast cancer cell line through a mechanism that involves MAPK phosphorylation and regulation of estrogen receptor (ERα) concentration. As a cul5 gene product, VACM‐1 protein is involved in ubiquitin ligase dependent protein degradation, but factors that regulate its expression and degradation have not been identified. In our search to identify factors/drugs that control VACM‐1 expression, we studied the effects of resveratrol (3,5,4′‐trihydroxy‐trans‐stilbene) which inhibits cellular functions associated with tumor initiation, promotion, and progression by a mechanism that involves inhibition of MAPK phosphorylation, ERα activation and cell cycle arrest. In our study, control and VACM‐1 cDNA transfected T47D breast cancer cells were treated with reservatol and cell growth and VACM‐1 levels were measured. Our data demonstrate that the dose and time dependent effect of resveratol on cellular proliferation is significantly enhanced in cells transfected with VACM‐1 cDNA. Further, our results indicate that expression of VACM‐1 protein is induced by resveratrol. Consequently, these results indicate that the antiproliferative effect of VACM‐1 can be further enhanced by resveratol. This work was supported by a grant from NCI (R15CA104014) and by Dept of Biology.
IQGAP2 and Neurite Outgrowth: AKAP220 Tying the Knots
Authors: Müllendorff Karin Andrea , Tunquist Brian , Scott John D.
Published: June 2021

Akaps are A‐Kinase Anchoring Proteins that tether Protein Kinase A (PKA) and other enzymes to regulate a wide variety of cellular events by means of phosphorylation. Recent studies have suggested that members of the IQ motif GTPase Activating Proteins (IQGAP) gene family are important modulators of the actin cytoskeleton that ultimately regulate processes such as polarity, cell migration and neurite outgrowth. Here we hypothesized that AKAP220 acts as a scaffolding protein that tethers PKA and IQGAP2 to form a complex that promotes neurite outgrowth in a phosphorylation dependent manner. To test this hypothesis, we transfected primary hippocampal neurons and PC12 cells with cDNAs encoding different IQGAP2 phosphorylation mutants and measured neurite outgrowth upon neuronal differentiation. We found that PKA phosphorylates IQGAP2 in Thr716, and this is dependent on AKAP220 since its knockdown abolishes not only the phosphorylation of IQGAP2 but also the complex formed between these three proteins. Furthermore, the depletion of AKAP220 reduced neurite outgrowth in a similar manner than the IQGAP2 T716A mutant in PC12 cells, and reduced the length of the primary longest axon in primary hippocampal neurons. Taken together, these results indicate that AKAP220 modulates neurite outgrowth by acting as a scaffolding protein that regulates PKA‐mediated phosphorylation of IQGAP2 during neuronal differentiation.
Amyloid precursor protein isoforms differentially modulate amyloid‐beta metabolism
Authors: Tan Tse Mien , Lim Mei Li , Chua Li Min , Wong Boon Seng
Published: June 2021

Alzheimer's disease (AD) is the most common form of age‐related dementia. Accumulation of amyloid‐beta (Aβ) is a key neuropathological hallmark and Aβ is proteolytically derived from amyloid precursor protein (APP). There are three major APP splice forms in the human brain; APP695, APP751 and APP770, but APP695 is the predominant transcript. In AD‐afflicted human brain tissues, APP751 and APP770 expression were elevated. But it is unclear if APP isoforms have different propensity to generate Aβ. Increased expression of APP in Down syndrome (DS) is likely to account for the development of AD‐like neurological features including Aβ deposition in older DS individual. However, Aβ accumulation was not detected in trisomic DS animal model that also over‐express APP. The mechanisms underlying this discrepancy are unknown. To better understand this conundrum, we created an inducible neuronal APP‐knockout cell line that express human APP695, APP751 or APP770. We observed that the longer APP isoforms is more prone to Aβ formation. Although APP expression is a major determinant to Aβ formation, their resultant accumulation is modulated by the expression and function of enzymes involved in Aβ metabolism.
Important role of peroxynitrite‐nitric oxide interaction for S‐, N‐ and C‐nitrosation reactions
Authors: Daiber Andreas , Schildknecht Stefan , Müller Johanna , Frein Daniel , Ullrich Volker
Published: June 2021

Biological nitrosations belong to a set of redox based chemical posttranslational modifications of proteins that are involved in cellular regulation. Peroxynitrite is a central player in redox signaling and is expected to play a role in nitrosation. In earlier work we and others have suggested that nitrosation is mediated by intermediates that arise by cellular fluxes of nitric oxide and superoxide but the nature of such intermediates remained elusive. In the present study we have investigated model systems for cellular nitrosation using phenol, diaminonaphthaline and isocitrate dehydrogenase as substrates for C‐, N‐, and S‐nitrosation. The thermal aerobic decay of Sin‐1 into equal amounts of NO and superoxide was used and NONOate was added as a NO source which lead to a nitrosation maximum at a NO:superoxide ratio of 3:1. When Cu, Zn‐SOD was present during the decay of Sin‐1 a gradual shift to NO formation was indicated by use of an NO‐sensitive electrode and a SOD dependent maximum of nitrosations was obtained also in this system. Qualitatively similar results were obtained when the SOD mimetic osmium tetroxide was used. We conclude from the various systems investigated that biological nitrosations can arise from fluxes of NO and superoxide at ratios of about 3:1. It is too early to speculate on the mechanism involved but the systems presented seem to be valid models to elucidate biological nitrosation reactions.
Role of the transcription factor E47 in uncommitted hematopoietic progenitors
Authors: Borghesi Lisa , Yang Qi , Aites Jennifer
Published: June 2021

Uncommitted bone marrow precursors replenish the immune system throughout life, and it is of great interest to understand the mechanisms that control their functional integrity. We show that E47 knockout (KO) mice have major defects in multipotent LSKs, a subset that contains self‐renewing hematopoietic progenitors. We have found that LSKs from E47 KO mice have skewed levels of the flk2/flt3 cytokine receptor as well as the cell cycle regulator p21. 70% of wild type LSKs are flk2+ versus only 30% of E47 KO LSKs. Moreover, p21 expression is reduced by 50% in the latter mice. Two populations thought to be downstream of LSKs, common lymphoid progenitors (CLPs) and early thymic progenitors (ETPs) are both reduced ~4–10‐fold in E47 KO animals. While V(D)J recombinase activity, an indicator of lymphoid lineage progression, is normally expressed in ~50% of CLPs and ETPs, interruption of E47 impairs this process. We have shown that loss of E47 ablates recombinase activity in CLPs and now show that loss of E47:HEB complexes ablates recombinase activity in ETPs. Specifically, inhibition of E47:HEB leads to a 40% reduction in recombination activity as assessed at the single cell level. Together, these data indicate a key role for E47 in the functional integrity of LSKs, and the ability of these cells to effectively repopulate downstream lymphoid compartments. Support from: NIH, Elsa U Pardee Cancer Research Foundation, and USIDNET.
Human CD4+ effector memory T cells are pre‐sensitized to Fas‐induced apoptosis due to more efficient receptor signaling
Authors: Cruz Anthony C , Ramaswamy Madhu , Siegel Richard M
Published: June 2021

Naïve T cells activated through acute antigen stimulation expand and contract independently of the presence of the death receptor Fas. However, Fas‐FasL interactions play a critical role in TCR‐induced apoptosis of activated CD4+ T cells. Fas deficiency prompts the accumulation of CD44+ memory phenotype T cells in mice, suggesting Fas may play a more prominent role in apoptosis of memory T cells. The sensitivity of cells derived from naïve versus memory CD4+ T cells to Fas‐induced apoptosis has not been addressed. We compared the susceptibility of purified naïve versus memory human CD4+ T cells, both directly ex vivo and after in vitro activation and expansion, to undergo Fas‐induced apoptosis.

Remarkably, effector memory phenotype (CD27− CCR7−) cells were the most sensitive to non‐crosslinked anti‐Fas induced cell death. We have previously shown a correlation between lipid raft‐associated Fas and susceptibility to undergo apoptosis. Memory cells have increased lipid raft‐associated Fas surface protein versus naïve cells, which could account for increased sensitivity to Fas. Memory cells have more efficient assembly and activation of proximal Fas signaling components, including FADD and Caspase 8. These results suggest that effector memory cells have more efficient proximal Fas signaling and are intrinsically more sensitive to Fas‐induced apoptosis, possibly due to lipid raft localiztion of Fas.
Changes in the mature antibody repertoire may impair heterosubtypic immunity to influenza A virus infection in the aged
Authors: Schroeder H , Buckley K , Nguyen H , Schelonka R , Zhuang Y , Wang D , Brown M , Mestecky J
Published: June 2021

ΔD‐iD mice express a CDR‐H3 repertoire enriched for charged amino acids. After a heterosubtypic challenge with influenza A virus, young homozygous ΔD‐iD suffer increased morbidity and mortality. To test dominance, we challenged ten heterozygous ΔD‐iD, ten homozygous ΔD‐DFL, and ten wild‐type mice. All of the ΔD‐DFL and wild‐type mice survived, whereas eight heterozygous ΔD‐iD mice died (p<0.001). Aged individuals are particularly susceptible to influenza. We evaluated CDR‐H3 repertoire development in the bone marrow of 26mo old mice. We found that developing B cells from these aged mice failed to demonstrate the progressive enrichment for tyrosine seen in the young. Instead, almost 10% of sequences from the mature, recirculating fraction of B cells in the aged mice retained an average hydrophobicity ≤‐0.68 (p<0.05). These findings suggest a possible relationship between failure to properly regulate the CDR‐H3 repertoire and immunosenescence.
Strong expression of CD99 on human plasma cells, triggering homotypic aggregation
Authors: Park Seo‐Jeong , Park Young S. , Kang Soon‐Suk , Kim Youngji , Park Seong Hoe , Park Chan‐Sik
Published: June 2021

Plasma cells are terminally differentiated antibody producing cells. Recently, signaling molecules controlling plasma cell functions have been recognized as a crucial component of humoral immune response. We have established ex‐vivo plasma cell culture system. CD99 is a 32kD type I transmembrane protein that is highly expressed on hematopoietic precursor cells and some malignant neoplasms. CD99 is known to be involved in cell adhesion, migration, apoptosis and secretion. We report that CD99 is strongly expressed on developing human plasma cells. These findings were also consistent with immunohistochemistry and flow cytometric results on in vivo plasma cells and plasmacytoma. Engagement of the CD99 molecule by monoclonal antibodies, DN16 and YG32, triggered homotypic aggregation of plasma cell line RPMI8226. These observations suggest that CD99 may play a critical role in humoral immune response by controlling adhesion or migration of plasma cells.

This study was supported by a grant of the Korea Health 21 R & D Project, Ministry of Health & Welfare, Republic of Korea. (Project No.: A040004)
CD4+CD25+Foxp3+ regulatory T cells maintain peripheral tolerance in the context of T cell LIP through preservation of TCR diversity
Authors: Winstead Colleen Jean , Khoruts Alex
Published: June 2021

T cell diversity is a key feature of the adaptive immune system. Lymphopenia‐induced proliferation (LIP) can trigger oligoclonal expansion of T cells reacting to foreign or self‐antigens. This can result in loss of T cell diversity and compromise immune fitness that can be achieved following reconstitution by LIP. We demonstrate that CD4+CD25+Fopx3+ regulatory T cells selectively inhibit the spontaneous (fast) form of LIP, which is associated with differentiation into memory/effector‐like cells, and is likely to contain the most reactive T cell clones. Selective suppression of these clones is likely to leave more resources for T cells undergoing the slower homeostatic form of LIP. Putting it all together, we hypothesize that one of the functions of Tregs is to maintain the T cell diversity and overall immune fitness during reconstitution from lymphopenia. We are currently testing this hypothesis directly through flow cytometric analysis of TCR V beta usage, PCR‐based TCR J beta spectratyping, and sampling antigen‐specific cells by means of magnetic bead pull‐down with MHC class I and II tetramers.
FoxP3 maintains Treg unresponsiveness by selectively inhibiting the promoter DNA‐binding activity of AP‐1
Authors: Fang Deyu , Lee Sang‐Myeong
Published: June 2021

Regulatory T cells (Tregs) have been shown to play a crucial role in maintaining self‐tolerance and suppressing autoimmunity. The forkhead transcription factor, FoxP3, is a key molecule necessary and sufficient for Tregs development and function. Here we found that the promoter DNA‐binding activity of AP‐1 transcription factors is selectively inhibited in the naturally occurring CD4+CD25+ Tregs from mice. The impaired AP‐1 DNA binding is not due to the decreased nuclear translocation of AP‐1 family transcription factors including c‐Jun, JunB and c‐Fos. FoxP3 sufficiently suppresses both the transcriptional activity and promoter DNA‐binding of AP‐1 by interacting with c‐Jun. The N‐terminus of FoxP3, but not its C‐terminus forkhead domain, specifically interacts with phosphorylated c‐Jun and alters c‐Jun subnuclear distribution. This N‐terminus of FoxP3 with nuclear localization signals (FoxP3N/NLS) is sufficient to suppress AP‐1 transcriptional activity. Ecotopic expression of FoxP3N/NLS sufficiently induced the unresponsiveness of mouse primary CD4+CD25‐ T cells, while the full‐length FoxP3 is required for the suppressive functions of Tregs. These findings uncover one of the mechanisms underlying how FoxP3 maintains the unresponsiveness of Tregs.
TGFb and IL2 maintain helminth‐induced FOXP3 expression and increase relative FOXP3 levels in murine gut lamina propria CD8 T cells
Authors: Stoyanoff Korynn , Blum Arthur , Hang Long , Setiawan Tommy , Weinstock Joel V.
Published: June 2021

Intro: The immune system limits gut inflammation. H. polygyrus is a murine intestinal helminth. In lamina propria (Lp), H. polygyrus infection up‐regulates a marker of Tregs, FOXP3, in CD8+T cells and induces Th2 and regulatory cytokines. This study examined if worm‐induced cytokines enhance FOXP3 expression.

Method: Reporter mice, which express a FOXP3‐EGFP fusion protein, were infected with H. polygyrus. After 2 wks, Lp mononuclear cells (LPMC) were isolated. Unfractionated LPMC and FACS sorted EGFP+ and EGFP− T cells were cultured 48hr with or without various cytokines (IL10, TGFb, IL2, IL4). Cells then were stained for CD8 and Thy1.2 and analyzed for FOXP3 expression with FACS. Controls included cells from littermates without worms.

Results: In LPMC from worm‐infected mice, TGFb/IL2 together increased FOXP3+CD8+ T cell number and degree of expression: 2.23 ± 0.21, p = 0.002, N = 5 fold increase in number, 1.78 ± 0.18, p = 0.0065, N = 3 fold change in degree of expression. Mesenteric lymph node or spleen cells were unaffected. EGFP− LPMC cultured with TGFb/IL2 did not turn +. EGFP+ cells cultured alone lose FOXP3. TGFb/IL2 added to these cultures maintained FOXP3 expression.

Discussion: H. polygyrus infection abrogates colitis. Lp CD8+ T cells limit gut inflammation. TGFb/IL2, induced in the gut by worms, maintain but do not induce high FOXP3 levels in CD8+ Tregs and may promote regulation. (DK38327, 058755, 034928)
The mechanism of synergy between cell‐mediated and humoral immune responses in protection against acute respiratory pathogens
Authors: Kirimanjeswara Girish Soorappa , Metzger Dennis W
Published: June 2021

To maintain the sterility of the lungs, cell‐mediated and humoral responses (CMI and HI) co‐operate to rapidly eliminate pathogens. The importance of interactions between the two arms of immune responses for protection against intracellular pathogens is well appreciated but not understood. Therefore, the objective of this study was to elucidate the mechanism of synergy between CMI and HI in enhancing microbicidal activities of phagocytes. Using a mouse model of respiratory tularemia, we observed that specific antibodies in concert with IFN‐γ rapidly eliminated intracellular Francisella tularensis. Similarly, IFN‐γ‐activated, but not resting alveolar macrophages (AMs) mediated rapid killing of opsonized bacteria. Further analysis revealed that both FcγR and IFN‐γR dependent signaling events were necessary and led to enhanced RNS and ROS generation, which were also essential for bacterial killing. In addition, differential trafficking of opsonized bacteria within AMs was found to be necessary. Studies to determine the compartment where opsonized bacteria are killed by activated AMs and the cellular events that result in the formation of these compartments are currently in progress. These results establish a potential mechanism to explain the synergy between CMI and HI and provide an opportunity for intervention to augment the efficacy of antibodies and vaccines.

PO1 AI056320

U54 AI057158

γ
Dysregulated expression patterns of pro‐and anti‐inflammatory biomarkers in the intestinal mucosa define phenotypic subsets of Crohn's Disease
Authors: Kaetzel Charlotte S , Bruno Maria E.C. , Stefka Andrew , McMahan Adam , Arsenescu Razvan
Published: June 2021

A dysregulated inflammatory response to gut microbes has been implicated in the etiology of Crohn's Disease (CD). Identifying specific biomarkers may enhance our understanding of CD and suggest targeted therapies. Mucosal biopsies from 30 CD patients and 18 normal controls were analyzed for mRNA for NF‐κB/RelA; A20, a negative regulator of NF‐κB; the polymeric immunoglobulin receptor (pIgR), TNF; IL‐8; and serum IgA, which is elevated when pIgR‐mediated epithelial transport is compromised. Principal component analysis identified an “anti‐inflammatory” factor, with high expression of RelA, A20 and pIgR, moderate TNF and IL‐8, and low serum IgA, and a “pro‐inflammatory” factor with high expression of IL‐8 and TNF and low RelA and A20. Individuals were grouped into Set 1 (high anti‐ and low pro‐inflammatory scores), Set 2 (low anti‐ and pro‐inflammatory scores) and Set 3 (high anti‐ and pro‐inflammatory scores). Set 1 comprised normal subjects and CD patients with mild disease and good responses to therapy. Patients in Set 2 had moderate to severe disease, with repeated flares and poor responses to immunosuppressive and anti‐TNF therapy. Patients in Set 3 presented with severe inflammatory disease, but responded promptly and durably to therapy. We conclude that classification of CD patients by these biomarkers may predict disease behavior and responses to therapy. Supported by the NIH, CCFA, and AstraZeneca.
TLR2 ligation reveals differences in cytokine secretion and signaling properties of dendritic cells from distinct anatomical sites and murine strains
Authors: Wilder Julie , Hernandez‐Hansen Valerie , Gott Katherine , Carter Mark , Crowder Brandy , Lipscomb Mary
Published: June 2021

Clearance of Cryptococcus neoformans (Cne) is genetically determined and influenced by the route of inoculation in mice. C.B‐17 mice clear the organism after pulmonary inoculation by mounting a T helper (Th) 1 response whereas C57BL/6 (and B10.D2) mice fail to clear Cne and develop a Th2 response. In contrast, C57BL/6 mice are resistant and C.B‐17 mice susceptible to systemic inoculation of Cne. As dendritic cells (DCs) are the initiators of the adaptive immune response, we compared lung and splenic DCs from C.B‐17 and B10.D2 mice. DCs were stimulated with the Toll‐like receptor 2 (TLR2) agonist fixed Staphylococcus aureus (SAC) to study their pattern of inflammatory cytokine secretion as well as their signaling properties. Splenic DCs from C.B‐17 and B10.D2 mice secreted comparable amounts of IL‐12 when stimulated with SAC, while lung DCs from C.B‐17 mice produced significantly higher amounts of IL‐12 compared to those from B10.D2 mice. In addition, the activity of phosphatidylinositol‐3‐kinase (PI3K) was significantly upregulated in B10.D2 lung DC compared to C.B‐17 lung DC. In contrast, C.B‐17 lung DC stimulated with SAC support greater phosphorylation of the proliferative kinase, extracellular‐regulated kinase 1/2 (ERK1/2). These data suggest that differentially regulated TLR signaling pathways in lung and spleen DCs may play a role in whether a Th1 or Th2 immune response is initiated.
T helper dependent CTL migration into the vaginal mucosa
Authors: Nakanishi Yusuke , Lu Bao , Gerard Craig , Iwasaki Akiko
Published: June 2021

Cytotoxixc T lymphocyte (CTLs) play a critical role in controlling virus infection. However, little is known regarding how CTLs migrate into the mucosal tissues and mediate antiviral host defense. Here, we addressed the mechanism of CTL migration into the mucosal site. By using mouse model of vaginal herpes simple virus (HSV) 2, we demonstrated that HSV‐specific CTLs were localized in the vaginal epithelia and migration of HSV‐specific CTLs depends on the presence of CD4 help. In addition, although division and IFN‐γ production by HSV‐specifc CTLs in the draining lymph node normally occurred in the absent of CD4 help, there was a decrease in the accumulation of these cells in the helpless mice. In the absence of CD4 T help, secretion of IFN‐γ and IFN‐inducible chemokines were diminished at the infection site. Importantly, HSV‐specific CTLs deficient in the chemokine receptor CXCR3 failed to migration into the vaginal mucosa after infection. These observations indicated that CD4 help is required to support CTL migration to the site of infection by inducing appropriate chemokines secretion in situ.
Dietary fat modulates intestinal absorption of luminal gut antigens
Authors: Eckhardt Erik , Ghoshal Sarbani , Ward Martin , Woodward Jerold , de Villiers Willem
Published: June 2021

BACKGROUND: Mesenteric lymph nodes (MLN) are important for proper immune responses to gut antigens. Dietary long‐chain fat stimulates mesenteric lymph flow and lymphatic secretion of chylomicrons (CM) which have been shown to bind lipopolysaccharides (LPS) and amphiphilic peptides.

HYPOTHESIS: CM formation promotes intestinal absorption of LPS and peptidic antigens.

METHODS: Antigen absorption was measured in mice and CaCo‐2 cells, absorbed ovalbumin (OVA) was detected by adoptive T‐cell (DO11.10) transfer.

RESULTS: Fasted mice gavaged with LPS absorbed 3‐times more LPS into blood and MLN when subsequently gavaged with triolein than with tributyrin. This was abolished by an inhibitor of CM formation, Pluronic L‐81. Absorbed LPS was associated with plasma CM remnants. CaCo‐2 cells pretreated with LPS secreted more LPS from cell‐associated pools into basolateral medium when stimulated with oleic acid than with butyric acid; this was also Pl‐81 sensitive. MLN P38 MAPK phosphorylation was higher after gavage with triolein than with tributyrin. Balb/C mice adoptively transfered with CFSE‐labeled DO11.10 CD4 T‐cells and gavaged with OVA and triolein had markedly more CFSE‐labeled cells in MLN and spleen than mice gavaged with OVA and tributyrin; T‐cell proliferation was also elevated.

CONCLUSION: Dietary fat may affect the intestinal immune system by modulating absorption and MLN delivery of gut antigens.
Retinal laser burn alters immune regulation of the eye
Authors: Lucas Kenyatta G. , Qiao Hong , Stein‐Streilein Joan
Published: June 2021

It is known that immune privilege allows for immune protection in the eye in the absence of inflammation. Only a few events have been reported to overcome the immune privileged mechanisms in the eye. Here we report the effect of retinal laser burn (RLB) on immune privilege in the anterior chamber. As early as 6hr post RLB and as late as 21D, antigen inoculation into the anterior chamber failed to induce ACAID induction in both the eye that received the RLB and the contralateral eye. Aqueous humor samples harvested from naïve eyes but not from either the burned and the non‐burned eye (24 hrs post RLB) were able to down regulate the expression of CD40 or up regulate IL‐10 mRNA in peritoneal exudates cells (PEC) and convert them to tolerogenic antigen presenting cell (APC). RTPCR analyses of mRNA extracted from experimental and control eyes, showed little or no change in TGFβ. Increasing use of lasers in both industrial and medical fields raises the risk of losing immune regulation in the eye and adds the subsequent risk of being a candidate for autoimmune uveitis. This work was supported in part by grants to JSS: DOD W81XWH and NIH EY11983; KL: NIH T32 EY07145.
Lytic granule‐mediated, noncytolytic CD8+ T cell inhibition of HSV‐1 reactivation from neuronal latency
Authors: Knickelbein Jared E. , Hendricks Robert L.
Published: June 2021

The purpose of our studies is to identify and characterize the mechanisms employed by HSV‐specific CD8+ T cells (HSV‐CD8) to block HSV‐1 reactivation from latency in sensory ganglia, potentially limiting recurrent herpetic disease. HSV‐CD8 can secrete IFN‐γ to inhibit reactivation in some, but not all neurons, suggesting involvement of additional effector mechanisms. We now show that the lytic granule components perforin (Pfn) and granzyme B (GrB) are necessary for optimal protection from HSV‐1 reactivation in sensory neurons both in vivo and in ex vivo trigeminal ganglia (TG) cultures. We further establish that HSV‐CD8 exhibit vectorial release of lytic granules into immune synapses formed with both susceptible fibroblasts and latently infected neurons in ex vivo TG cultures. Importantly, while lytic granule release activates the caspase system of fibroblasts leading to morphologic signs of apoptosis, similar lytic granule release does not induce caspase activation or morphologic signs of apoptosis in neurons. Together our findings establish that directed lytic granule release by HSV‐CD8 is required to block neuronal HSV‐1 reactivation, that lytic granules block HSV‐1 reactivation in neurons without activating caspases or inducing apoptosis, and that GrB is one (but apparently not the only) lytic granule component required to block HSV‐1 reactivation.

Supported by NIH, Eye & Ear Foundation, & RPB.
The vaccinia virus A35R protein inhibits antigen presentation and suppresses cytokine synthesis by responding CD4+ T cells
Authors: Rehm Kristina Elizabeth , Connor Ramsey , Jones Gwendolyn , Mannie Mark , Roper Rachel L
Published: June 2021

An adaptive immune response is initiated when an antigen presenting cell (APC) presents foreign antigen via major histocompatibility complex class II (MHC II) molecules to antigen‐specific CD4+ T cells. Many viruses, such as poxviruses, have evolved strategies to evade the host adaptive immune response. The poxvirus A35R protein is conserved in all 98 sequenced mammalian tropic poxviruses and has little homology to any non‐pox protein, suggesting an important and novel function. The A35R protein was not required for viral replication in any cell tested thus far, but was required for full virulence in the mouse model. It was therefore hypothesized that the A35R protein regulated aspects of the host immune response. In a model antigen presentation system, the presence of A35R caused a decrease in the amount of numerous cytokines that are produced during antigen presentation. Further data indicated that it is the APCs, and not the T‐cells, that are directly affected by A35R. Furthermore, A35R appears to localize to an endosomal compartment within the APC, where it can regulate many aspects of the MHC II antigen processing and presentation pathway. Because A35R is immunosuppressive, its removal from live virus vaccines should create more immunogenic vaccine strains.
Long‐Distance Tunneling Nanotubules Shuttle Immunoglobulin Class Switch‐Suppressing Factors from HIV‐Infected Macrophages to B Cells
Authors: Xu Weifeng , Santini Paul A , Shan Meimei , Chen Kang , Ball Susan C , Chiu April , Kingsley Lawrence A , Rinaldo Charles R , Chadburn Amy , Knowles Daniel M , He Bing , Cerutti Andrea
Published: June 2021

Class switching from IgM to IgG and IgA is essential for systemic and mucosal immune responses against viruses and requires activation of follicular B cells by CD4+ T cells through CD40‐CD40L interaction. The mechanism by which HIV evades virus‐specific IgG and IgA responses remains unclear. We found that lymphoid follicles from HIV‐infected patients contained Nef, an early viral protein involved in AIDS development. In these follicles, long‐distance membrane tethers shuttled Nef from HIV‐infected macrophages to B cells. This process was associated with decreased signaling of CD40 via NF‐κB and with loss of activation‐induced cytidine deaminase (AID), a CD40‐inducible enzyme essential for class switching. In vitro studies showed that macrophages productively infected by HIV or transfected with a Nef‐GFP‐expressing construct were capable of translocating Nef to B cells via actin‐propelled and ATP‐dependent membrane channels, including long‐range tunneling nanotubules. Transfer of Nef to B cells required a clathrin‐dependent pathway that involved the guanine nucleotide exchange factor Vav as well as downstream Rho, Rac and Cdc42 small GTPases. Once in B cells, Nef inhibited CD40L‐induced AID expression and subsequent IgG and IgA class switching. These studies indicate that HIV hijacks a physiological intercellular connectivity network to generate B cell‐intrinsic defects and evade humoral immunity.
A Novel Immune Evasion Mechanism by Human Alphaherpesviruses
Authors: Ku Chia‐Chi , Che Xi‐Bing , Rajamani Jaya , Marvin Sommer H. , Shaap‐Nutt Anne , Reichelt Mike , Huang Ke‐Jung , Tu Chao‐Jung , Arvin Ann
Published: June 2021

Type I interferon induced MxA has antiviral activity against RNA viruses. Here we demonstrate a previously unrecognized MxA isoform induced by herpes simplex virus‐1 (HSV‐1) in human fibroblasts. Transcript encoding MxA splice variant has a deletion in Exons 14–16 which encode a central interactive domain associated with recognition of viral nucleocapsids and is predicted to encode a novel peptide sequence at the C‐terminal region as compared with IFN ‐induced MxA prototype. Polyclonal antiserum raised in rabbit against C‐terminal half of MxA splice variant demonstrated that HSV‐1 induced variant MxA undergoes nuclear translocation associated with viral replication compartment. Infection of variant MxA‐overexpressing fibroblasts with HSV‐1 resulted in the enhancement of viral yields but the yields were decreased in MxA‐knockdown cell by siRNA, suggesting that HSV‐1 induced MxA splice variant enhances viral replication. Both IFN‐ α and HSV‐1 are able to activate the promoter of MxA in luciferase reporter assay; however, only the MxA variant is translated in virus‐infected cells. Thus, differential expression of MxA controlled by IFN‐α or HSV‐1 could result in the increase of host resistance or favoring viral replication, depending on stimulating event. In summary, modulation of cellular mRNA could be a novel mechanism for human alphaherpesviruses to avoid innate immune detection.
Delayed antigen‐specific responses and increased disease activity in lupus‐prone mice following influenza infection
Authors: Crowe Sherry R , Ash Linda L , James Judith A
Published: June 2021

Influenza virus infections result in significant morbidity and mortality especially in individuals with autoimmune disorders, such as systemic lupus erythematosus. It is unclear, however, whether this is due to innate immune defects or immunosuppressive drugs. To address this question, we utilized the MRL/lpr‐fas mouse model of disease and examined the susceptibility, antigen‐specific response, and disease development following intranasal influenza infection. The lupus‐prone mice had significantly increased weight loss and lung pathology following infection as compared to controls indicating increased susceptibility. Additionally, the lupus‐prone mice had delayed kinetics of anti‐influenza antibodies and a significant delay in the anti‐influenza responses as measured by IFNγ production. While there was not a significant increase in the concentration of anti‐ANA or dsDNA antibodies in the MRL/lpr‐fas mice, there was a dramatic increase in infiltrating cells in the kidneys. These data indicate that there is a delay in antigen‐specific immune responses and increased disease activity following infection of the MRL/lpr‐fas mouse strain. Current studies are underway to examine these parameters in other lupus‐prone strains, such as BXSB and NZM2410. These models will provide a unique system to dissect the immune‐pathogen interaction in an autoimmune disease setting. NCRR: 5P20RR020143
Analysis of germinal center responses after intranasal and intraperitoneal challenge with influenza virus
Authors: Boyden Alexander , Scroggins Sabrina , Legge Kevin , Waldschmidt Thomas
Published: June 2021

The primary germinal center (GC) response in the murine spleen is characterized by a steady state reaction. The ratio of non‐switched (IgM+) to switched (IgM−) GC B cells and the percentage of founder GC B cells remains steady over the course of the response. To date however, we have examined GC responses only after intraperitoneal challenge with experimental antigens (SRBC, NP‐KLH and Phycoerythrin) under Th2 conditions. To test the universality of the steady state GC model, splenic responses were examined after challenge with influenza virus. Not only is influenza a natural antigen, but it activates B cells under Th1 conditions. BALB/c mice were therefore immunized with H1N1 influenza virus using either an intranasal or intraperitoneal route, followed by multiparameter analysis of splenic GC B cells at selected time points. To date, results have demonstrated that splenic GC responses after intraperitoneal challenge exhibit steady state characteristics, similar to GCs induced with other experimental antigens. However, splenic GC responses after intranasal challenge exhibit a more dynamic pattern at later time points. These results suggest that route of viral challenge influences the regulatory environment in the spleen and the behavior of GC responses.
Multigenic mechanisms ensure T cell contraction and prevent mortality during persistent infections
Authors: Schmidt Nathan , Khanolkar Aaruni , Harty John
Published: June 2021

After infection or vaccination, antigen(Ag)‐specific T cells undergo a proliferative expansion phase that is followed by an abrupt contraction or death phase where 90–95% of the expanded populations are lost. Although the molecular details are unclear, T cell contraction is thought to preserve immune homeostasis by preventing the accumulation of memory T cells specific for one or a few pathogens. In addition, contraction may prevent immunopathology that could occur with prolonged or persistent infections, although this point has not been substantiated by experimental evidence. Recent studies demonstrate that contraction in T cell numbers is largely unaffected by treatments that shorten the duration of infection or under circumstances of persistent infection. However, it is not known if the mechanisms that regulate T cell contraction are similar after acute and persistent infections. Here we show that, in contrast to acute infections, prolonged and persistent infections stimulate continued T cell proliferation during the contraction phase. However, the actions of either perforin or TNF are sufficient to ensure T cell contraction and to prevent immunopathology in response to a persistent infection. The commitment of multigenic mechanisms to ensure T cell contraction underscores the importance of this process to prevent immunopathology during persistent infection.