Publications by authors named "Tomozumi Imamichi"

59 Publications

Interleukin-27 promotes autophagy in human serum-induced primary macrophages via an mTOR- and LC3-independent pathway.

Sci Rep 2021 Jul 21;11(1):14898. Epub 2021 Jul 21.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Building 550, Room 126, P.O. Box B, Frederick, MD, 21702, USA.

Interleukin-27 (IL-27) is a cytokine that suppresses human immunodeficiency virus (HIV)-1 infection in macrophages and is considered as an immunotherapeutic reagent for infectious diseases. It is reported that IL-27 suppresses autophagy in Mycobacterium tuberculosis-infected macrophages; however, a role for IL-27 on autophagy induction has been less studied. In this study, we investigated the impact of IL-27 in both autophagy induction and HIV-1 infection in macrophages. Primary human monocytes were differentiated into macrophages using human AB serum (huAB) alone, macrophage-colony stimulating factor (M-CSF) alone, or a combination of IL-27 with huAB or M-CSF. Electron microscopy and immunofluorescence staining demonstrated that a 20-fold increase in autophagosome formation was only detected in IL-27 + huAB-induced macrophages. Western blot analysis indicated that the autophagosome induction was not linked to either dephosphorylation of the mammalian target of rapamycin (mTOR) or lipidation of microtubule-associated protein 1A/1B-light chain 3 (LC3), an autophagosomal marker, implying that IL-27 can induce autophagy through a novel non-canonical pathway. Here we show for the first time that IL-27 induces autophagy during monocyte-to-macrophage differentiation in a subtype-dependent manner.
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http://dx.doi.org/10.1038/s41598-021-94061-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295388PMC
July 2021

Correction: Hu et al. Profiles of Long Non-Coding RNAs and mRNA Expression in Human Macrophages Regulated by Interleukin-27. 2019, , 6207.

Int J Mol Sci 2021 May 12;22(10). Epub 2021 May 12.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

The authors wish to make the following corrections to this paper [...].
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http://dx.doi.org/10.3390/ijms22105129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151633PMC
May 2021

Cytoplasmic-translocated Ku70 senses intracellular DNA and mediates interferon-lambda1 induction.

Immunology 2021 Jul 7;163(3):323-337. Epub 2021 Mar 7.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD, USA.

We have previously identified that human Ku70, a nuclear protein, serves as a cytosolic DNA sensor. Upon transfection with DNA or infection with DNA virus, Ku70 translocates from the nucleus into the cytoplasm and then predominately induces interferon lambda1 (IFN-λ1) rather than IFN-alpha or IFN-beta, through a STING-dependent signalling pathway. However, a detailed mechanism for Ku70 cytoplasmic translocation and its correlation with IFN-λ1 induction have not been fully elucidated. Here, we observed that cytoplasmic translocation of Ku70 only occurred in DNA-triggered IFN-λ1-inducible cells. Additionally, infection by Herpes simplex virus type-1 (HSV-1), a DNA virus, induces cytoplasmic translocation of Ku70 and IFN-λ1 induction in a strain-dependent manner: the translocation and IFN-λ1 induction were detected upon infection by HSV-1 McKrae, but not MacIntyre, strain. A kinetic analysis indicated that cytoplasmic translocation of Ku70 was initiated right after DNA transfection and was peaked at 6 hr after DNA stimulation. Furthermore, treatment with leptomycin B, a nuclear export inhibitor, inhibited both Ku70 translocation and IFN-λ1 induction, suggesting that Ku70 translocation is an essential and early event for its cytosolic DNA sensing. We further confirmed that enhancing the acetylation status of the cells promotes Ku70's cytoplasmic accumulation, and therefore increases DNA-mediated IFN-λ1 induction. These findings provide insights into the molecular mechanism by which the versatile sensor detects pathogenic DNA in a localization-dependent manner.
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http://dx.doi.org/10.1111/imm.13318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8207419PMC
July 2021

MicroRNA Profiles in Monocyte-Derived Macrophages Generated by Interleukin-27 and Human Serum: Identification of a Novel HIV-Inhibiting and Autophagy-Inducing MicroRNA.

Int J Mol Sci 2021 Jan 28;22(3). Epub 2021 Jan 28.

Laboratory of Human Retrovirology and Immunoinformatics, Applied and Development Research Directorate, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

Interleukin-27 (IL-27) is a pleiotropic cytokine that influences the innate and adaptive immune systems. It inhibits viral infection and regulates the expression of microRNAs (miRNAs). We recently reported that macrophages differentiated from human primary monocytes in the presence of IL-27 and human AB serum resisted human immunodeficiency virus (HIV) infection and showed significant autophagy induction. In the current study, the miRNA profiles in these cells were investigated, especially focusing on the identification of novel miRNAs regulated by IL-27-treatment. The miRNA sequencing analysis detected 38 novel miRNAs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that IL-27 differentially regulated the expression of 16 of the 38 miRNAs. Overexpression of the synthesized miRNA mimics by transfection revealed that miRAB40 had potent HIV-inhibiting and autophagy-inducing properties. B18R, an interferon (IFN)-neutralization protein, partially suppressed both activities, indicating that the two functions were induced via IFN-dependent and -independent pathways. Although the target mRNA(s) of miRAB40 involving in the induction of both functions was unable to identify in this study, the discovery of miRAB40, a potential HIV-inhibiting and autophagy inducing miRNA, may provide novel insights into the miRNA (small none-coding RNA)-mediated regulation of HIV inhibition and autophagy induction as an innate immune response.
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http://dx.doi.org/10.3390/ijms22031290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865382PMC
January 2021

Profiles of MicroRNAs in Interleukin-27-Induced HIV-Resistant T Cells: Identification of a Novel Antiviral MicroRNA.

J Acquir Immune Defic Syndr 2021 Mar;86(3):378-387

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD.

Objectives: Interleukin-27 (IL-27) is known as an anti-HIV cytokine. We have recently demonstrated that IL-27-pretreatment promotes phytohemagglutinin-stimulated CD4(+) T cells into HIV-1-resistant cells by inhibiting an uncoating step.

Purpose: To further characterize the function of the HIV resistant T cells, we investigated profiles of microRNA in the cells using microRNA sequencing (miRNA-seq) and assessed anti-HIV effect of the microRNAs.

Methods: Phytohemagglutinin-stimulated CD4(+) T cells were treated with or without IL-27 for 3 days. MicroRNA profiles were analyzed using miRNA-seq. To assess anti-HIV effect, T cells or macrophages were transfected with synthesized microRNA mimics and then infected with HIVNL4.3 or HIVAD8. Anti-HIV effect was monitored by a p24 antigen enzyme-linked immunosorbent assay kit. interferon (IFN)-α, IFN-β, or IFN-λ production was quantified using each subtype-specific enzyme-linked immunosorbent assay kit.

Results: A comparative analysis of microRNA profiles indicated that expression of known miRNAs was not significantly changed in IL-27-treated cells compared with untreated T cells; however, a total of 15 novel microRNAs (miRTC1 ∼ miRTC15) were identified. Anti-HIV assay using overexpression of each novel microRNA revealed that 10 nM miRTC14 (GenBank accession number: MF281439) remarkably suppressed HIV infection by (99.3 ± 0.27%, n = 9) in macrophages but not in T cells. The inhibition was associated through induction of >1000 pg/mL of IFN-αs and IFN-λ1.

Conclusion: We discovered a total of 15 novel microRNAs in T cells and characterized that miRTC14, one of the novel microRNAs, was a potent IFN-inducing anti-HIV miRNA, implicating that regulation of the expression of miRTC14 may be a potent therapeutic tool for not only HIV but also other virus infection.
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http://dx.doi.org/10.1097/QAI.0000000000002565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879852PMC
March 2021

Genome-wide association study of high-sensitivity C-reactive protein, D-dimer, and interleukin-6 levels in multiethnic HIV+ cohorts.

AIDS 2021 02;35(2):193-204

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland.

Objectives: Elevated levels of interleukin-6 (IL-6), D-dimer, and C-reactive protein (hsCRP) are associated with increased incidence of comorbid disease and mortality among people living with HIV (PLWH). Prior studies suggest a genetic basis for these biomarker elevations in the general population. The study objectives are to identify the genetic basis for these biomarkers among PLWH.

Methods: Baseline levels of hsCRP, D-dimer, and IL-6, and single nucleotide polymorphisms (SNPs) were determined for 7768 participants in three HIV treatment trials. Single variant analysis was performed for each biomarker on samples from each of three ethnic groups [African (AFR), Admixed American (AMR), European (EUR)] within each trial including covariates relevant to biomarker levels. For each ethnic group, the results were pooled across trials, then further pooled across ethnicities.

Results: The transethnic analysis identified three, two, and one known loci associated with hsCRP, D-dimer, and IL-6 levels, respectively, and two novel loci, FGB and GCNT1, associated with D-dimer levels. Lead SNPs exhibited similar effects across ethnicities. Additionally, three novel, ethnic-specific loci were identified: CATSPERG associated with D-dimer in AFR and PROX1-AS1 and TRAPPC9 associated with IL-6 in AFR and AMR, respectively.

Conclusion: Eleven loci associated with three biomarker levels were identified in PLWH from the three studies including six loci known in the general population and five novel loci associated with D-dimer and IL-6 levels. These findings support the hypothesis that host genetics may partially contribute to chronic inflammation in PLWH and help to identify potential targets for intervention of serious non-AIDS complications.
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http://dx.doi.org/10.1097/QAD.0000000000002738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789909PMC
February 2021

A pull-down assay using DNA/RNA-conjugated beads with a customized competition strategy: An effective approach to identify DNA/RNA binding proteins.

MethodsX 2020 13;7:100890. Epub 2020 Apr 13.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Bldg. 550, Rm 126,1050 Boyles Street, Frederick, MD 21702 United States.

Innate immune response is insisted upon detection of foreign intracellular DNA or RNA derived from viruses and bacteria. This reaction is important to initiate an effective protective response for the host cells. This crucial step is induced by cytosolic nucleic acids sensors/binding proteins, which triggers the production of type I or type III interferons (IFNs) and proinflammatory cytokines such as Interleukin 6 (IL-6). The identification of these cytosolic DNA or RNA sensors is a key step in understanding the signaling pathways triggered by those pathogens. Here we describe an effective approach to identify potential known/novel DNA or RNA sensors: a pull-down assay using DNA/RNA-conjugated beads with a customized competition strategy, which conferred a more effective and efficient way to determine the interaction between DNA/RNA and the sensor protein(s), therefore greatly improves the progress to investigate potential novel cytosolic DNA or RNA sensors/ binding proteins •The customized method makes the traditional pull-down assays more effective and efficient to identify DNA/RNA binding protein(s).•With the competitor of your choice, the method provides specific information about the competitive binding between DNA/RNA and binding proteins.
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http://dx.doi.org/10.1016/j.mex.2020.100890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7195543PMC
April 2020

HIV influences microtubule associated protein-2: potential marker of HIV-associated neurocognitive disorders.

AIDS 2020 06;34(7):979-988

Laboratory of Preclinical Neurobiology, Department of Neuroscience.

Objective: Postmortem brains of patients diagnosed with HIV-1-associated neurocognitive disorders (HAND) exhibit loss of dendrites. However, the mechanisms by which synapses are damaged are not fully understood.

Design: Dendrite length and remodeling occurs via microtubules, the dynamics of which are regulated by microtubule-binding proteins, including microtubule-associated protein 2 (MAP2). The HIV protein gp120 is neurotoxic and interferes with neuronal microtubules. We measured MAP2 concentrations in human cerebrospinal fluid (CSF) and MAP2 immunoreactivity in rat cortical neurons exposed to HIV and gp120.

Methods: First, we examined whether HIV affects MAP2 levels by analyzing the CSF of 27 persons living with HIV (PLH) whose neurocognitive performance had been characterized. We then used rat cortical neurons to study the mechanisms of HIV-mediated dendritic loss.

Results: PLH who had HAND had greater MAP2 concentrations within the CSF than cognitive normal PLH. In cortical neurons, the deleterious effect of HIV on MAP2-positive dendrites occurred through a gp120-mediated mechanism. The neurotoxic effect of HIV was blocked by a CCR5 antagonist and prevented by Helix-A, a peptide that displaces gp120 from binding to microtubules, conjugated to a nanolipoprotein particle delivery platform.

Conclusion: Our findings support that HIV at least partially effects its neurotoxicity via neuronal cytoskeleton modifications and provide evidence of a new therapeutic compound that could be used to prevent the HIV-associated neuropathology.
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http://dx.doi.org/10.1097/QAD.0000000000002509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210064PMC
June 2020

Defective HIV-1 proviruses produce viral proteins.

Proc Natl Acad Sci U S A 2020 02 6;117(7):3704-3710. Epub 2020 Feb 6.

Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;

HIV-1 proviruses persist in the CD4 T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as "defective" by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant "graveyard" of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4 T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4 T cells and T cell-cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.
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http://dx.doi.org/10.1073/pnas.1917876117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035625PMC
February 2020

Profiles of Long Non-Coding RNAs and mRNA Expression in Human Macrophages Regulated by Interleukin-27.

Int J Mol Sci 2019 Dec 9;20(24). Epub 2019 Dec 9.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

Macrophages play an essential role in the immune system. Recent studies have shown that long non-coding RNAs (lncRNAs) can regulate genes encoding products involved in the immune response. Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines with broad anti-viral effects that inhibits human immunodeficiency virus (HIV) type-1 and herpes simplex virus (HSV). However, little is known about the role of lncRNAs in macrophages affected by IL-27. Therefore, we investigated the expression profiles of mRNA and lncRNA in human monocyte-derived macrophages (MDMs) regulated by IL-27. Monocytes were differentiated in the presence of macrophage-colony stimulatory factor (M-CSF)- or human AB serum with or without IL-27, and these cells were the subject for the profile analysis using RNA-Seq. We identified 146 lncRNAs (including 88 novel ones) and 434 coding genes were differentially regulated by IL-27 in both M-CSF- and AB serum-induced macrophages. Using weighted gene co-expression network analysis, we obtained four modules. The immune system, cell cycle, and regulation of complement cascade pathways were enriched in different modules. The network of mRNAs and lncRNAs in the pathways suggest that lncRNAs might regulate immune activity in macrophages. This study provides potential insight into the roles of lncRNA in macrophages regulated by IL-27.
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http://dx.doi.org/10.3390/ijms20246207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941108PMC
December 2019

Complete Genome Sequence of Herpes Simplex Virus 1 Strain McKrae.

Microbiol Resour Announc 2019 Sep 26;8(39). Epub 2019 Sep 26.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

Herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent and relatively neuroinvasive in animal models compared with other wild-type HSV-1 strains. To identify the genetic determinants that lead to the unique phenotypes of the McKrae strain, we sequenced its genome with PacBio single-molecule real-time (SMRT) technology and resolved the complete sequence.
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http://dx.doi.org/10.1128/MRA.00993-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763650PMC
September 2019

Complete Genome Sequence of Herpes Simplex Virus 1 Strain MacIntyre.

Microbiol Resour Announc 2019 Sep 12;8(37). Epub 2019 Sep 12.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

Herpes simplex virus type 1 (HSV-1) strain MacIntyre has a severe defect in the anterograde spread after replication in the nucleus. To better understand and identify the genetic determinants that lead to the unique phenotypes of the MacIntyre strain, we sequenced its genome with PacBio single-molecule real-time sequencing technology and resolved the complete sequence.
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http://dx.doi.org/10.1128/MRA.00895-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742799PMC
September 2019

siRNA containing a unique 5-nucleotide motif acts as a quencher of IFI16-mediated innate immune response.

Mol Immunol 2019 10 21;114:330-340. Epub 2019 Aug 21.

Laboratory of Human Retrovirology and Immunoinformatics, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA. Electronic address:

We previously reported that some small interfering RNA (siRNA) enhances DNA or DNA virus mediated-interferon (IFN)-λ1(a type III IFN) induction through the crosstalk between retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) signalling pathway. Here we provide further evidence of a new role for siRNA. siRNA containing a 5-nucleotide (nt) motif sequence suppresses DNA-mediated not only type III IFNs, but also type I IFNs and inflammatory cytokines. We define that motif siRNA inhibits the induction when the motif is located at the 3' or 5'-terminus of siRNA. Using THP1-Lucia ISG cells with various DNA stimulants, we reveal that motif siRNA inhibits DNA or DNA virus but not RNA virus-mediated signalling. Motif siRNA specifically interrupts IFI16 but not cyclic GMP-AMP synthase (cGAS) binding to DNA and has 2.5-fold higher affinity to IFI16 than that of siRNA without the motif. We further confirm that motif siRNA potently suppresses HSV-1 virus-mediated IFNs and inflammatory cytokines, such as IFNL1, IFNB and TNFA, in human primary immature dendritic cells. Collectively, these findings may shed light on a novel function of siRNA with the unique 5-nt motif as a quencher of innate immunity and facilitate the development of potential therapeutics to regulate innate immune cascades.
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http://dx.doi.org/10.1016/j.molimm.2019.08.007DOI Listing
October 2019

IL-27 posttranslationally regulates Y-box binding protein-1 to inhibit HIV-1 replication in human CD4+ T cells.

AIDS 2019 10;33(12):1819-1830

Laboratory of Human Retrovirology and Immunoinformatics.

Objectives: IL-27 is known as an antiviral cytokine that inhibits HIV, hepatitis C virus, and other viruses. We have previously demonstrated that, IL-27 posttreatment after HIV-infection inhibits viral replication in primary CD4 T cells.

Design: Here, we evaluated the anti-HIV effect of IL-27 pretreatment in CD4 T cells from healthy donors prior to HIV infection with HIVNL4.3 or vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-luciferase virus (HIV-LUC-V).

Methods: IL-27-treated CD4 T cells were infected with HIVNL4.3 or HIV-LUC-V and assessed the anti-HIV effect. HIV infection was monitored by p24 antigen ELISA or luciferase assay. HIV fusion/entry and uncoating were determined by BlaM-Vpr assay and HIV fate of capsid and/or HIV Entry/Uncoating assay based on core-packaged RNA availability and Translation assay, respectively. HIV proviral copy number was determined by real-time PCR. Gene expression profile from IL-27-pretreated CD4 T cells was determined using Genechip array. Posttranslational modification of global proteins from IL-27-pretreated CD4 T cells was determined by a combination of 2-dimensional difference-in-gel-electrophoresis (2D-DIG), western-blot and protein mass spectrometry.

Results: IL-27 pretreatment inhibited HIVNL4.3 and HIV-LUC-V infection in CD4 T cells. HIV copy assay demonstrated that IL-27-treatment suppressed an early step of reverse transcription during HIV infection. A combination of 2D-DIG-electrophoresis and western blot assays demonstrated that IL-27-treatment induces a change in posttranslational modification of Y box binding protein-1 (YB-1). Overexpression of domain negative YB-1 mutants illustrated that a residue Lysine at 118 plays a key role in supporting HIV infection in CD4 T cells.

Conclusion: IL-27-pretreatment inhibits HIV-1 infection by suppressing an HIV-reverse transcription product formation/uncoating step by suppressing the acetylation of YB-1 in primary CD4 T cells.
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http://dx.doi.org/10.1097/QAD.0000000000002288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731144PMC
October 2019

A novel microRNA, hsa-miR-6852 differentially regulated by Interleukin-27 induces necrosis in cervical cancer cells by downregulating the FoxM1 expression.

Sci Rep 2018 01 17;8(1):900. Epub 2018 Jan 17.

Laboratory of Human Retrovirology and Immunoinformatics, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, 21702, USA.

We have previously demonstrated that Interleukin-27 differentially regulates the expression of seven novel microRNAs. Here we elucidate the functional significance of these novel microRNAs. Of the seven microRNAs, over expression of miRNA-6852 (miR-SX4) mimic induces cell cycle arrest at G2/M phase and induces necrosis in HEK293 and panel of cervical cancer cells (Human Papilloma Virus (HPV) infected cell lines; HeLa, CaSki and SiHa cells). To define the mechanism of the miR-SX4-mediated G2/M arrest, a microarray gene chip array and western blot analysis were performed. FoxM1, a transcription factor is identified as a key protein down-regulated by miR-SX4, even though the miR-SX4 does not target 3'UTR of FoxM1. Knock down of FoxM1 using si-RNA demonstrate that FoxM1 silenced cell induces G2/M cell cycle arrest and necrosis. Our data demonstrated for the first time that miR-SX4 could be a potent anti-cancer microRNA.
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http://dx.doi.org/10.1038/s41598-018-19259-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772045PMC
January 2018

Corrigendum: Induction of Monocyte Chemoattractant Proteins in Macrophages the Production of Granulocyte/Macrophage Colony-Stimulating Factor by Breast Cancer Cells.

Front Immunol 2017 13;8:1524. Epub 2017 Nov 13.

Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, United States.

[This corrects the article on p. 2 in vol. 7, PMID: 26834744.].
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http://dx.doi.org/10.3389/fimmu.2017.01524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5693906PMC
November 2017

STING is an essential mediator of the Ku70-mediated production of IFN-λ1 in response to exogenous DNA.

Sci Signal 2017 Jul 18;10(488). Epub 2017 Jul 18.

Laboratory of Human Retrovirology and Immunoinformatics, Applied and Developmental Research Directorate, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

We previously identified Ku70, a subunit of a DNA repair protein complex, as a cytosolic DNA sensor that induces the production of interferon-λ1 (IFN-λ1) by human primary cells and cell lines. IFN-λ1 is a type III IFN and has similar antiviral activity to that of the type I IFNs (IFN-α and IFN-β). We observed that human embryonic kidney (HEK) 293T cells, which are deficient in the innate immune adaptor protein STING (stimulator of IFN genes), did not produce IFN-λ1 in response to DNA unless they were reconstituted with STING. Conversely, parental HEK 293 cells produced IFN-λ1 after they were exposed to exogenous DNA; however, when STING was knocked out in the HEK 293 cells through the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system, they lost this response. Through confocal microscopy, we demonstrated that endogenous Ku70 was located in the nucleus and then translocated to the cytoplasm upon DNA exposure to form a complex with STING. Additionally, the DNA binding domain of Ku70 was essential for formation of the Ku70-STING complex. Knocking down STING in primary human macrophages inhibited their ability to produce IFN-λ1 in response to transfection with DNA or infection with the DNA virus HSV-2 (herpes simplex virus-2). Together, these data suggest that STING mediates the Ku70-mediated IFN-λ1 innate immune response to exogenous DNA or DNA virus infection.
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http://dx.doi.org/10.1126/scisignal.aah5054DOI Listing
July 2017

Genome-Wide Analyses of MicroRNA Profiling in Interleukin-27 Treated Monocyte-Derived Human Dendritic Cells Using Deep Sequencing: A Pilot Study.

Int J Mol Sci 2017 Apr 28;18(5). Epub 2017 Apr 28.

Laboratory of Human Retrovirology and Immunoinformatics, Applied and Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

MicroRNAs (miRNAs) regulate gene expression and thereby influence cell fate and function. Recent studies suggest that an abundant class of miRNAs play important roles in immune cells, such as T cells, natural killer (NK) cells, B cells, and dendritic cells (DCs). Interleukin (IL)-27 is a member of the IL-12 family of cytokines with broad anti-viral effects. It is a potent inhibitor of HIV-1 infection in CD4+ T cells and macrophages, as well as monocyte-derived immature dendritic cells (iDCs). This pilot study compared miRNA profiles between iDCs and IL-27-treated iDCs (27DCs) using deep sequencing methods and identified 46 known miRNAs that were significantly differentially expressed in 27DCs: 36 were upregulated and 10 downregulated by IL-27. Many of the potential target genes of these miRNAs are involved in IL-27 associated pathways, such as JAK/STAT, MAPKs, and PI3K and several were also previously reported to be involved in the regulation of human DC function. This study found that these miRNAs also potentially target several viral genomes and therefore may have antiviral effects. Four of these differential miRNAs (miR-99a-5p, miR-222-3p, miR-138-5p, and miR-125b-5p) were validated using quantitative reverse transcription PCR (RT-qPCR). Twenty-two novel miRNAs were discovered from deep sequencing and confirmed using RT-qPCR. This study furthers the understanding of the role of IL-27 in immunity and lays a foundation for future characterization of the role of specific miRNAs in DCs.
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http://dx.doi.org/10.3390/ijms18050925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454838PMC
April 2017

Interleukin-27 Enhances the Potential of Reactive Oxygen Species Generation from Monocyte-derived Macrophages and Dendritic cells by Induction of p47.

Sci Rep 2017 02 27;7:43441. Epub 2017 Feb 27.

Laboratory of Human Retrovirology and Immunoinformatics, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA.

Interleukin (IL)-27, a member of the IL-12 cytokine family, plays an important and diverse role in the function of the immune system. We have previously demonstrated that IL-27 is an anti-viral cytokine which inhibits HIV-1, HIV-2, Influenza virus and herpes simplex virus infection, and enhances the potential of reactive oxygen species (ROS) generating activity during differentiation of monocytes to macrophages. In this study, we further investigated the mechanism of the enhanced potential for ROS generation by IL-27. Real time PCR, western blot and knock down assays demonstrate that IL-27 is able to enhance the potential of superoxide production not only during differentiation but also in terminally differentiated-macrophages and immature dendritic cells (iDC) in association with the induction of p47, a cytosolic component of the ROS producing enzyme, NADPH oxidase, and the increase in amounts of phosphorylated p47 upon stimulation. We also demonstrate that IL-27 is able to induce extracellular superoxide dismutase during differentiation of monocytes but not in terminal differentiated macrophages. Since ROS plays an important role in a variety of inflammation, our data demonstrate that IL-27 is a potent regulator of ROS induction and may be a novel therapeutic target.
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http://dx.doi.org/10.1038/srep43441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5327488PMC
February 2017

Interleukin-15 (IL-15) Strongly Correlates with Increasing HIV-1 Viremia and Markers of Inflammation.

PLoS One 2016 23;11(11):e0167091. Epub 2016 Nov 23.

Applied and Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, United States of America.

Objective: IL-15 has been postulated to play an important role in HIV-1 infection, yet there are conflicting reports regarding its expression levels in these patients. We sought to measure the level of IL-15 in a large, well characterised cohort of HIV-1 infected patients and correlate this with well known markers of inflammation, including CRP, D-dimer, sCD163 and sCD14.

Design And Methods: IL-15 levels were measured in 501 people (460 patients with HIV-1 infection and 41 uninfected controls). The HIV-1 infected patients were divided into 4 groups based on viral load: <50 copies/ml, 51-10,000 copies/ml, 10,001-100,000 copies/ml and >100,000 copies/ml. The Mann Whitney test (non-parametric) was used to identify significant relationships between different patient groups.

Results: IL-15 levels were significantly higher in patients with viral loads >100,000 copies/ml (3.02 ± 1.53 pg/ml) compared to both uninfected controls (1.69 ± 0.37 pg/ml, p<0.001) or patients with a viral load <50 copies/ml (1.59 ± 0.40 pg/ml (p<0.001). There was a significant correlation between HIV-1 viremia and IL-15 levels (Spearman r = 0.54, p<0.001) and between CD4+ T cell counts and IL-15 levels (Spearman r = -0.56, p<0.001).

Conclusions: IL-15 levels are significantly elevated in HIV-1 infected patients with viral loads >100,000 copies/ml compared to uninfected controls, with a significant direct correlation noted between IL-15 and HIV-1 viremia and an inverse correlation between IL-15 levels and CD4+ T cell counts. These data support a potential role for IL-15 in the pathogenesis of HIV-associated immune activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167091PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120855PMC
June 2017

Distinguishing highly similar gene isoforms with a clustering-based bioinformatics analysis of PacBio single-molecule long reads.

BioData Min 2016 5;9:13. Epub 2016 Apr 5.

Leidos BioMedical Research, Inc., Frederick National Laboratory for Cancer Research, NIH, Frederick, MD USA.

Background: Gene isoforms are commonly found in both prokaryotes and eukaryotes. Since each isoform may perform a specific function in response to changing environmental conditions, studying the dynamics of gene isoforms is important in understanding biological processes and disease conditions. However, genome-wide identification of gene isoforms is technically challenging due to the high degree of sequence identity among isoforms. Traditional targeted sequencing approach, involving Sanger sequencing of plasmid-cloned PCR products, has low throughput and is very tedious and time-consuming. Next-generation sequencing technologies such as Illumina and 454 achieve high throughput but their short read lengths are a critical barrier to accurate assembly of highly similar gene isoforms, and may result in ambiguities and false joining during sequence assembly. More recently, the third generation sequencer represented by the PacBio platform offers sufficient throughput and long reads covering the full length of typical genes, thus providing a potential to reliably profile gene isoforms. However, the PacBio long reads are error-prone and cannot be effectively analyzed by traditional assembly programs.

Results: We present a clustering-based analysis pipeline integrated with PacBio sequencing data for profiling highly similar gene isoforms. This approach was first evaluated in comparison to de novo assembly of 454 reads using a benchmark admixture containing 10 known, cloned msg genes encoding the major surface glycoprotein of Pneumocystis jirovecii. All 10 msg isoforms were successfully reconstructed with the expected length (~1.5 kb) and correct sequence by the new approach, while 454 reads could not be correctly assembled using various assembly programs. When using an additional benchmark admixture containing 22 known P. jirovecii msg isoforms, this approach accurately reconstructed all but 4 these isoforms in their full-length (~3 kb); these 4 isoforms were present in low concentrations in the admixture. Finally, when applied to the original clinical sample from which the 22 known msg isoforms were cloned, this approach successfully identified not only all known isoforms accurately (~3 kb each) but also 48 novel isoforms.

Conclusions: PacBio sequencing integrated with the clustering-based analysis pipeline achieves high-throughput and high-resolution discrimination of highly similar sequences, and can serve as a new approach for genome-wide characterization of gene isoforms and other highly repetitive sequences.
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http://dx.doi.org/10.1186/s13040-016-0090-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820869PMC
April 2016

The effects of storage temperature on PBMC gene expression.

BMC Immunol 2016 Mar 15;17. Epub 2016 Mar 15.

Biogen, 125 Broadway, Cambridge, MA, 02142, USA.

Background: Cryopreservation of peripheral blood mononuclear cells (PBMCs) is a common and essential practice in conducting research. There are different reports in the literature as to whether cryopreserved PBMCs need to only be stored ≤ -150 °C or can be stored for a specified time at -80 °C. Therefore, we performed gene expression analysis on cryopreserved PBMCs stored at both temperatures for 14 months and PBMCs that underwent temperature cycling 104 times between these 2 storage temperatures. Real-time RT-PCR was performed to confirm the involvement of specific genes associated with identified cellular pathways. All cryopreserved/stored samples were compared to freshly isolated PBMCs and between storage conditions.

Results: We identified a total of 1,367 genes whose expression after 14 months of storage was affected >3 fold in PBMCs following isolation, cryopreservation and thawing as compared to freshly isolated PBMC aliquots that did not undergo cryopreservation. Sixty-six of these genes were shared among two or more major stress-related cellular pathways (stress responses, immune activation and cell death). Thirteen genes involved in these pathways were tested by real-time RT-PCR and the results agreed with the corresponding microarray data. There was no significant change on the gene expression if the PBMCs experienced brief but repetitive temperature cycling as compared to those that were constantly kept ≤ -150 °C. However, there were 18 genes identified to be different when PBMCs were stored at -80 °C but did not change when stored < -150 °C. A correlation was also found between the expressions of 2'-5'- oligoadenylate synthetase (OAS2), a known interferon stimulated gene (IFSG), and poor PBMC recovery post-thaw. PBMC recovery and viability were better when the cells were stored ≤ -150 °C as compared to -80 °C.

Conclusions: Not only is the viability and recovery of PBMCs affected during cryopreservation but also their gene expression pattern, as compared to freshly isolated PBMCs. Different storage temperature of PBMCs can activate or suppress different genes, but the cycling between -80 °C and -150 °C did not produce significant alterations in gene expression when compared to PBMCs stored ≤ -150 °C. Further analysis by gene expression of various PBMC processing and cryopreservation procedures is currently underway, as is identifying possible molecular mechanisms.
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http://dx.doi.org/10.1186/s12865-016-0144-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791795PMC
March 2016

Towards Better Precision Medicine: PacBio Single-Molecule Long Reads Resolve the Interpretation of HIV Drug Resistant Mutation Profiles at Explicit Quasispecies (Haplotype) Level.

J Data Mining Genomics Proteomics 2016 Jan 8;7(1). Epub 2015 Nov 8.

Applied and Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, MD 21702, USA.

Development of HIV-1 drug resistance mutations (HDRMs) is one of the major reasons for the clinical failure of antiretroviral therapy. Treatment success rates can be improved by applying personalized anti-HIV regimens based on a patient's HDRM profile. However, the sensitivity and specificity of the HDRM profile is limited by the methods used for detection. Sanger-based sequencing technology has traditionally been used for determining HDRM profiles at the single nucleotide variant (SNV) level, but with a sensitivity of only ≥ 20% in the HIV population of a patient. Next Generation Sequencing (NGS) technologies offer greater detection sensitivity (~ 1%) and larger scope (hundreds of samples per run). However, NGS technologies produce reads that are too short to enable the detection of the physical linkages of individual SNVs across the haplotype of each HIV strain present. In this article, we demonstrate that the single-molecule long reads generated using the Third Generation Sequencer (TGS), PacBio RS II, along with the appropriate bioinformatics analysis method, can resolve the HDRM profile at a more advanced quasispecies level. The case studies on patients' HIV samples showed that the quasispecies view produced using the PacBio method offered greater detection sensitivity and was more comprehensive for understanding HDRM situations, which is complement to both Sanger and NGS technologies. In conclusion, the PacBio method, providing a promising new quasispecies level of HDRM profiling, may effect an important change in the field of HIV drug resistance research.
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http://dx.doi.org/10.4172/2153-0602.1000182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775093PMC
January 2016

Induction of Monocyte Chemoattractant Proteins in Macrophages via the Production of Granulocyte/Macrophage Colony-Stimulating Factor by Breast Cancer Cells.

Front Immunol 2016 20;7. Epub 2016 Jan 20.

Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute , Frederick, MD , USA.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 plays an important role in the initiation and progression of cancer. We previously reported that in 4T1 murine breast cancer, non-tumor stromal cells, including macrophages, were the major source of MCP-1. In the present study, we analyzed the potential mechanisms by which MCP-1 is upregulated in macrophages infiltrating 4T1 tumors. We found that cell-free culture supernatants of 4T1 cells (4T1-sup) markedly upregulated MCP-1 production by peritoneal inflammatory macrophages. 4T1-sup also upregulated other MCPs, such as MCP-3/CCL7 and MCP-5/CCL12, but modestly upregulated neutrophil chemotactic chemokines, such as KC/CXCL1 or MIP-2/CXCL2. Physicochemical analysis indicated that an approximately 20-30 kDa 4T1 cell product was responsible for the capacity of 4T1-sup to upregulate MCP-1 expression by macrophages. A neutralizing antibody against granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage CSF, almost completely abrogated MCP-1-inducing activity of 4T1-sup, and recombinant GM-CSF potently upregulated MCP-1 production by macrophages. The expression levels of GM-CSF in 4T1 tumors in vivo were higher than other tumors, such as Lewis lung carcinoma. Treatment of mice with anti-GM-CSF antibody significantly reduced the growth of 4T1 tumors at the injection sites but did not reduce MCP-1 production or lung metastasis in tumor-bearing mice. These results indicate that 4T1 cells have the capacity to directly upregulate MCP-1 production by macrophages by releasing GM-CSF; however, other mechanisms are also involved in increased MCP-1 levels in the 4T1 tumor microenvironment.
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http://dx.doi.org/10.3389/fimmu.2016.00002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718995PMC
February 2016

HIV-1 Treated Patients with Undetectable Viral Loads have Lower Levels of Innate Immune Responses via Cytosolic DNA Sensing Systems Compared with Healthy Uninfected Controls.

J AIDS Clin Res 2014 Jun;5(6)

Applied and Developmental Research Directorate, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21702, USA.

Objectives: After DNA or RNA virus infection, cytosolic foreign DNA or RNA derived from the infecting viruses is recognized by intracellular pathogen recognition receptors (PRRs) and induces activation of the innate immune system. Transfection of DNA has been used as an experimental model for DNA virus-mediated innate responses. We have previously reported that DNA transfection preferentially induces Type-III IFN (IFN-λ1) rather than Type-I IFN (IFN-β). In this study, we compared the DNA-mediated immune response between healthy controls and HIV-1 infected patients with undetectable viral loads and assessed potential innate immune responses in these patients.

Methods: The study consisted of 50 HIV-1 negative healthy donors, 46 patients on combination antiretroviral therapy with HIV-1 viral loads <50 copies/ml and 7 long term non-progressors (LTNPs). PBMCs were isolated from whole blood using Ficoll-Paque. DNA transfection was performed using Lipofectamine 2000. After 22 hours incubation, total cellular RNA was extracted and real time RT-PCR was performed to determine gene expression level of IFN-λ1, IFN-β and RANTES. Gene induction was compared by fold change.

Results: Baseline levels of endogenous gene expression of IFN-λ1, IFN-β and RANTES in HIV-1 patients were higher than in controls. Following DNA transfection, both HIV infected patients and healthy controls induced gene induction, however, the induction in HIV-1 patients was at a significantly lower level compared to uninfected controls.

Conclusion: HIV-1 treated patients with undetectable viral loads have lower levels of innate immune responses via cytosolic DNA sensing systems. This may be caused by persistent immune activation.
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http://dx.doi.org/10.4172/2155-6113.1000315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444065PMC
June 2014

Activated platelet-T-cell conjugates in peripheral blood of patients with HIV infection: coupling coagulation/inflammation and T cells.

AIDS 2015 Jul;29(11):1297-308

aCMRS/Laboratory of Immunoregulation bResearch Technology Branch, NIAID, NIH, Bethesda cElectron Microscopy Laboratory, Cancer Research Technology Program dLaboratory of Human Retrovirology, Leidos Biomedical Research, Inc. Frederick eLaboratory of Pathology, NCI, NIH fBiostatistics Research Branch, DCR, NIAID, NIH, Bethesda gAIDS Monitoring Laboratory, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.

Background: Despite successfully suppressed viremia by treatment, patients with high levels of biomarkers of coagulation/inflammation are at an increased risk of developing non-AIDS defining serious illnesses such as cardiovascular diseases. Thus, there is a relationship between persistent immune activation and coagulation/inflammation, although the mechanisms are poorly understood. Platelets play an important role in this process. Although interactions between platelets and elements of the innate immune system, such as monocytes, are well described, little is known about the interaction between platelets and the adaptive immune system.

Design: We investigated the interaction of a component of the coagulation system, platelets, and the adaptive immune system T cells.

Methods: Healthy controls and combination antiretroviral therapy (cART)-treated HIV-infected patients with viral loads of less than 40 copies/ml for more than 15 months were analysed for platelet-T-cell conjugate formation.

Results: Platelets can form conjugates with T cells and were preferentially seen in CD4 and CD8 T-cell subsets with more differentiated phenotypes [memory, memory/effector and terminal effector memory (TEM)]. Compared with healthy controls, these conjugates in patients with HIV infection were more frequent, more often composed of activated platelets (CD42bCD62P), and were significantly associated with the D-dimer serum levels.

Conclusion: These data support a model in which platelet-T-cell conjugates may play a critical role in the fast recruitment of antigen-experienced T cells to the place of injury. This mechanism can contribute in maintaining a state of coagulation/inflammation observed in these patients contributing to the pathology of the disease.
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http://dx.doi.org/10.1097/QAD.0000000000000701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502988PMC
July 2015

Construction of an isonucleoside on a 2,6-dioxobicyclo[3.2.0]-heptane skeleton.

Molecules 2015 Mar 12;20(3):4623-34. Epub 2015 Mar 12.

Laboratory of Human Retrovirology, Leidos Biochemical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

We have built a new isonucleoside derivative on a 2,6-dioxobicyclo[3.2.0]heptane skeleton as a potential anti-HIV agent. To synthesize the target compound, an acetal-protected dihydroxyacetone was first converted to a 2,3-epoxy-tetrahydrofuran derivative. Introduction of an azide group, followed by the formation of an oxetane ring, gave a pseudosugar derivative with a 2,6-dioxobicyclo[3.2.0]heptane skeleton. The desired isonucleoside was obtained by constructing a purine base moiety on the scaffold, followed by amination.
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http://dx.doi.org/10.3390/molecules20034623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272333PMC
March 2015

S100A9 induced inflammatory responses are mediated by distinct damage associated molecular patterns (DAMP) receptors in vitro and in vivo.

PLoS One 2015 23;10(2):e0115828. Epub 2015 Feb 23.

MedImmune LLC, One MedImmune Way, Gaithersburg, Maryland 20878, United States of America.

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4338059PMC
December 2015

Plasma interleukin-27 (IL-27) levels are not modulated in patients with chronic HIV-1 infection.

PLoS One 2014 4;9(6):e98989. Epub 2014 Jun 4.

Applied and Developmental Research Directorate, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.

Objective: IL-27 is an immunomodulatory cytokine with potent anti-HIV properties in PBMCs, CD4+ T cells, macrophages and immature dendritic cells. Previous smaller studies have suggested that HIV-1 infection may alter IL-27 and influence HIV-1 pathogenesis. The aim of this study was to examine the relationship between plasma IL-27 levels in a well-characterised cohort of HIV-1 infected patients.

Methods: Patients were stratified into four groups based on HIV-1 viral load and matched according to age, gender and those receiving antiretroviral treatment. IL-27 levels and C-reactive protein (CRP) were measured using electrochemiluminescence assays. D-dimer and CD4+ T cell counts were measured using an Enzyme Linked Fluorescence Assay and FACS, respectively. sCD14 and sCD163 were measured using ELISA. HIV-1 viral load was measured by bDNA or qRT-PCR assays.

Results: Plasma IL-27 levels were measured in 505 patients (462 HIV+, 43 controls). The mean level (±SEM) of IL-27 in controls was 2990.7±682.1 pg/ml, in the <50 copies/ml group it was 2008.0±274.8 pg/ml, in the 51-10,000 copies group it was 1468.7±172.3 pg/ml, in the 10,001-100,000 copies/ml group it was 1237.9±127.3 pg/ml and in the >100,000 copies/ml group it was 1590.1±223.7 pg/ml. No statistically significant difference in IL-27 levels between groups were seen. There were no correlations noted between IL-27 and HIV-1 viral load or CD4+ T cell counts. There was a small correlation noted between D-dimer and IL-27 (Spearman r = 0.09, p = 0.03) and sCD163 and IL-27 (Spearman r = 0.12, p = 0.005). No correlation was observed between IL-27 and CRP or sCD14 levels.

Conclusions: This is the largest study examining the levels of plasma IL-27 in HIV-1 infection. While IL-27 levels are not significantly altered in HIV-1 infection compared to uninfected controls there may be a small association between IL-27 and D-dimer levels and IL-27 and sCD163 levels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098989PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4045808PMC
October 2015

Lifespan of effector memory CD4+ T cells determined by replication-incompetent integrated HIV-1 provirus.

AIDS 2014 May;28(8):1091-9

aClinical and Molecular Retrovirology Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda bClinical Services Program, Applied and Development Research Directorate, Leidos Biomedical Research Incorporated, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.

Objective: Determining the precise lifespan of human T-cell is challenging due to the inability of standard techniques to distinguish between dividing and dying cells. Here, we measured the lifespan of a pool of T cells that were derived from a single cell 'naturally' labelled with a single integrated clone of a replication-incompetent HIV-1 provirus.

Design/methods: Utilizing a combination of techniques, we were able to sequence/map an integration site of a unique provirus with a stop codon at position 42 of the HIV-1 protease. In-vitro reconstruction of this provirus into an infectious clone confirmed its inability to replicate. By combining cell separation and integration site-specific PCR, we were able to follow the fate of this single provirus in multiple T-cell subsets over a 20-year period. As controls, a number of additional integrated proviruses were also sequenced.

Results: The replication-incompetent HIV-1 provirus was solely contained in the pool of effector memory CD4 T cells for 17 years. The percentage of the total effector memory CD4 T cells containing the replication-incompetent provirus peaked at 1% with a functional half-life of 11.1 months. In the process of sequencing multiple proviruses, we also observed high levels of lethal mutations in the peripheral blood pool of proviruses.

Conclusion: These data indicate that human effector memory CD4 T cells are able to persist in vivo for more than 17 years without detectably reverting to a central memory phenotype. A secondary observation is that the fraction of the pool of integrated HIV-1 proviruses capable of replicating may be considerably less than the 12% currently noted in the literature.
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http://dx.doi.org/10.1097/QAD.0000000000000223DOI Listing
May 2014
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