MiRNA profiles in unstimulated and TLR stimulated MDMs
ABSTRACT: Macrophages play a crucial role in HIV-1 pathogenesis. Toll-like receptors (TLRs) are fundamental for innate and adaptive immune responses, but their role in HIV-1 infection is still incompletely understood. The TLR3 and TLR4 ligands poly(I:C) and LPS are known to modulate HIV-1 infection of and replication in monocyte-derived macrophages (MDMs), but the mechanism is incompletely understood. We found that MDMs stimulation with poly(I:C) or LPS abrogated infection by CCR5-using, macrophage-tropic HIV-1, or by VSV-G-pseudotyped HIV-1 virions, while TLR7 and TLR9 agonists Imiquimod and CpG only reduced infection to varying extent. Suppression of infection, or lack thereof, did not correlate with differential effects on CD4 or CCR5 expression, type I interferon induction, or production of pro-inflammatory cytokines. Furthermore, integrated pro-viruses were readily detected in unstimulated, TLR7- and TLR9-stimulated cells, but not in TLR3- or TLR4-stimulated MDMs, suggesting the alteration of post-entry, pre-integration event(s). MicroRNA (miRNA) microarray and real time PCR demonstrated increased miR-155 levels in MDMs upon TLR3/4, but not TLR7, stimulation, and a miR-155 inhibitor partially restored infectivity in poly(I:C)-stimulated MDMs. Finally, miR-155 over-expression in MDMs and cell lines remarkably diminished HIV-1 infection, inducing an accumulation of late reverse transcription products, concurrently with a decrease in mRNA levels of several HIV-1 dependency factors involved in nuclear import of pre-integration complexes. Our results suggest that miR-155 may target mRNA(s) for host cell protein(s) that either participate in or facilitate post-entry, pre-integration events, resulting in severely diminished HIV-1 infection. miRNA profiles were investigated in total RNA isolated from unstimulated and TLR3-, TLR4- and TLR7-stimulated human MDMs from a single normal donor
Project description:In the activated B-cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), the most frequent gain-of-function mutations target MyD88, a signaling adapter for Tolllike receptors (TLRs). The most prevalent oncogenic mutant, MyD88 L265P, occurs in 29% of cases and is the most active in engaging the NF-kappaB pathway. Here we show that MyD88 mutants do not function autonomously, but rather require TLR7, TLR9, and to a lesser extent, TLR4 to promote the survival of ABC DLBCL cells. Unlike wild type MyD88, MyD88 mutants associate constitutively with TLR7 and TLR9 in ABC DLBCL cells. Like ligand-induced TLR7/9 signaling in normal immune cells, the survival of ABC DLBCL cell lines depends upon translocation of TLR7 and TLR9 to acidic endolysosomes, where proteolytic processing of their ligand binding ectodomains is required for their oncogenic signaling. ABC DLBCL viability also depends upon CD14, a co-receptor for TLR7 and TLR9 that promotes engagement of nucleic acid ligands by these receptors. Point mutations in the TLR7 or TLR9 ectodomains that abrogate ligand binding and/or signaling were incapable of sustaining ABC DLBCL survival. An inhibitory oligonucleotide that suppresses TLR9 responses in normal B cells blocked NF-kappaB signaling and survival of ABC DLBCL lines. Together, these data suggest that an endogenous TLR ligand may play a pathogenic role in ABC DLBCL and provide a rationale for targeting TLR signaling to improve therapy of this aggressive lymphoma. Gene expression was analyzed using Agilent human 2-color 4X44K oligo gene expression arrays. Cell line, TMD8 ABC-DLBCL, was infected with control (shControl, Cy3), shLTR7 (Cy5) or shLTR9 (Cy5) and changes in gene expression were monitored on day 1 and day 2 after induction of the shRNA with doxycycline, co-hybridizing control and experimental samples (Cy3+Cy5), for a total of 4 arrays.
Project description:TLR3 and TLR7 are critical recognition receptor which reacts with viruses. TLR3 and TLR7 contribute to many immunological pathway. Especially, TLR3 and TLR7 are clearly involved in the regulation of innate immunity in the gut. To identify the downstream target of TLR 3 and TLR7 in the IBD, we carried out microarray. The colon tissues were isolated from four different groups. None-no treatment, after DSS treated, after DSS+antiviral treatment, after DSS+TLR3+7 agonists.
Project description:Goal of the experiment was to examine the effects of different Toll-like receptor agonists on the human astrocyte response, in termsof levels of transcripts encoding cytokines, chemokines and theirreceptors. Stathmin and poly IC are considered as agonist for TLR3, LPSas an agonist for TLR4. The manuscript we are about to submit to Journal of Immunology contains these data, and identifies stathmin, aprotein, as a novel TLR3 agonist. We all know that poly IC is, and bycomparing the transcript responses in astrocytes to either poly IC andstathmin, and observing the similarity in these responses, the experiment adds to the evidence that stathmin indeed activates a verysimilar cellular response as poly IC. The LPS-induced response,mediated by TLR4, is included as a reference, to illustrate thatanother TLR-mediated reaction produces quite something different.
Project description:Bicuspid aortic valve (BAV) is a common congenital cardiac anomaly, with an estimated incidence of 1-2%. It is responsible for the greatest burden of aortic valve disease in patients younger than 70 years in North America. We performed microRNA profiling in end-stage valve leaflets with BAV and TAV. Patients undergoing elective aortic valve replacement for aortic stenosis at St. Michael’s Hospital, University of Toronto, between June 2010 and June 2011 were enrolled. Aortic valve leaflets were obtained intraoperatively from patients with congenital bicuspid (BAV; N=10) and tricuspid aortic valves (TAV; N=10) at the time of valve replacement. Leaflets were flash frozen in liquid nitrogen. MiRNA was isolated using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer`s instructions. For miRNA microarray analysis, total RNA was directly labeled with biotin and hybridized to the GenoExplorer microRNA human array containing 1583 human miRNA probes (Genosensor, Tempe, AZ) and the fluorescent signals were then scanned using a GenePix 4000b Biochip. The average of 3 mean fluorescence signal intensities for each miRNA probe was normalized to that for tRNAmet. Precursor miRNAs detected at 2-fold greater than background were considered to be expressed. Data were analyzed with GenePix 5.0 software, provided by GenoSensor Corp.
Project description:Lack of a standard method for stratifying advanced-stage NSCLC patients receiving platinum combination therapy often results in a number of patients that do not derive benefit yet are still exposed to treatment toxicity. We hypothesized that miRNAs in pre-treatment serum and/or plasma could be used to differentiate non-small cell lung cancer (NSCLC) patients who would have disease progression to first-line carboplatin and gemcitabine chemotherapy at first response assessment. miRNA profiling of mature and precursor miRNAs was performed on total RNA isolated from the pre-treatment serum and plasma of 24 NSCLC patients. Single validated candidates or combinations thereof were selected based on specificity and sensitivity to segregate patients with disease progression at first radiologic response (PD) vs. those without progressed disease (nonPD). Two precursor miRNA were significantly over-expressed in serum (but not plasma) of PD patients: pre-miR-518b and pre-miR-598. Serum miRNAs may serve as a screening tool in predicting chemoresistance to platinum-based combination chemotherapy. miRNA microarray was performed on RNA extracted from matched human serum or plasma obtained from NSCLC patients
Project description:Cells were transduced with a lentiviral Lenti-miR library composed of 661 miRNAs (System Biosciences) at a low multiplicity of infection (MOI) such that each cell over-expressed a single miRNA. The transduced population was then injected intra-hepatically into NOD-SCID mice for in vivo selection of miRNAs that when over-expressed, either promoted or suppressed metastatic liver colonization. Genomic DNA PCR amplication and recovery of lenti-viral miRNA inserts was performed on cells prior to injection, and from liver nodules, according to the manufacturer’s protocol. miRNA array profiling allowed for miRNA insert quantification prior to and subsequent to in vivo selection. To use an unbiased approach to identify regulators of colon cancer metastasis, we transduced a population of colon cancer cells, WiDR, with a lenti-miR library comprising of 661 human miRNAs, such that each individual cells over-express a single miRNA. The heterogenous population is then injected into the liver of immunodeficient mice. We theorized that cells over-expressing miRNAs that modulate colon cancer metastais will have differential representation in the whole population compared to a reference pool of cells. miRNAs that suppressed colon cancer metastasis will be lost in the resulting pool, while miRNAs that promote metastasis will be over-represented compared to the reference pool. Replicate pools of cells were transduced and injected into mice. After a period of 3-5 weeks, liver nodules were taken out and processed for lenti-viral derived miRNA profiling (Post samples) and compared to the reference pool of cells that were not injected into the mice (Pre Samples). The changes in miRNA representation between cells that underwent liver colonization were analyzed.
Project description:Cells were transduced with a lentiviral Lenti-miR library composed of 661 miRNAs (System Biosciences) at a low multiplicity of infection (MOI) such that each cell over-expressed a single miRNA. The transduced population was then injected intra-hepatically into NOD-SCID mice for in vivo selection of miRNAs that when over-expressed, either promoted or suppressed metastatic liver colonization. Genomic DNA PCR amplication and recovery of lenti-viral miRNA inserts was performed on cells prior to injection, and from liver nodules, according to the manufacturer’s protocol. miRNA array profiling allowed for miRNA insert quantification prior to and subsequent to in vivo selection. To use an unbiased approach to identify regulators of colon cancer metastasis, we transduced a population of colon cancer cells, SW620, with a lenti-miR library comprising of 661 human miRNAs, such that each individual cells over-express a single miRNA. The heterogenous population is then injected into the liver of immunodeficient mice. We theorized that cells over-expressing miRNAs that modulate colon cancer metastais will have differential representation in the whole population compared to a reference pool of cells. miRNAs that suppressed colon cancer metastasis will be lost in the resulting pool, while miRNAs that promote metastasis will be over-represented compared to the reference pool. Replicate pools of cells were transduced and injected into mice. After a period of 3-5 weeks, liver nodules were taken out and processed for lenti-viral derived miRNA profiling (Post samples) and compared to the reference pool of cells that were not injected into the mice (Pre Samples). The changes in miRNA representation between cells that underwent liver colonization were analyzed.
Project description:The goal of this study is to compare gene expression of mouse cultured cortical and hippocampal neurons in response to TLR3, TLR7 and TLR8 activation Overall design: At 4 day in vitro, wild-type mouse cortical and hippocampal neurons were treated with vehicle control, poly IC (for TLR3 activation) and CL075 (for TLR7 activation). Tlr7-/- neurons were treated with vehicle control and CL075/poly dT (for TLR8 activation). Six hr after treatment, total RNAs were extracted for deep sequencing, in triplicate, using the NextSeq500 High-output v2 (75 cycle) sequencing kit on the Illumina NextSeq500 instrument.
Project description:HIV is able to outpace the innate immune response, including the response mediated by interferon (IFN), to establish a productive infection. However, monocyte derived macrophages (MDMs) may be protected from HIV infection by treatment with type I IFN before virus exposure. The ability of HIV to modulate the type I IFN-mediated innate immune response when it encounters a cell that has already been exposed to IFN was investigated. To investigate the presence of HIV on an established IFN response, MDMs were subjected to four different conditions: (1) IFN-treated only, (2) IFN-treated followed by HIV infection, (3) HIV infected only, and (4) a mock-treated and mock-infected control. Microarray gene expression analysis was performed on a total of 24 samples derived from the 4 conditions assessed at 3 time points (1, 4 and 8 days following treatment/infection) for both IFN-α2 or -ω. Initially, ISGs were identified as those that were upregulated greater than 2-fold by IFN alone (condition 1) at both Days 4 and 8. Then, the IFN-treated condition was compared to the IFN-treated followed by HIV-infection condition in order to identify those ISGs that were downregulated at least 1.5-fold by the presence of HIV at both days. Assuming that it would be counterproductive for HIV infection by itself to induce the expression of ISGs with putative anti-HIV effects, those ISGs that were upregulated greater than 2-fold in the HIV control were removed. Finally, ISGs that passed these filters and were concordant with both IFN-treatments (IFN-α2 and -ω) were identified and corresponded to the following 8 ISGs: AXL receptor tyrosine kinase (AXL), interferon-alpha inducible protein 27 (IFI27), interferon-induced protein 44 (IFI44), interferon-induced protein 44-like (IFI44L), ISG15, OAS1, OAS3 and XIAP associated factor 1 (XAF1). It should be noted that the IFN-α2 and -ω microarray experiments were performed in different batches but batch effects were not corrected since genes were identified by the filtering approach just described within each batch.
Project description:Toll like receptors (TLRs) sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). Since pathogens may contain several agonists we asked whether different TLRs may synergize in DC activation. We report that in human and mouse DC TLR3 or TLR4 potently synergize with TLR7, TLR8 or TLR9 in the induction of selected cytokine genes. Upon synergistic stimulation, IL-12, IL-23 and Delta-4 are induced at levels 50-100 fold higher than those induced by optimal concentrations of single agonists, leading to enhanced and sustained TH1 polarizing capacity. Using microarray analysis we show that only 1.5% of the transcripts induced by single TLR agonists are synergistically regulated by combinations of TLR4 and TLR8 agonists.. These results identify a combinatorial code by which DCs discriminate pathogens and provide (suggest) a rationale to design adjuvants for TH1 responses. Series_overall_design: 3 untreated, 3 treated with LPS at 2h, 3 treated with LPS at 8h, 3 treated with R848 at 2h, 3 treated with R848 at 8h, 3 treated with LPS + R848 at 2h, 3 treated with LPS + R848 at 8h