Novel HIV-1 MiRNAs Stimulate TNFα Release in Human Macrophages via TLR8 Signaling Pathway

Purpose To determine whether HIV-1 produces microRNAs and elucidate whether these miRNAs can induce inflammatory response in macrophages (independent of the conventional miRNA function in RNA interference) leading to chronic immune activation. Methods: Using sensitive quantitative Real Time RT-PCR and sequencing, we detected novel HIV-derived miRNAs in the sera of HIV+ persons, and associated with exosomes. Release of TNFα by macrophages challenged with HIV miRNAs was measured by ELISA. Results: HIV infection of primary alveolar macrophages produced elevated levels of viral microRNAs vmiR88, vmiR99 and vmiR-TAR in cell extracts and in exosome preparations from conditioned medium. Furthermore, these miRNAs were also detected in exosome fraction of sera from HIV-infected persons. Importantly, vmiR88 and vmiR99 (but not vmiR-TAR) stimulated human macrophage TNFα release, which is dependent on macrophage TLR8 expression. These data support a potential role for HIV-derived vmiRNAs released from infected macrophages as contributing to chronic immune activation in HIV-infected persons, and may represent a novel therapeutic target to limit AIDS pathogenesis. Conclusion: Novel HIV vmiR88 and vmiR99 are present in the systemic circulation of HIV+ persons and could exhibit biological function (independent of gene silencing) as ligands for TLR8 signaling that promote macrophage TNFα release, and may contribute to chronic immune activation. Targeting novel HIV-derived miRNAs may represent a therapeutic strategy to limit chronic immune activation and AIDS progression

Novel Developments in the Epidemic of Human Immunodeficiency Virus and Tuberculosis Coinfection

Tuberculosis (TB) disease remains one of the highest causes of mortality in HIV-infected individuals, and HIV–TB coinfection continues to grow at alarming rates, especially in sub-Saharan Africa. Surprisingly, a number of important areas regarding coinfection remain unclear. For example, increased risk of TB disease begins early in the course of HIV infection; however, the mechanism by which HIV increases this risk is not well understood. In addition, there is lack of consensus on the optimal way to diagnose latent TB infection and to manage active disease in those who are HIV infected. Furthermore, effective point-of-care testing for TB disease remains elusive. This review discusses key areas in the epidemiology, pathogenesis, diagnosis, and management of active and latent TB in those infected with HIV, focusing attention on issues related to high- and low-burden areas. Particular emphasis is placed on controversial areas where there are gaps in knowledge and on future directions of study

Identification of candidate miRNAs encoded by GU-rich tract in HIV LTR and is highly conserved in HIV-1.

<p><b>A</b>: Small RNAs processed from HIV-1 LTR region observed by SOLiD Deep Sequencing. Left peak shows small RNAs derived from TAR stem (miR TAR). Right peak shows a hotspot for small RNAs derived from R and U5 stem region. The GU-rich tract (46 nt) encodes a family of viral miRs including vmiR88 and vmiR99. Modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Schopman1" target="_blank">[14]</a>. <b>B</b>: shRNA mirs are intermediates in biogenesis of mature vmiRs. shRNA reported for 43/9175 TAR (left). UNAFold software predicts folding of shRNAs vmir88 (middle) and vmir99 (right), which suggests the structures of intermediates in the biogenesis of the mature vmiR-TAR (black rectangle), vmiR88 (blue rectangle) and vmiR99 (red rectangle). UNAFold's thermodynamic calculations predict that all three shRNAs fold spontaneously (Δ<i>G</i><0) into stable hairpins (high melting temperature, <i>T</i>_M>53.8°C in 1M Na⁺). <b>C</b>: To delineate the boundaries and sequences of mature viral miRNAs, cell extracts and exosomal extracts were analyzed. Sample cell extracts were <i>in vitro</i>-infected AM (healthy AM+HIV), HIV-positive U1 macrophages stimulated by PMA (U1+PMA). Exosomal extracts were from exosomes of HIV+ human serum (HIV+ serum 10 b). Total RNA was amplified by qRT-PCR, cloned into pCR4-TOPO vector and DNA was sequenced. Sequences of vmiR88 and vmiR99 PCR products were aligned with sequences of plasmid (vector) and HIV-BaL strain. The polyadenylation signal (PA signal) and polyadenylation site (PA site) were reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Bhnlein1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106006#pone.0106006-Berkhout1" target="_blank">[36]</a>.</p

Novel HIV-produced miRNAs are released by HIV-infected human macrophages and associated with exosomes <i>in vitro</i>, and detected in sera from asymptomatic HIV+ persons.

<p>Quantitative PCR measurement of HIV miRNA from exosomal preparation of cultured supernatants from adherent (<b>A</b>) human macrophage cell lines U937 and HIV+U1, and (<b>B</b>) human alveolar macrophages (established <i>in vitro</i> HIV infection, or from asymptomatic HIV+ person), incubated in the absence or presence of PMA for 24 h. Western blot immediately beneath each bar graph demonstrates exosomal marker CD63 associated with corresponding sample. Data reflect a minimum of 4 experiments performed in duplicate. (<b>C</b>) Quantitative PCR measurement of HIV miRNA in exosomal preparations from archived sera of asymptomatic HIV+ persons with peripheral blood CD4+ T-lymphocyte count <200 cells/mm³. Data reflect measurements performed in duplicate. Results shown include exosome preparations isolated from HIV+ sera (<i>n</i> = 14) sampled from HIV+ patients (numbered 1–13). Serum samples “10a” and “10b” were drawn on separate days from Patient 10. (<b>D</b>) Exosomes were isolated from serum (HIV⁺ serum from Patient 9 or healthy serum) using ExoQuick-TC reagent and resuspended in the original volume of PBS (divalent cation-free). THP-1 macrophages cultured in medium (1.0 mL) were treated with 10 µL of exosome suspension (24 h, 37°C, 5% CO₂). (<b>E</b>) THP-1 macrophages were treated (24 h) with 500 µL of conditioned medium (healthy parental U937 macrophages that had been cultured 6 d) or medium-suspended exosomes that had been isolated from 500 µL HIV⁺ conditioned medium (U1 macrophages cultured 6 d). Conditioned medium was analyzed by ELISA for TNFα. *, p<0.05.</p

Oligoribonucleotides in quantitative Real Time RT-PCR and melting analysis of PCR products.

<p>“*”: phosphorothioate linkage</p><p>“m”: 2′-O-methyl modification</p><p>Oligoribonucleotides in quantitative Real Time RT-PCR and melting analysis of PCR products.</p

Sequence alignment of HIV vmiRs with consensus genomic sequence from HIV-1 subtypes and absolute quantitation of miRNAs by Real Time RT-PCR.

<p><b>A</b>: Alignment of vmiR sequences of GU-rich tract is consistent with consensus genomic sequence from HIV-1 subtypes A-J, 533 isolates. <b>B</b>: VmiR99 is 90–100% identical to 96% of HIV-1 sequences. Sequences within the GU tract, vmiR88, vmiR99 and ssRNA40 were aligned with 196, 201, 254 and 272 genome sequences, respectively. <b>C</b>: Genomic RNA of HIV-1 BaL strain was scanned for every 21-bp RNA segment and the distribution of base compositions (46.5±11.8% G+U) is shown. VmiR-TAR is GU-poor (35%). VmiR88 and vmiR99 are GU-rich (71% and 76% G+U, respectively). Absolute quantitation of miRNAs was determined by Real Time RT-PCR. After first strand cDNA synthesis, amplification (<b>D, F, H</b>) and absolute quantitation (<b>E, G, I</b>) of vmiR-TAR (<b>D–E</b>) vmiR88 (<b>F–G</b>), vmiR99 (<b>H–I</b>) and RNA40 (I) was standardized using synthetic miR oligonucleotides in the miRCURY LNA Universal RT microRNA PCR method (Exiqon) on an ABI 7900HT FAST Real Time PCR system. ΔR_n is the change in normalized reporter fluorescence intensity. <i>C</i>_T is the threshold cycle in which the amplification curve crosses the dashed horizontal line. Data depict a representative experiment done in duplicate.</p

Viral miRNAs stimulate THP-1 macrophages to release TNFα rapidly in a vmiR sequence-dependent manner, and release is inhibited by antagomirs.

<p><b>A</b>: THP-1 macrophages were treated with vmiR99 (1.0 µg/mL) at the indicated time points (hr) or with Lipid A or ssRNA40 (24 h). Conditioned medium was analyzed by ELISA. Total RNA was isolated from cell extracts, and expression of <i>TNF</i> (normalized by <i>GAPD</i>) was analyzed by qRT-PCR. Results are the average of three independent experiments done in duplicate. <b>B</b>: THP-1 macrophages were pre-treated with antagomir (5.0 µg/mL for 1 h) followed by treatment with ssRNA40 (2.5 µg/mL), vmiR88 (5.0 µg/mL) or vmiR99 (5.0 µg/mL for 24 h), and conditioned medium analyzed by ELISA. <b>C</b>: Sequence variants of vmiR88 and vmiR99 can elicit TNFα release by THP-1 macrophages. Variants of vmiR88 or vmiR99 were chemically synthesized by substituting the uridine residues of U-rich motifs (boxed regions) for adenine residues. VmiRs and variants (5.0 µg/mL) were applied to cells for 24 h. Supernatants of conditioned medium were assayed for TNFα by ELISA. *, p<0.05.</p

Novel HIV-produced miRNAs are detected in HIV-infected human macrophages, and stimulate macrophage TNFα release <i>in vitro</i>.

<p><b>A</b>: AM were exposed to HIV-1 particles, BaL strain (10 ng/0.1 mL Gag p24/106 cells for 3 h) and washed. HIV p24 levels were assayed by ELISA. Data presented are AM infected with HIV-1 done in duplicate (<i>n</i> = 4 subjects). Quantitative PCR measurement of HIV miRNA from cell extracts of adherent (<b>B</b>) human macrophage cell lines U937 and HIV+U1, and (<b>C</b>) human primary alveolar macrophages (established <i>in vitro</i> HIV infection, or from asymptomatic HIV+ person), incubated in the absence or presence of PMA for 24 hr. <b>D</b>: TNFα measurement (ELISA) in culture supernatants in AM from healthy volunteers following 24 h incubation with HIV miRNA (vmiR-TAR, vmiR88, or vmir99) over a concentration range (0.01–1.0 µg/mL), lipid A (10 µg/mL), or control GU-rich ssRNA40 or AU-rich ssRNA41. <b>E</b>: TNFα measurement (ELISA) in culture supernatants from adherent human alveolar macrophages from healthy volunteers was treated with antagomir99 (1 h) followed by vmiR99 (24 h). At right, adherent AM were treated with pre-annealed antagomir99•vmiR99 duplex (**) for 24 h. <b>F</b>: TNFα measurement (ELISA) in culture supernatants from adherent human alveolar macrophages from healthy volunteers, in the presence of targeted TLR8 gene silencing (TLR8 siRNA) compared to control non-silencing RNAi (Control siRNA) following 24 h incubation with novel HIV vmiR99 (1.0 µg/mL in LyoVec), Lipid A (10 µg/mL) or unstimulated (US; LyoVec vehicle control). Cell extracts were analyzed by Western blot for TLR8 knockdown using anti-TLR8 antibody and for well loading using anti-β-actin antibody. Data for each figure reflect a minimum of 4 experiments, performed in duplicate. *, p<0.05.</p