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. 2010 Jan;87(1):25-34.
doi: 10.1189/jlb.0409251.

Pivotal advance: Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded microRNA specifically induce IL-6 and IL-10 secretion by macrophages and monocytes

Zhiqiang Qin  1 , Patricia Kearney, Karlie Plaisance, Chris H Parsons
Affiliations

Pivotal advance: Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded microRNA specifically induce IL-6 and IL-10 secretion by macrophages and monocytes

Zhiqiang Qin et al. J Leukoc Biol. 2010 Jan.

Abstract

Macrophages are an important source of inflammatory cytokines generated during the innate immune response,but in the microenvironment of certain tumors,macrophages promote tumor progression through their preferential secretion of cytokines that support tumor cell growth and suppress antitumoral immune responses. KSHV is the causative agent of KS and lymphomas preferentially arising in immuno compromised patients, and specific cytokines, including IL-6 and IL-10, have been implicated in KSHV-associated cancer pathogenesis. However, the contribution of KSHV-infected macrophages to the cytokine milieu within KSHV-related tumors is unclear. We found that individual KSHV-encoded miRNA induce IL-6 and IL-10 secretion independently and additively by murine macrophages and human myelomonocytic cells. Bioinformatics analysis identified KSHV miRNA binding sites formiR-K12-3 and miR-K12-7 within the 3'UTR of the basic region/leucine zipper motif transcription factor C/EBPbeta, a known regulator of IL-6 and IL-10 transcriptional activation.Subsequent immunoblot analyses revealed that miR-K12-3 and miR-K12-7 preferentially reduce expression of C/EBPbeta p20 (LIP), an isoform of C/EBPbeta known to function as a negative transcription regulator. In addition,RNA interference specifically targeting LIP induced basal secretion of IL-6 and IL-10 by macrophages.Taken together, these data support a role for KSHV miRNA in the programming of macrophage cytokine responses in favor of KSHV-related tumor progression.

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Figures

Figure 1.
Figure 1.
Murine macrophages express KSHV miRNA following transient transfection. RAW cells were transiently cotransfected with combinations of 1 μg empty control vector (pcDNA), 1 μg of a construct encoding 10 individual KSHV miRNA (pcDNA-miRNAs), 0.5 μg respective miRNA luciferase reporter constructs (pGL3-miRNA sensor), and 300 pmol 2′OMe RNA antagomirs targeting miR-K12-1 (mi1), miR-K12-3, miR-K12-7, miR-K12-9, miR-K12-11, or miR-K12-12 (the latter as a negative control, as miR-K12-12 is not expressed in the pcDNA-miRNA construct). Forty-eight hours after transfection, cells were lysed for quantitation of luciferase expression normalized to total protein content (RLU). A reduction in luciferase expression denotes expression of mature miRNA. Error bars represent the sem for two independent experiments.
Figure 2.
Figure 2.
KSHV miRNA induce secretion of IL-6 and IL-10 by macrophages. RAW cells were transiently transfected with pcDNA or pcDNA-miRNA as above. (A) Twenty-four hours later, cytokines were quantified in culture supernatants using a flow cytometry-based bead array, per the manufacturer’s instructions. (B) In subsequent experiments, IL-6 and IL-10 secretion was quantified in culture supernatants by ELISA at different time-points following transfection. (C and D) Transcripts for IL-6, IL-10, and their respective receptor components were amplified by real-time RT-PCR 8 h (C) or 24 h (D) after transfection of RAW cells with pcDNA or pcDNA-miRNA. Normalized fold change, using no-RT samples and β-actin amplification for loading controls, was calculated using automated Bio-Rad iQ5 v2.0 software. Error bars represent the sem for two (A and B) or three (C and D) independent experiments. *, P < 0.05; **, P < 0.01.
Figure 3.
Figure 3.
KSHV miRNA induction of IL-6 and IL-10 secretion by macrophages is independent of TLR pathways. (A) RAW cells were transiently transfected with pcDNA or pcDNA-miRNA for 24 h, then incubated with 10 mM 2-AP or vehicle control for 3 h, and incubated for an additional 24 h. (B) In parallel, RAW cells were transfected as in A and then incubated with 100 μM of a control peptide or MyD88 inhibitor peptide for 24 h. Cytokines were quantified within culture supernatants by ELISA. Error bars represent the sem for three independent experiments. *, P < 0.05.
Figure 4.
Figure 4.
Individual KSHV miRNA differentially regulate IL-6 and IL-10 secretion by macrophages. RAW cells were transiently transfected with pcDNA or pcDNA-miRNA with additional transfection media (solid bars, “mock”) or inhibitors of individual KSHV miRNA (open bars; 2′OMe miX, where X=numeric designation of KSHV miRNA). Of note, the 2′OMe RNA antagomir targeting KSHV miR-K12-12, a miRNA not encoded by the pcDNA-miRNA construct, was used as a negative control. Forty-eight hours following transfection, cytokines were quantified in supernatants by ELISA. Error bars represent the sem for three independent experiments. *, P < 0.05; **, P < 0.01.
Figure 5.
Figure 5.
KSHV miRNA induce IL-6 and IL-10 secretion following de novo infection of macrophages by KSHV. (A) For IFA, RAW cells were incubated with purified KSHV (K+) or UV-inactivated KSHV (K–) for 2 h (MOI ∼10), and then cells were fixed 16 h later, prior to their incubation with an anti-LANA mAb and a secondary antibody conjugated to Texas Red to identify the typical punctate intranuclear expression of KSHV LANA (red dots). (B) Sixteen hours after viral incubation, transcripts for LANA and vFLIP were amplified by RT-PCR, and β-actin was amplified as an internal control. (C) In parallel experiments, RAW cells were transfected with 0.5 μg pGL-miRNA sensors (mi-X=pGL sensor plasmid encoding compliment for miR-K12-X) overnight and then incubated with purified KSHV or UV-KSHV as above. Luciferase expression was quantified subsequently for cells collected 2 h or 24 h following viral incubation (p.i.), as outlined in Materials and Methods. (D and E) RAW cells were transfected with 300 pmol 2′OMe RNA targeting miRNA (miX=antagomir for miR-K12-X) for 48 h and then infected with purified KSHV. IL-6 (D) and IL-10 (E) were quantified within culture supernatants 24 h after viral incubation by ELISA. Error bars represent the sem for three independent experiments. *, P < 0.05; **, P < 0.01.
Figure 6.
Figure 6.
KSHV miRNA induce IL-6 and IL-10 secretion by human monocytes. MM6 cells were transiently cotransfected with 1 μg pcDNA or pcDNA-miRNA and 300 pmol 2′OMe miX, and the 2′OMe mi12 was used as a negative control as above. Supernatants were collected 48 h later and IL-6 (A) and IL-10 (B) quantified by ELISA. Error bars represent the sem for two independent experiments. *, P < 0.05; **, P < 0.01 relative to pcDNA miRNA transfectants.
Figure 7.
Figure 7.
KSHV miRNA induce macrophage secretion of IL-6 and IL-10 through the down-regulation of C/EBPβ. (A) KSHV miRNA binding sites in the 3′UTR region of C/EBPβ. KSHV miRNA nucleotides with matching base pairs within the C/EBPβ 3′UTR (1–481 bp) are depicted in capital letters. (B) RAW cells were transfected with pcDNA (pc) or pcDNA-miRNA (miRNA) or cotransfected with pcDNA-miRNA and 2′OMe antagomirs (miX; X=miR-K12-X targeted by the antagomir). For Western blot analyses 24 h later, cell lysates were incubated with a C/EBPβ-specific antibody recognizing three independent isoforms of this protein (p42, p35, and LIP), and the intensity of immunoreactive bands for LIP was quantified relative to pcDNA control transfectants using Image-J software. (C) RAW cells were incubated with transfection reagent alone (mock) or control, nontarget siRNA (n-siRNA) or C/EBPβ-specific siRNA (c-siRNA). Twenty-four hours later, Western blot quantified C/EBPβ isoform expression as in B. β-Actin was quantified for loading controls for all Western blots. (D and E) IL-6 and IL-10 were quantified within supernatants from cultures in C by ELISA. Error bars represent the sem for two independent experiments. *, P < 0.05; **, P < 0.01.
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