1. Volume 21, Issue 1, Pages 141-153 (October 2017)
Host MicroRNAs-221 and -222 Inhibit HIV-1 Entry in Macrophages by Targeting the CD4 Viral Receptor 
Robert Lodge, Jérémy A. Ferreira Barbosa, Félix Lombard-Vadnais, Julian C. Gilmore, Alexandre Deshiere, Annie Gosselin, Tomas Raul Wiche Salinas, Mariana G. Bego, Christopher Power, Jean-Pierre Routy, Petronela Ancuta, Michel J. Tremblay, Éric A. Cohen 
Cell Reports 
Volume 21, Issue 1, Pages (October 2017)
DOI: /j.celrep
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2. Cell Reports 2017 21, 141-153DOI: (10.1016/j.celrep.2017.09.030)
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3. Figure 1
MiR-221 and miR-222 Are Enhanced in Bystander MDMs of HIV-1-Infected Macrophage Cultures
(A) Heatmap comparing the relative expression of the 30 most highly expressed miRNAs as obtained by miRNA-seq in sorted GFP+ve (GFP-POS) or GFP−ve (GFP-NEG) macrophages following 6-day of VSV-G-pseudotyped HIV-1 infection, versus uninfected (mock) cells. Data are pooled from 3 blood donors. Arrows identify miR-221 and miR-222.
(B) MiR-221, miR-222, miR-186, and miR-151a expression was determined in sorted macrophages derived from 8 blood donors by real-time qPCR. Bars shown are the mean fold change compared to mock (n = from 4 to 8 donors).
See also Figure S1 and Table S1.
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4. Figure 2 The 3′UTR of CD4 Is a Target of miR-221 and miR-222
(A) Nucleotide sequence of miR-221 and miR-222 with the CD4 seed sequence underlined. Sequence of the 3′UTR of CD4 recognized by miR-221 and miR-222 and the CD4 mutated sequence (stars) used in the target validation assay are shown. Nucleotide annotation is from gb: M
(B) Target validation assay in wild-type (WT) or mutant Luc-3′UTR(CD4) transfected 293T cells treated with either miR-221 or miR-222 mimics. Results were plotted as mean firefly Luc activity (relative light units [RLUs]) standardized to control Renilla Luc activity ± SD. Statistical significance was determined using Student’s t test (n = 4 technical replicates).
(C) Real-time qPCR analysis of CD4 mRNA expression in HIV-1-infected sorted MDMs of 8 donors versus uninfected (mock). Bars represent mean fold change (n = 8 donors).
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5. Figure 3
Effect of Transfected miR-221 and/or miR-222 Mimics on CD4 Expression and HIV-1 Infection in MDMs
MDMs from several blood donors were transfected with the indicated mimics or controls and cultured for 48 hr.
(A) CD4 mRNA was quantified by real-time qPCR and compared to control-transfected levels. Bars are the mean fold change as compared to mock (n = 6 donors).
(B) Surface CD4 was measured by flow cytometry (left). Geometric mean fluorescence intensity (MFI) is shown for each condition, for a representative blood donor. Expression of surface CD4 (right), as determined by MFI, is shown relative to control-transfected cells. Bars are the mean relative MFIs (n = 9 donors).
(C) Surface CCR5 was measured by flow cytometry. Geometric MFI for each condition is shown for a representative blood donor (n = 4).
(D) MDMs isolated from 4 distinct donors were treated with the indicated mimics or controls, infected with HIV-Env (ADA) or VSV-G-pseudotyped Luciferase-encoding HIV and F-Luc activity determined in lysates. RLU’s were normalized to F-Luc obtained in VSV-G-pseudotyped virus-infected cells. Mean relative F-Luc activity ± SD are shown (n = 4 donors).
See also Figure S2.
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6. Figure 4
MiR-221 and miR-222 Antagomirs Enhance HIV-1 Spread in MDM Populations
(A) Representative dot plot illustrating the transfection efficiency of fluorescent antagomirs in MDMs.
(B) MDMs obtained from several blood donors were transfected with either a control or a mix of miR-221 and miR-222 antagomirs (inhibitors) and infected the next day with GFP-expressing HIV-1. The percentage of GFP+ve cells and HIV-1 p24 (± SD) in supernatants were then assessed at the indicated time points. Representative experiments of infected MDMs from 3 donors are shown.
(C) Percentage of infected cells (GFP+ve) and levels of virus (HIV p24) produced at day 12 from infected MDMs exposed to control or a mix of miR-221/miR-222 antagomirs are shown (n = 8 using MDMs from 5 distinct donors). Red dots indicate data from less responsive MDMs (for example, donor 196).
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7. Figure 5
MiR-221 and/or miR-222 Antagomirs Counteract TNF-α-Driven Down-Modulation of CD4 and Restore HIV-1 Entry
MDMs derived from several blood donors were treated with 10 ng/mL of TNF-α for 24 hr and then transfected with the indicated antagomirs for the following 24 hr. See also Figure S3.
(A) CD4 mRNA from activated MDMs was quantified by real-time qPCR. Bars represent the mean CD4 mRNA fold change (n = 4 donors).
(B) Surface CD4 in activated MDMs was measured by flow cytometry (representative data are shown with geometric MFIs for each condition). Expression of surface CD4 (right), as determined by relative MFI, is compared to the control-transfected cells. Bars represent mean relative MFIs (n = 6 donors).
(C) Surface CCR5 was measured by flow cytofluorometry. MFI for each condition is shown for a representative blood donor (n = 4 donors).
(D) MDMs isolated from 4 donors were treated with TNF-α (10 ng/mL) (or not), transfected with the indicated control or antagomirs, and infected with the indicated Blam-Vpr containing viruses. Graph represents mean viral fusion efficiency (%) ± SEM (n = 4 donors). Representative data with cells derived from a blood donor are shown in Figure S4.
(E) Differentiated THP-1-CD4R cells were treated with TNF-α (10 ng/mL) and infected with the indicated Blam-Vpr containing viruses. The graph represents mean viral fusion efficiency (%) ± SD (n = 3 technical replicates). Statistics in were performed using Student’s t test.
See also Figures S2, S3, and S4.
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8. Figure 6
TNF-α Secreted in HIV-1-Infected MDM Supernatants Enhances miR-221/miR-222 Expression in Macrophages
(A) MDMs from 4 blood donors were infected with HIV-1 and the levels of TNF-α in their supernatants following 3, 6, or 15 days of infection was compared to that of uninfected cells by ELISA analysis. Bars represent mean TNF-α concentration ± SD (n = 4 technical replicates). Note that bars in red correspond to infected MDMs shown in red in Figure 4C.
(B) Virus-cleared supernatant from infected macrophages (left: mean TNF-α concentration ± SEM; n = 4 technical replicates) was added to new MDM cultures from 4 distinct donors, prior to infection with the indicated Luc-encoding pseudotyped HIV-1 viruses, and Luc activity in cell lysates was measured (right). In some cases, the conditioned supernatant was treated with neutralizing anti-TNF-α or control goat anti-human IgGs before its addition to MDM cultures. Shown are mean Luc activity ± SD (n = 4 donors).
(C) The RNAs of the supernatant-treated macrophages in (B) were extracted and the expression of miR-221 or miR-222 determined by real-time qPCR. Bars represent mean fold change (n = 4 donors).
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9. Figure 7
MiR-221 Is Enhanced in Myeloid Cells of the Colon as Compared to Peripheral Blood Myeloid Cells
(A) The gating strategy used to define myeloid cell populations by flow cytofluorometry is shown.
(B) Cells of the myeloid lineage were sorted from matched sigmoid intestine biopsies and blood of 3 HIV-negative study participants (A, B, and C). The expression of miR-221 in the blood and colon myeloid cells was determined by real-time qPCR, and normalized relative to that of blood myeloid cells. Shown are the fold changes for each donor in a box and whiskers graph (n = 4–7 technical replicates/donor).
Cell Reports  , DOI: ( /j.celrep )
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