FIG 2.
Transfer of cGAMP to macrophages is connexin-dependent and propagates antiviral responses. (A) IP-10 was measured from the supernatants of PMA-treated THP-1 cells cultured for 18 h with HEK or HEK-cGAS cells or alone and in the presence of conditioned medium (CM) from HEK or HEK-cGAS cells. Data shown are averaged from three independent experiments conducted in biological triplicate (± the SEM and unpaired t tests comparing coculture with CM conditions). (B) WT THP-1 cells pretreated or not with PMA for 2 h were rinsed and cocultured overnight with HEK or HEK-cGAS cells, and the IP-10 levels were measured by ELISA. The data shown are averaged from three independent experiments conducted in biological triplicate (± the SEM and unpaired t tests comparing NT with PMA conditions). PMA-treated WT THP-1 cells (C) or human blood-derived monocytes (mono) (D) were cocultured overnight with HEK or HEK-cGAS cells alone, in the presence of 100 μM carbonoxolone (CBX) or 100 μM meclofenamate (Meclo) to inhibit connexins. (C and D) Data are averaged from two independent experiments conducted in biological duplicate (Meclo) or three (CBX) independent experiments conducted in biological triplicate (± the SEM and unpaired t test results). (E) CX43 and CX45 mRNA levels upon cotransfection of targeting (or control) siRNAs for 24 h were measured by RT-qPCR relative to 18S. Data shown are averaged from two independent experiments conducted in biological duplicate. (F) IP-10 levels were measured by ELISA in supernatants from overnight culture of PMA-treated WT or cGAS−/− THP-1 cells with HEK-cGAS cells previously transfected with siRNAs targeting CX43 and CX45. Data shown are averaged from at least two independent experiments conducted in biological triplicate (± the SEM and unpaired t tests compared to the si-control). (G) HEK-CX43/45WT (CX43/45 WT) or HEK-CX43/45−/− (CX43/45–/–) cells were transfected with a plasmid encoding cGAS-GFP or GFP (used as control) for 1 h prior to their coculture with cGAS−/− THP-1 cells (THP-1) for 18 h. Data shown are averaged from two independent experiments conducted in biological triplicate. (H, I) HEK-Blue cells were transfected for 24 h prior washing and overnight coculture with PMA-treated THP-1 cGAS–/– (see Fig. S2A). CBX (100 μM) or SFZ (0.5 mM) was added at the time of coculture where indicated. Coculture supernatants were analyzed for IP-10 production (H) and SEAP (reflective of IFN activation of the HEK-Blue) (I). (H and I) Data shown are averaged from two independent experiments conducted in biological triplicate (I). (J) Cells treated as for panels H and I were infected for 24 h with influenza A virus (IAV) (strain A/WSN/1933[H1N1]) (MOI of 5). Then, 10-fold dilutions of supernatants were made in PBS and added to a 96-well tissue culture plate containing Vero cells in growth medium. The data shown are averaged from three independent experiments conducted in biological triplicate, reported to the mean of condition HEK-Blue expressing GFP or cGAS (± the SEM and unpaired t tests shown). (A to J) Each point represents the mean data for each independent experiment, the column representing the mean of the experiments.*, P ≤ 0.05; **, P ≤ 0.01; ns, not significant; nd, not detected. Detailed materials and methods are provided in Text S1.