Fig. 5: Hypercapnia-induced suppression of the macrophage antiviral response is mediated by Akt1.

AMØs obtained by BAL from untreated mice were immunostained with isoform-specific antibodies for Akt1 (red), Akt2 (white) and Akt3 (green); nuclei were stained with DAPI (blue), n=5, results representative of at least 3 independent experiments (A). Differentiated THP-1 MØ were exposed to 5% CO₂ (NC) or 15% CO₂ (HC) in the absence or presence of specific inhibitors of Akt1 or Akt2, A674563 (50 nM) and CCT128930 (1 μM), respectively, and infected with IAV (MOI 2). Expression of RIG-I (B) and viperin (C) protein was assessed by immunoblot, and viral titers in culture supernatants were assessed by plaque assay (D); mean ± SEM, n = 4 from 4 independent experiments, *p<0.05 vs NC NI; **p<0.05 vs NC + IAV; ***p<0.05 vs HC + IAV. In additional experiments, mice were infected intranasally with lentivirus containing non-silencing (NS) shRNA or Akt1-targeted shRNA, and 21 days later AMØs were collected by BAL and immunostained for Akt1 (red) and F4/80 (green, MØ marker) (E) or lysed to measure Akt1 expression by immunoblot (F), n=4, results representative of at least 2 independent experiments. Alternatively, AMØs from the shRNA-treated animals were cultured in NC or HC, infected with IAV (MOI 2), and 18 h later processed for quantitation of viral NS1 protein by immunoblot (G); n=4, results representative of at least 2 independent experiments, *p<0.05 vs NS shRNA, ##p<0.05 vs NS shRNA + IAV in NC, ### p<0.05 vs NS shRNA + IAV in HC.