Fig 5. Progesterone (P4) increases amphiregulin (AREG) expression and administration of recombinant AREG protects P4-depleted female mice against IAV infection.
Adult female mice were ovariectomized, treated with placebo (-P4) or exogenous P4 (+P4), and inoculated with a sublethal dose of IAV or mock-infected. The expression of amphiregulin (Areg) mRNA (A) and protein concentrations (B) in the lungs were quantified at 3, 5, 7, 9 and 14 dpi (n = 8-10/treatment/dpi). Gene expression was normalized to Gapdh and mock-infected controls using the ΔΔCt method. Ovariectomized mice were treated with placebo (-P4), placebo and recombinant amphiregulin (-P4 +rAREG), or P4 (+P4) and inoculated with a sublethal dose of IAV. To confirm AREG replacement, pulmonary concentrations of AREG were measured at 14 dpi (C). Mice were monitored daily for changes in body temperature (D) and clinical disease (E) (n = 9-10/treatment). H&E stained lung sections collected at 14 dpi were scored for inflammation as a cumulative score of perivasculitis, vasculitis, bronchiolitis, alveolitis, edema, consolidation, and necrosis (F). Representative images of overall inflammation (2X magnification) and focused areas (10X magnification) with cellular infiltration and edema are shown (G) (n = 3-5/treatment, with 10 fields per animal). Pulmonary function tests were performed at 14 dpi and lung diffusing capacity (DFCO; H), lung compliance (Crs; I), and resistance (Rrs; J) were measured (n = 8-10/treatment). The dotted lines represent the value (means ±SEM) for mock-infected mice and bars and circles represent means ±SEM for IAV-infected mice from 2 independent experiments, with significant differences represented by asterisks (*).