Fig. 4. Germ-free mice do not mount a proinflammatory response upon bleomycin treatment.

Mice were housed in germfree, ABSL-1, or ABSL-2 facilities and intranasally inoculated with 75 μl containing 0.04 Units of bleomycin nor saline (control). 14 days postinoculation, mice were sacrificed and single-cell lung suspension prepared form the lungs. Flow cytometric analysis of T cells from single cell were conducted for CD4 + a IL-6, b gp130, c PD-1+ IL-17A + , d gating strategy, e representative histograms from ABSL 2 cohort and f pSTAT3Y⁷⁰⁵ expression on T cells. g Relative expression of STAT3 mRNA in lung tissue. Comparisons between cohorts were performed using two-way ANOVA with Tukey’s post-hoc. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: no significance. Bars are mean ± SD; each symbol represents an individual mouse, n = 6–29 mice. The experiments were independently conducted an average of two times.