Fig 2: Expression of Il11 in smooth muscle cells is associated with body weight loss, elevated organ weights and spontaneous death.(a) Schematic diagram of the targeted expression of Il11 in Myh11+ve SMC. In Rosa26-Il11 mice, a floxed cassette containing both the neomycin (neo) resistance and stop elements is positioned before the murine Il11 transgene cassette, which undergoes tamoxifen (tam) initiated Cre-mediated recombination when crossed to the Myh11-Cre/ERT2 mouse. (b) Breeding scheme to generate Myh11Cre/+Rosa26Il11/+ (Il11SMC) and Myh11Cre/+Rosa26+/+ (CreSMC) offspring mice. Note that the Myh11-Cre gene is expressed on the Y chromosome and therefore only male offspring carry the transgene. (c) Genotyping of tail biopsy DNA. A 287 bp band indicates the presence of the Cre transgene whereas the 180 bp band determines the presence of the internal positive control (top gel). Polymerase chain reaction with the Rosa26-Il11 primer set detects a 270 bp band indicative of the Rosa26-Il11 transgene whereas the 727 bp band indicates the presence of the wild-type transgene (bottom gel). Uncropped blots are presented in S2 Fig. (d) Survival curve of Il11SMC mice treated with tam (n = 35) and corn oil vehicle (veh; n = 12) mice following tamoxifen initiation at day 0 and followed until day 14. Survival curves were compared using the log-rank Mantel-Cox test. (e) Body weight changes (expressed as percentage of day 0 body weight) in Il11SMC mice treated with tam or veh (n = 8 per group). Green arrows denote individual injections. Statistical analyses by two-way ANOVA with Sidak multiple comparisons; data expressed as mean ± standard deviation. (f) Collated body weights (left) and (g) body lengths (right) of Il11SMC mice treated with tam or veh measured at d14 post initial tamoxifen dose (n = 12–13 per group). (h) Organ weights of the heart, (i) lung and (j) kidney normalized to body weight in Il11SMC mice treated with tam or veh (n = 12–13 per group). All comparisons were conducted in mice 14 days post-veh and tam treatment. Statistical analyses by two-tailed unpaired t-test; data expressed as median ± IQR, whiskers represent the minimum and maximum values.

Fig 3: SiRNA-IL-11 transiently transfection attenuates bleomycin-induced lung fibrosis and pulmonary hypertension in transgenic Tie2-GFP mice. Wild-type (WT) siRNA(-) Tie2-GFP mice and IL-11-KO siRNA-IL-11 Tie2-GFP mice received a single intratracheal dose of bleomycin (1.5 U/kg) on day 1 (n = 11) during 14 days. siRNA-IL-11 was administered intravenously and intranasally three times a week from day 1 to day 14. At day 14 the following parameters were measured. A Masson’s trichrome histological images are showed. Scale bar: 100 µm. B Ashcroft score lung fibrotic index, C hydroxyproline amount in lung tissue D right ventricular systolic pressure (RVSP) mmHg, E right ventricular (RV) hypertrophy measured by the ratio of RV/left ventricular (LV) + septo in mg/mg, F pulmonary artery remodeling and G inflammatory cells in bronchoalveolar lavage fluid (BALF) were measured. H Immunohistochemical analysis of aSMA, IL-11 and IL-11Ra. Scale bar: 50 µm. Black arrows show pulmonary arteries. I Co-immunofluorescence of aSMA/Tie2-GFP. Scale bar: 25 µm. White arrows indicates co-localizations. Data are presented as scatter dot blot with median and interquartile range values. P-values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Fig 4: Il11 expression in smooth muscle cells leads to lymphoid cell aggregates, villi distortion and ganglionic hyperplasia.(a) Peyer’s patch of tam-treated Il11SMC mice showed increased expression of IL11, CD45, LAMP2 and LGALS3 compared to veh-treated controls. (b) Cross- and longitudinal sections of the mucosa region of the colon in tam-treated Il11SMC mice have inflammatory cell infiltrates that extend from the submucosa to the mucosa region resulting in distortion of villi architecture. (c) Ganglionic hyperplasia and fibrosis in the myenteric plexus of tam-treated Il11SMC mice compared to vehicle controls. White arrowheads denote ganglionic cells. Black boxes were imaged at 400X magnification. All scale bars represent 200 µm.

Fig 5: IL-11 and IL-11Ra are localized and secreted by human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A Human lung tissue from control subjects, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF was immune-stained with IL-11, IL-11Ra and aSMA and with secondary fluorescence antibodies. Representative images are showed. White colour represents co-localization of both antibodies. Yellow arrows indicate endothelial cells. B HPAECs and C HPASMCs were isolated from pulmonary arteries of control subjects, IPF and PH associated to IPF patients and cultured until passage 1. Cell culture supernatants were collected to measure IL-11 by ELISA. Data are presented as scatter dot blot with median and interquartile range values of n = 6 patients in each group. P-values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison