Figure 7: Anakinra promotes the autophagy and proteasomal degradation pathway. | Nature Communications

Figure 7: Anakinra promotes the autophagy and proteasomal degradation pathway.

From: IL-1 receptor antagonist ameliorates inflammasome-dependent inflammation in murine and human cystic fibrosis

Figure 7

(a) Autophagy on purified lung macrophages from C57BL/6 or Cftr−/−mice stimulated with Aspergillus conidia or P. aeruginosa in the presence of 10 μg ml−1 anakinra and incubated with anti-LC3 antibody followed by PE secondary antibody. Representative images (original magnification, × 40) are shown. DAPI was used to detect nuclei. Numbers refer to % positive cells. Scale bar, 12.5 μm. (b) Density of bands from western blots of LC3b-I and II in homogenates of lungs of naive, Aspergillus- or Pseudomonas-infected untreated (none), or infected and anakinra-treated mice at 7 dpi. (c) Conidiocidal activity of RAW264.7 cells pretreated with DPI, 3-MA, lactacystin or chloroquine before the exposure to live A. fumigatus resting conidia for 2 h in the presence of anakinra. Conidiocidal activity refers to inhibition of CFUs (expressed as mean percentage±s.d. for all bar graphs, data pooled from three experiments). Each treatment had no effects on fungal growth in the absence of effector cells. *P<0.05, ****P<0.0001, pretreated versus anakinra 10 μg ml−1 only, one-way ANOVA, Bonferroni post hoc test. (d) Immunofluorescence imaging of purified alveolar macrophages from lungs of C57BL/6 or Cftr−/−mice after in vitro exposure to GFP-conidia at 37 °C for 2 h and chasing for 15 and 45 min. Formaldehyde-fixed cells were incubated with primary antibodies against Lamp1 or 20S followed by secondary anti-rabbit IgG–TRITC antibody. Nuclei were counterstained with DAPI. Images were acquired using EVOS FL Color Imaging System with × 40 objective. Shown are merged images of cells (a single cell is magnified in the inset) pulsed with GFP-conidia and red-stained for each compartment. Shown are representative data from three independent experiments.(e,f) RAW 247.3 (e) and HBE (f) cells from control or CF patients were stimulated with Aspergillus conidia in the presence of anakinra for 4 h and the amount of pmols of proteolytic activity was analysed with the proteasome activity assay kit (Abcam) Shown in the insets the relative slope with error bars representing the mean±s.d. *P<0.05, ***P<0.001, None versus anakinra, Student’s t-test.

Back to article page