Fig 1.
Effect of E. multilocularis infection on the expression of proteins involved in UPR and ER redox functions.
Western blotting and semi-quantitative analysis by densitometry (graphs of densitometry data are shown in S2 Fig) of protein/phospho-protein levels of A) GRP78, B) PERK, eIF2α, p-eIF2α and ATF4, C) ATF6, CHOP, and ERp72, D) IRE1α and p-IRE1α, E) CNX and CRT, and F) H6PD in mock-infected control mice (CTRL), E. multilocularis infected mice (AE), infected mice treated with ABZ (AE-ABZ) or uninfected mice treated with ABZ (ABZ). One representative blot (of two) containing samples from three different mice is shown. Lamin B1 served as loading control. G) Bands corresponding to the respective protein/phospho-protein were analyzed by densitometry (animals per group n = 6). Numbers represent the expression of protein/phospho-protein levels normalized to those of the control (CTRL) group (mean ± SD). Significantly decreased protein/phospho-protein levels are highlighted in red and increased levels in blue. Symbols indicate significant differences (p≤0.05) between groups: *, compared to CTRL; §, compared to ABZ; #, compared to AE-ABZ. No outliers were detected/excluded. Non-parametric, Kruskal-Wallis test followed by Dunn’s Multiple Comparison post-test.
Fig 2.
Albendazole decreases proinflammatory cytokine levels in the liver that were elevated by E. multilocularis infection.
Cytokine levels of IL1β, IL6, TNFα, and IFNγ in liver tissue samples of mock-infected control mice (CTRL), E. multilocularis infected mice (AE), infected mice treated with ABZ (AE-ABZ) or uninfected mice treated with ABZ (ABZ) (animals per group n = 6). No outliers were detected/excluded. Non-parametric, Kruskal-Wallis test followed by Dunn’s Multiple Comparison post-test. *P≤0.05; **p≤0.01; ***p≤0.001.
Fig 3.
Effect of E. multilocularis infection and αPD-L1 treatment on the expression of proteins involved in UPR.
Western blotting and semi-quantitative analysis by densitometry of protein levels of ATF4, CHOP, and IRE1α in mock-infected control mice (CTRL), E. multilocularis infected mice (AE), infected mice treated with αPD-L1 (AE-αPD-L1) or uninfected mice treated with αPD-L1 (αPD-L1) (animals per group n = 6). One representative blot (of two) containing samples from three different mice is shown. Lamin B1 served as loading control (animals per group n = 6). Densitometry results represent data from the two blots on samples from six mice (mean ± SD), normalized to Lamin B1 control and with CTRL set as 1. No outliers were detected/excluded. Non-parametric, Kruskal-Wallis test followed by Dunn’s Multiple Comparison post-test. *P≤0.05; **p≤0.01; ***p≤0.001.
Fig 4.
Schematic overview of ERS signaling pathways under basal and E. multilocularis infection stressed conditions.
The ER chaperone GRP78 binds to unfolded luminal proteins and dissociates from the three major ERS sensors: A) ATF6, B) IRE1α and C) PERK. A) Loss of GRP78 binding leads to the translocation of ATF6 to the Golgi apparatus, where it is cleaved by proteases. The cleaved form of ATF6 translocates into the nucleus to act as a transcription factor for ER chaperons (e.g. ERp72) and ERS related genes. B) ERS promotes IRE1α dimerization and autophosphorylation, which activates the endoribonuclease activity resulting in the splicing and thereby activation of XBP1. XBP1-s promotes the expression of ERAD related genes and chaperones (e.g. GRP78). C) Activation of PERK is initiated by dimerization and self-phosphorylation. Activated PERK phosphorylates eIF2α, leading to eIF2α-mediated inhibition of global protein translation in order to decrease the luminal protein load. Besides, phosphorylated eIF2α increases the transcription of ATF4, which in turn upregulates expression of genes related to cell homeostasis restoration. If prolonged ERS occurs and pro-adaptive UPR fails, ATF4 induces genes (including CHOP) leading to apoptosis. During ERS, increased levels of miR-1839-5p potentially control the expression of the IRE1α gene, which contains a predicted target site in its 3’-UTR for this miR, thereby affecting the cellular ERS response. By suppressing the propagation of E. multilocularis infection, through yet poorly defined molecular mechanisms, ABZ and αPD-L1 treatment decrease inflammation and ERS.