Dysregulated signal transducer and activator of transcription 3 drives intestinal permeability and may contribute to acute-on-chronic liver failure

Figure 1 Intestinal tissue of mouse models shows increased signal transducer and activator of transcription 3 expression and altered epithelial integrity during progression from liver steatosis to acute-on-chronic liver failure. A-C: Paraffin embedded intestinal tissue of mouse models generating steatosis, cirrhosis, and acute-on-chronic liver failure (ACLF) was used for: Hematoxylin and eosin (HE) staining (A); Immunohistochemical analysis of signal transducer and activator of transcription 3 (STAT3) and phospho-STAT3 (pSTAT3) and compared to tissue of healthy controls, respectively (B and C); D and E: STAT3 and pSTAT3 protein levels of immunohistochemical staining were quantified using Fiji (ImageJ) by color deconvolution, followed by signal quantification and graphical representation in GraphPad Prism; F: Disease stage-dependent changes of STAT3 messenger RNA levels from cirrhosis to ACLF were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and compared. HE staining demonstrated a progressive disruption of intestinal epithelial architecture during disease progression. Immunohistochemistry revealed a significant upregulation of STAT3 expression in intestinal tissue from ACLF mice compared with healthy controls, which was confirmed by RT-qPCR. Statistical analysis of immunohistochemical staining was performed using one-way analysis of variance followed by Tukey’s multiple comparisons test. Differences in STAT3 messenger RNA expression were assessed by unpaired Student’s t-test. P values are two-sided (n = 3-6). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ACLF: Acute-on-chronic liver failure; STAT3: Signal transducer and activator of transcription 3; pSTAT3: Phospho-signal transducer and activator of transcription 3; mRNA: Messenger RNA; NS: Not significant.

Figure 2 Successful generation of differential signal transducer and activator of transcription 3 expressing cell lines. Signal transducer and activator of transcription 3 (STAT3) expression was modified by generation of differential, stable, lentiviral transduced Caco-2 or T84 cell lines. Reduction of STAT3 (STAT3 -) was produced via short hairpin RNA (shRNA) targeting STAT3. Enhanced STAT3 expression (STAT3 +) was induced by transduction of STAT3 expression plasmids. A non-mammalian shRNA expressing pLKO.1 vector was used as a control. Differential STAT3 expression was validated frequently from the start to the end of the experiments. Total protein was extracted, and proteins of interest were analyzed. A: Western blot; B: Quantified using Fiji (ImageJ distribution; version 2.14.0/1.54f). Values were normalized to Caco-2 or T84 control cell lines. Bars indicate mean ± SD. P values were calculated using one-way analysis of variance and post hoc Tukey analysis (n = 4). ^bP < 0.01. ^dP < 0.0001. STAT3: Signal transducer and activator of transcription 3.

Figure 3 Signal transducer and activator of transcription 3 reduction as well as enhanced signal transducer and activator of transcription 3 expression impairs permeability of epithelial cell layers. To determine the influence of signal transducer and activator of transcription 3 (STAT3) on epithelial permeability, monolayers consisting of Caco-2 or T84 cells with different levels of STAT3 expression were generated and epithelial permeability was analyzed by transepithelial electrical resistance (TEER) measurements over 28 days and compared to controls. A and B: Graphs show the differences of TEER values after 4 days, 7 days, 11 days, 14 days, 18 days, 21 days, 25 days and 28 days compared with endogenous STAT3 expressing (pLKO.1 transduced) controls; C and D: TEER values at day 28 were calculated in relation to controls in percent and shown as bar diagrams. Reduced (STAT3 -) as well as enhanced STAT3 (STAT3 +) expression resulted in a significant reduction in monolayer resistance values of Caco-2 and T84 monolayers. Data are expressed as mean ± SEM as appropriate. Differences between mean resistance values were assessed for statistical significance using one-way analysis of variance followed by Dunnett multiple comparisons test. P values are two-sided (n = 3-5). ^dP < 0.0001. STAT3: Signal transducer and activator of transcription 3; TEER: Transepithelial electrical resistance.

Figure 4 Expression of relevant tight junction proteins is significantly modified by differential signal transducer and activator of transcription 3 expression in Caco-2 and T84 monolayers. Monolayers consisting of Caco-2 or T84 cells with different levels of signal transducer and activator of transcription 3 (STAT3) expression were generated and epithelial permeability was determined by transepithelial electrical resistance measurements over 28 days. A and B: Whole protein extracts were analyzed via western blot (an exemplary blot is shown on which the protein extracts from three independently performed test series were applied next to each other; C and D: Quantified using Fiji (ImageJ distribution; version 2.14.0/1.54f). Values were normalized to Caco-2 or T84 control cell lines. Bars indicate mean ± SD. Western blots were performed with specific primary antibodies against STAT3, phospho-STAT3, STAT1, phospho-STAT1, Ocln, Cldn1, Cldn2, Cldn3 and zonulin 1, while β-actin served as a housekeeping protein. As shown, reduced (STAT3 -) as well as enhanced STAT3 (STAT3 +) expression had an important impact on the activation of STAT1 as well as the expression of tight junction proteins. P values were calculated using two-way analysis of variance followed by Dunnett’s multiple comparisons test. The data shown are mean ± SD, and the following P values were considered statistically significant (n ≥ 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. STAT3: Signal transducer and activator of transcription 3; pSTAT3: Phospho-signal transducer and activator of transcription 3; STAT1: Signal transducer and activator of transcription 1; pSTAT1: Phospho-signal transducer and activator of transcription 1; ZO-1: Zonula occludens-1.

Figure 5 Immunofluorescence staining and transmission electron microscopy verify deconstructed monolayer architecture in enhanced as well as reduced signal transducer and activator of transcription 3 expressing Caco-2 and T84 monolayers. Differential signal transducer and activator of transcription 3 (STAT3) expressing Caco-2 and T84 cell lines were grown over a period of 28 days and the formation of Caco-2 and T84 monolayers was verified during this period. A and B: Caco-2 (A) T84 (B) monolayers were fixed, permeabilized, incubated with anti zonula occludens-1, and subsequently stained with a fluorescent-labeled antibody. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole. Imaging was performed using a Keyence BZ X810 fluorescence microscope; C and D: In parallel, Caco-2 (C) and T84 (D) monolayers were prepared for transmission electron microscopy (TEM). Immunofluorescence as well as TEM showed the deconstructed architecture in enhanced STAT3 (STAT3 +) as well as reduced STAT3 (STAT3 -) expressing monolayers. Orange arrows = gaps at cell boundaries, black arrows = location of tight junctions. STAT3: Signal transducer and activator of transcription 3; DAPI: 4’,6-diamidino-2-phenylindole; ZO-1: Zonula occludens-1.

Figure 6 Identification of signal transducer and activator of transcription 3-dependent differences of lipid species by liquid chromatography high-resolution mass spectrometry analysis. Caco-2 and T84 cell monolayers were generated over a period of 28 days. Thereafter, cells were harvested and lipidomic analysis was performed. A and B: Venn diagrams illustrate the number of significantly altered lipid species in decreased signal transducer and activator of transcription 3 (STAT3) (STAT3 -) and enhanced STAT3 (STAT3 +) expressing Caco-2 and T84 cell monolayers compared to the controls. The overlapping area represents lipids significantly regulated in both epithelial models, indicating STAT3-dependent lipid remodeling; C and D: Bars show all lipid species that were significantly changed in Caco-2 as well as T84 monolayers (see also A and B), respectively. Bars represent mean log₂ fold change ± SEM. Cross-validation across two independent intestinal epithelial models ensures robustness and minimizes false-positive discovery. P < 0.05, two-sided Welch’s t-test (n = 5). STAT3: Signal transducer and activator of transcription 3; PG: Phosphatidylglycerol; LPC: Lysophosphatidylcholines; PC: Phosphatidylcholine; TG: Triacylglycerol; SM: Sphingomyelin; PC O: Ether-linked phosphatidylcholine; TG O: Ether-linked triacylglycerol.