p62/Sequestosome-1 (p62) is a multifunctional adaptor protein and is also a constant component of disease-associated protein aggregates, including Mallory–Denk bodies (MDBs), in steatohepatitis and hepatocellular carcinoma. We investigated the interaction of the two human p62 isoforms, p62-H1 (full-length isoform) and p62-H2 (partly devoid of PB1 domain), with keratins 8 and 18, the major components of MDBs. In human liver, p62-H2 is expressed two-fold higher compared to p62-H1 at the mRNA level and is present in slightly but not significantly higher concentrations at the protein level. Co-transfection studies in CHO-K1 cells, PLC/PRF/5 cells as well as p62− total-knockout and wild-type mouse fibroblasts revealed marked differences in the cytoplasmic distribution and aggregation behavior of the two p62 isoforms. Transfection-induced overexpression of p62-H2 generated large cytoplasmic aggregates in PLC/PRF/5 and CHO-K1 cells that mostly co-localized with transfected keratins resembling MDBs or (transfection without keratins) intracytoplasmic hyaline bodies. In fibroblasts, however, transfected p62-H2 was predominantly diffusely distributed in the cytoplasm. Aggregation of p62-H2 and p62ΔSH2 as well as the interaction with K8 (but not with K18) involves acquisition of cross-β-sheet conformation as revealed by staining with luminescent conjugated oligothiophenes. These results indicate the importance of considering p62 isoforms in protein aggregation disease.

Chronic intoxication of mice with the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) leads to morphological and metabolic changes closely resembling steatohepatitis, a severe form of metabolic liver disease in humans. Since human steatohepatitis (both the alcoholic and non-alcoholic type) is characterized by reduced expression of PPARα and disturbed lipid metabolism we investigated the role of this ligand-activated receptor in the development of DDC-induced liver injury. Acute DDC-intoxication was accompanied by early significant downregulation of Pparα mRNA expression along with PPARα-controlled stress-response and lipid metabolism genes that persisted in the chronic stage. Administration of the specific PPARα agonist fenofibrate together with DDC prevented the downregulation of PPARα-associated genes and also improved the stress response of Nrf2-dependent redox-regulating genes. Moreover, oxidative stress and inflammation were strongly reduced by DDC/fenofibrate co-treatment. In addition, fenofibrate prevented the disruption of hepatocyte intermediate filament cytoskeleton and the formation of Mallory-Denk bodies at late stages of DDC intoxication. Our findings show that, like in human steatohepatitis, PPARα is downregulated in the DDC model of steatohepatitis-like hepatocellular damage. Its downregulation and the pathomorphologic features of steatohepatitis are prevented by co-administration of fenofibrate.

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Trans-urocanic acid (UCA) is a natural photoreceptor present in the stratum corneum of the skin. Upon exposure to ultraviolet-radiation (UV-R), trans-UCA is isomerized to cis-UCA. Several studies indicate that cis-UCA induces local and systemic immune suppression via various underlying mechanisms. However, microbes are established all over the surface of the skin and the interplay between cis-UCA and the skin microbiome is not completely understood. In this study, we investigated the effects of cis-UCA on the skin microbiome and antimicrobial peptides (AMPs) expression using mouse models. We employed HPLC to determine quantitative isomerization of trans-UCA to cis-UCA by UV-R. We further made use of the model of contact allergy to assess the percentage of immune suppression by UV-A, UV-B, PUVA and cis-UCA to the contact allergen DNFB. Next, we treated mice with UV-A, UV-B, PUVA and cis-UCA and performed 16S rRNA gene sequencing for microbiome analysis and qPCR for AMPs gene expression. We noted that UV-B (p¼0.002) and PUVA (p¼0.023) significantly increased the formation of cis-UCA, whereas UV-A exposure alone showed no significant formation of cis-UCA in the skin. Utilizing the contact allergy model, we observed a dose-dependent increase in immune suppression (by up to 100%) against the contact allergen DNFB, when mice were pretreated with cis-UCA. Furthermore, application of cis-UCA on the skin altered the microbial landscape of the skin both at 8h and 24h, correlating with a change in expression of various AMPs. Collectively our results suggest that cis-UCA alters the skin microbial landscape and AMP expression. This imbalance in the skin microbial landscape and altered AMP expression may be crucial in immune suppression upon UV-R exposure mediated through cis-UCA.

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