Anti-inflammatory control of human skin keratinocytes by targeting nuclear transport checkpoint.

Background: In the two common inflammatory skin diseases, Atopic Dermatitis (AD) and Psoriasis (Ps), keratinocytes (KCs) respond to immune insults through activation of proinflammatory transcription factors (TFs) and their translocation to the cell's nucleus. Therein, the TFs induce expression of genes encoding mediators of skin inflammation. The Nuclear Transport Checkpoint Inhibitors (NTCIs) were developed to regulate nuclear translocation of activated TFs, the essential step of inflammatory response. This new class of cell-penetrating peptide therapeutics controls inflammation caused by allergic, autoimmune, metabolic, and microbial insults. In preclinical model of AD, the treatment with NTCI, cSN50.1 peptide, suppressed the expression of Thymic Stromal Lymphopoietin (TSLP), the key gene in the development of allergic inflammation, among the 15 genes silenced by the NTCI. Here, we report the mechanism of anti-inflammatory action of NTCI in human skin-derived KCs. Objectives: We aimed to determine whether the NTCI treatment can protect human KCs from harmful inflammatory insults. Methods: Human primary KCs were pretreated with NTCI and challenged with the mix of cytokines Tumour Necrosis Factor alpha (TNF-α) and Interleukin (IL)-17A, or with Phorbol 12-Myristate 13-Acetate (PMA), and analysed for nuclear content of TFs and the expression of genes encoding mediators of inflammation. Results: The nuclear import of TFs, Nuclear Factor ĸB (NF-ĸB) and Signal Transduction and Activator of Transcription 3 (STAT3), was inhibited in cells treated with NTCI. The expression of TSLP, along with genes encoding the core mediators of inflammation (TNF, IL1B, and IL6) was suppressed by NTCI. Noteworthy, NTCI silenced genes encoding Granulocyte-Macrophage Colony-Stimulating Factor (CSF2), and chemokine IL-8 (CXCL8), responsible for skin infiltration by the eosinophils and other myelomonocytic cells. Conclusion: The control of inflammatory response in human KCs by NTCI is attributed to the inhibition of nuclear import of proinflammatory TFs. The protection of human KCs by NTCI, adds new perspectives to the completed Phase two clinical trial of the NTCI (AMTX-100 CF) for AD (NCT04313400).

Cytoplasmic retention of the DNA/RNA-binding protein FUS ameliorates organ fibrosis in mice.

Uncontrolled accumulation of extracellular matrix leads to tissue fibrosis and loss of organ function. We previously demonstrated in vitro that the DNA/RNA-binding protein fused in sarcoma (FUS) promotes fibrotic responses by translocating to the nucleus, where it initiates collagen gene transcription. However, it is still not known whether FUS is profibrotic in vivo and whether preventing its nuclear translocation might inhibit development of fibrosis following injury. We now demonstrate that levels of nuclear FUS are significantly increased in mouse models of kidney and liver fibrosis. To evaluate the direct role of FUS nuclear translocation in fibrosis, we used mice that carry a mutation in the FUS nuclear localization sequence (FUSR521G) and the cell-penetrating peptide CP-FUS-NLS that we previously showed inhibits FUS nuclear translocation in vitro. We provide evidence that FUSR521G mice or CP-FUS-NLS-treated mice showed reduced nuclear FUS and fibrosis following injury. Finally, differential gene expression analysis and immunohistochemistry of tissues from individuals with focal segmental glomerulosclerosis or nonalcoholic steatohepatitis revealed significant upregulation of FUS and/or collagen genes and FUS protein nuclear localization in diseased organs. These results demonstrate that injury-induced nuclear translocation of FUS contributes to fibrosis and highlight CP-FUS-NLS as a promising therapeutic option for organ fibrosis.