Probes for INS

Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders.

Psoriasis is a chronic, inflammatory skin disease, frequently associated with dyslipidemia. Lipid disturbance in psoriasis affects both circulatory system and cutaneous tissue. Epidermal Langerhans cells (LCs) are tissue-resident DCs that maintain skin immune surveillance and mediate various cutaneous disorders, including psoriasis. However, the role of LCs in psoriasis development and their lipid metabolic alternation remains unclear. Here, we demonstrate that epidermal LCs of psoriasis patients enlarge with longer dendrites and possess elevated IL-23p19 mRNA and a higher level of neutral lipids when compared with normal LCs of healthy individuals. Accordantly, epidermal LCs from imiquimod-induced psoriasis-like dermatitis in mice display overmaturation, enhanced phagocytosis, and excessive secretion of IL-23. Remarkably, these altered immune properties in lesional LCs are tightly correlated with elevated neutral lipid levels. Moreover, the increased lipid content of psoriatic LCs might result from impaired autophagy of lipids. Bulk RNA-Seq analysis identifies dysregulated genes involved in lipid metabolism, autophagy, and immunofunctions in murine LCs. Overall, our data suggest that dysregulated lipid metabolism influences LC immunofunction, which contributes to the development of psoriasis, and therapeutic manipulation of this metabolic process might provide an effective measurement for psoriasis.

Maintaining tissue homeostasis depends on a balance of cell proliferation, differentiation and apoptosis. Within the epidermis the levels of the polyamines putrescine, spermidine and spermine are altered in many different skin conditions yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, AMD1, as a crucial regulator of keratinocyte differentiation. AMD1 protein is upregulated on differentiation and highly expressed in the suprabasal layers of the human epidermis. During keratinocyte differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown/inhibition of AMD1 results in reduced spermine levels and inhibition of keratinocyte differentiation. Supplementing AMD1-knockdown keratinocytes with exogenous spermidine/spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signalling proteins that drive keratinocyte differentiation including KLF4 and ZNF750. These findings demonstrate that human keratinocytes use controlled changes in polyamine levels to modulate gene expression to drive cellular behaviour changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or be used in the treatment of hyper-proliferative skin disorders.

Cognitive impairment caused by systemic chemotherapy is a critical question that perplexes the effective implementation of clinical treatment, but related molecular events are poorly understood. Herein, we show that bortezomib exposure leads to microglia activation and cognitive impairment, this occurs along with decreased nuclear translocation of TFEB (transcription factor EB), which is linked to macroautophagy/autophagy disorder, STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL23A (interleukin 23 subunit alpha) expression. Pharmacological enhancement of TFEB nuclear translocation by digoxin restores lysosomal function and reduces STAT3-dependent endothelial IL23A secretion. As a consequence, we found that brain endothelial-specific ablation of Il23a ameliorated both microglia activation and cognitive dysfunction. Thus, the endothelial TFEB-STAT3-IL23A axis in the brain represents a critical cellular event for initiating bortezomib-mediated aberrant microglial activation and synapse engulfment. Our results suggest the reversal of TFEB nuclear translocation may provide a novel therapeutic approach to prevent symptoms of cognitive dysfunction during clinical use of bortezomib.Abbreviations: AAV: adeno-associated virus; BBB: blood-brain barrier; BTZ: bortezomib; DG: digoxin; DGs: dentate gyrus; DLG4/PSD95: discs large MAGUK scaffold protein 4; HBMECs: human brain microvascular endothelial cells; HP: hippocampus; IL23A: interleukin 23 subunit alpha; MBVECs: mouse brain vascular endothelial cells; mPFC: medial prefrontal cortex; NORT: novel object recognition test; OLT: object location test; PLX5622: 6-fluoro-N-([5-fluoro-2-methoxypyridin-3-yl]methyl)-5-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3- yl)methyl; PPP3/calcineurin: protein phosphatase 3; SBEs: STAT3 binding elements; shRNA: small hairpin RNA; SLC17A7/VGLUT1: solute carrier family 17 member 7; SLC32A1/VGAT: solute carrier family 32 member 1; STAT3: signal transducer and activator of transcription 3, TFEB: transcription factor EB; Ub: ubiquitin.