Cytokine RNA In Situ Hybridization Permits Individualized Molecular Phenotyping in Biopsies of Psoriasis and Atopic Dermatitis

Abstract

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 ¼ 20) and atopic dermatitis (n ¼ 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-a). 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.

Inhibition of type 1 immunity with tofacitinib is associated with marked improvement in longstanding sarcoidosis

“…As BAL might not fully reflect granulomas in the lung parenchyma, we also studied immunologic patterns in tissue containing parenchymal pulmonary granulomas from sarcoidosis patients ( n = 10) and compared them to cutaneous sarcoidosis ( n = 10) and controls ( Supplementary Table 6 ) . We utilized RNA in situ hybridization to stain for key cytokine markers of immune polarization including Th1 ( IFNG ), Th2 ( IL13 ), and Th17 ( IL17A ) 35 . We also included cutaneous inflammatory disorders with canonical Th2 (atopic dermatitis) and Th17 (psoriasis) polarization as positive controls to contextualize patterns seen in sarcoidosis 35 .…”

“…We utilized RNA in situ hybridization to stain for key cytokine markers of immune polarization including Th1 ( IFNG ), Th2 ( IL13 ), and Th17 ( IL17A ) 35 . We also included cutaneous inflammatory disorders with canonical Th2 (atopic dermatitis) and Th17 (psoriasis) polarization as positive controls to contextualize patterns seen in sarcoidosis 35 . We found significant expression of IFNG in lymphocytes in the sarcoidosis cases, but not normal lung or skin (Fig.…”

“…RNA in situ hybridization was performed using the RNAscope platform (Bio-techne) as previously described in detail 35 . Probes for IFNG (Cat #310501), IL12B (Cat #402071), IL13 (Cat #586241), and IL17A (Cat # 310931) are pre-diluted, were purchased directly from the manufacturer (Bio-techne), and used according to the manufacturer’s specifications.…”

“…Positive cells having scores of 2, 3, or 4 and were included in the analysis. Cells with scores of 0–1 were not quantitated and in our experience represent background staining 35 . The total number of positive cells per mm 2 of tissue was calculated for each case.…”

Comment on: Development of psoriasis during treatment with dupilumab: A systematic review

“…Specifically, by measuring the expression of interleukin 4, interleukin 13 versus interleukin 23, and interleukin 17A and F transcripts, we have found that we can distinguish AD from psoriasis. 2 We have also recently used this approach to evaluate a patient who was treated with dupilumab for moderate-to-severe AD and who had an excellent response to but in whom psoriasiform dermatitis subsequently developed 8 months after dupilumab initiation. Cytokine RISH was performed on a biopsy specimen taken before the patient started dupilumab as well as on a specimen of the subsequent psoriasiform dermatitis.…”