Differential regulation of 5-lipoxygenase and leukotriene-C4-synthase expression by IFNγ, IL-4 and IL-13 in human monocytes and macrophages from patients with atopic dermatitis

Abstract Introduction Skin lesions in atopic dermatitis (AD) are characterized by elevated levels of both Th1 and Th2 cytokines, along with increased concentrations of inflammatory mediators such as cysteinyl leukotrienes (CysLTs). The enzymes 5-lipoxygenase (5-LO/ALOX5) and leukotriene-C4-synthase (LTC4S), which are essential in CysLT biosynthesis, are highly expressed by macrophages. We aimed to investigate the expression of 5-LO/ALOX5 and LTC4S mRNA in lesional AD skin at single cell level. Furthermore, we analyzed the regulatory effects of the Th1 cytokine interferon gamma (IFNγ) and the Th2 cytokines IL-4, IL-13 on 5-LO/ALOX5 and LTC4S expression in monocytes and macrophages derived from AD patients and healthy volunteers. Methods Single-cell RNA sequencing (scRNA-seq) data from lesional AD skin biopsies, as reported in a previously published study, were re-analyzed to evaluate 5-LO/ALOX5 and LTC4S mRNA expression. PBMCs from AD patients, healthy volunteers and anonymous donors were used to isolate monocytes. Macrophages were generated in the presence of GM-CSF or M-CSF for 10 days. Cells were stimulated with IFNγ, IL-4 or IL-13. 5-LO/ALOX5 and LTC4S mRNA expressions were quantified by q-PCR. Intracellular 5-LO/ALOX5 expression was assessed by immunocytochemistry. Results Re-analysis of scRNA-seq data revealed high levels of 5-LO/ALOX5 and LTC4S transcripts in monocytes and macrophages. In-vitro, IFNγ induced 5-LO/ALOX5 mRNA expression in blood derived monocytes and macrophages from AD patients, and protein levels in both monocytes and macrophages from anonymous donors, whereas IL-4 and IL-13 suppressed its expression. Vice versa LTC4S mRNA expression was downregulated by IFNγ but upregulated by IL-13. Higher baseline mRNA expressions of 5-LO/ALOX5 and LTC4S were observed in blood derived monocytes from AD patients compared to cells from healthy volunteers . Conclusion We demonstrate that IFNγ enhances the 5-LO/ALOX5-catalyzed stage of CysLT synthesis, whereas IL-13 promotes the LTC4S dependent pathway in human monocytes or macrophages. These findings suggest a potential role for these cells in driving CysLT production in acute and chronic AD lesions and may give rise for future therapeutic interventions targeting this pathway. Graphical abstract Single-cell RNA sequencing (scRNA-seq) data from lesional atopic dermatitis (AD) skin biopsies, as reported in a previously published study, were re-analyzed to assess the expression of 5-lipoxygenase (5-LO/ALOX5) and leukotriene-C4-synthase (LTC4S). In parallel, peripheral blood mononuclear cell (PBMC) derived monocytes from AD patients, healthy volunteers (HV), and anonymous donors were differentiated into macrophages in the presence of either M-CSF or GM-CSF, and the expression of 5-LO/ALOX5 and LTC4S was evaluated. The major enzymes involved in cysteinyl leukotriene (CysLT) generation are 5-LO/ALOX5 (activated by the 5-LO/ALOX5-activating protein FLAP), which converts arachidonic acid to leukotriene A4 (LTA4) and LTC4S, which catalyzes the conjugation of LTA4 with glutathione to form leukotriene C4 (LTC4). LTC4 is subsequently metabolized extracellularly to LTD4 and LTE4. ScRNA-seq analysis revealed that both 5-LO/ALOX5 and LTC4S were highly expressed in monocyte/macrophage clusters. Specifically, 5-LO/ALOX5 was strongly expressed in GM-CSF–differentiated (M1-like) macrophages, while LTC4S expression was upregulated in M-CSF–differentiated (M2-like) macrophages. These patterns were confirmed in blood-derived macrophages from anonymous donors. Interferon gamma (IFNγ) induced 5-LO/ALOX5 mRNA expression in blood-derived monocytes and macrophages from AD patients, as well as 5-LO/ALOX5 protein in cells from anonymous blood donors. LTC4S mRNA expression was upregulated by interleukin 13 (IL-13) in cells from HVs. Monocytes from AD patients displayed higher baseline mRNA levels of 5-LO/ALOX5 and, more strikingly, of LTC4S compared with cells from HVs. NS = non-stimulated.