CysLTs are potent proinflammatory mediators that play a crucial role in the pathogenesis of various inflammatory diseases, including asthma [23] and AD [7, 8]. Recent studies have shown that elevated levels of CysLTs are present in the skin of AD patients [8]. Moreover, urinary LTE4 levels are significantly increased in moderate and severe AD compared to mild AD and a positive correlation was observed between urinary LTE4 and disease severity scores, absolute eosinophilic counts, and serum total IgE [24, 25].

Functional relevance for the role of CysLTs in the pathogenesis of AD was further supported by studies of murine models of AD, which identified CysLTs as mediators of skin fibrosis and itch [9, 10].

Kiekens et al. described increased numbers of macrophages in lesional skin compared to non lesional skin in AD patients or to healthy skin. Accumulation of macrophages in both acutely and chronically inflamed AD skin highlights their prominent role in the disease pathogenesis [12].

Analysis of single-cell RNA sequencing results in a data set obtained from lesional skin of AD patients of our previous publication [4] revealed higher expression of 5-LO/ALOX5 and LTC4S transcripts in monocytes and macrophages compared to other skin cells, with M1-like macrophages showing elevated 5-LO/ALOX5 transcript levels compared to M2-like macrophages. This observation was further corroborated by our in vitro experiments showing that GM-CSF-derived M1-like macrophages exhibited higher baseline 5-LO/ALOX5 mRNA expression than M-CSF-derived M2-like macrophages and monocytes. These results demonstrate a remarkable concordance between in vivo data from affected patients and in vitro generated data. 5-LO/ALOX5 mRNA expression and the presence of 5-LO/ALOX5 protein in murine bone-marrow-derived macrophages was observed by Sorgi et al. [26]. However, despite its expression the enzyme is mostly inactive showing little to no leukotriene expression. Addition of exogenous AA can activate 5-LO/ALOX5 and induce leukotriene expression [26].

To mimic the inflammatory milieu present in AD skin we examined the expression profiles of 5-LO/ALOX5 and LTC4S in monocytes and monocyte-derived M-CSF macrophages from AD patients and HVs, following stimulation with the AD-associated cytokines IFNγ, IL-4 and IL-13 [1].

We found that 5-LO/ALOX5 and LTC4S mRNA expressions were differentially regulated by the Th1 cytokine IFNγ being overexpressed in chronically inflamed skin in AD and by Th2 cytokines IL-4 and IL-13. 5-LO/ALOX5 mRNA expression was upregulated by IFNγ in monocytes and macrophages from AD patients.

We propose that epigenetic priming may contribute to the upregulation of 5-LO/ALOX5 mRNA expression observed exclusively in cells derived from AD patients. Cells from AD patients often show altered chromatin accessibility and DNA methylation patterns, making certain genes more responsive to pro-inflammatory signals. IFNγ may more effectively induce 5-LO/ALOX5 in AD patient cells because these cells have been primed by chronic inflammation, increasing promoter or enhancer accessibility for IFNγ-driven transcription factors. Also, monocytes and macrophages from AD patients are likely due to systemic inflammatory mediators or repeated exposure to skin-derived signals in an enhanced basal activation state which may sensitize the cells to cytokines like IFNγ [27].

The study from Li et al. [28] provides strong support for differential expression of 5-LO/ALOX5 pathway genes in disease states compared to controls [28]. They observed that elevated 5-LO/ALOX5 expression is linked to oxidative stress and inflammation and can be confirmed in macrophage populations under pathological conditions [28].

IL-4 and IL-13 downregulated 5-LO/ALOX5 mRNA expression in monocytes and macrophages from HVs and AD patients.

The stimulating effect of IFNγ on 5-LO/ALOX5 mRNA expression is in accordance with previous results from Ebert et al. [29] showing an upregulation of 5-LO/ALOX5 mRNA expression in M1 macrophages after a combined stimulation with GM-CSF for 7 d and IFNγ for 48 h, while differentiation of M2 macrophages by M-CSF for 7 d receiving IL-4 in the last 48 h resulted in lower 5-LO/ALOX5 mRNA expression compared to the medium control [29].

We observed that IFNγ upregulated 5-LO/ALOX5 protein both in monocytes and in macrophages from healthy anonymous donors, but no significant increase of mRNA levels in response to IFNγ. This discrepancy may result from translational repression in macrophages under normal/healthy conditions, where mRNA is present, but protein synthesis is blocked until stimulated. This aligns with Schott et al., [30] who showed that proinflammatory mRNAs in macrophages are constitutively expressed but their protein translation is repressed until activation [30]. Thus, IFNγ likely lifts this repression, causing stronger protein upregulation than mRNA increase in macrophages.

The downregulation of 5-LO/ALOX5 mRNA expression in response to IL-4 and IL-13 stimulation in monocyte-derived cells was previously documented by Spanbroek et al. [31]. Their results demonstrated a significant finding: During the process of monocytes-to-DCs transdifferentiation induced by these Th2 cytokines, there was a concurrent downregulation of 5-LO/ALOX5 mRNA expression detectable in developing DCs [31]. However, it is surprising that IL-4 and IL-13, while being key drivers of atopic dermatitis pathology, suppress 5-LO/ALOX5 expression. This may reflect their complex, context-dependent roles on distinct steps of inflammatory pathways. Depending on cell type and environment, IL-4 and IL-13 can either induce or suppress leukotriene-synthesizing enzymes. In macrophages, their suppression of 5-LO/ALOX5 likely highlights their nuanced function in shaping the overall inflammatory milieu.

In human mast cells from AD patients, significantly higher levels of 5-LO/ALOX5 were detected compared to cells from HVs. However, unlike our findings in human monocytes and macrophages, no regulation of mRNA expression for 5-LO/ALOX5 by Th2 cytokines was observed in mast cells of both groups [32]. In contrast to the regulation of 5-LO/ALOX5 expression LTC4S mRNA expression was downregulated by IFNγ in monocytes and macrophages from AD patients and HVs.

Remarkably, we detected a significant upregulation of LTC4S mRNA by IL-13 in monocytes from HVs and by trend only in monocytes from AD patients.

This observation may be explained by the elevated baseline expression of this enzyme in monocytes from patients with AD compared to the baseline expression in cells from HVs. The increased baseline expression of LTC4S in monocytes from AD patients could be driven through endogenous increased exposure of the cells to Th2 cytokines.

The differential regulation of LTC4S, upregulation by IL-13 and no significant regulation by IL-4, although both cytokines signal through the type II IL-4 receptor (IL-4Rα with IL-13Rα1) may be explained that only IL-4 can also use the type I receptor (IL-4Rα with the γc chain). Macrophages express these receptor subunits at varying levels which influences their responsiveness [33]. It is possible that IL-4 elicits a different effect when signaling through the type I receptor compared to the type II receptor, resulting in an overall reduction in LTC4S production.

Furthermore, IL-4 and IL-13 activate distinct patterns of intracellular signaling, and these differences can lead to divergent gene regulation, potentially supporting or opposing LTC4S expression [34].

The differential regulation of the CysLT pathway by Th1 and Th2 cytokines in monocytes and macrophages from AD patients compared to HVs could be attributed to several factors: In acute and chronic phases of AD specific sections of the CysLT pathway in monocytes and macrophages may be selectively activated. Our findings suggest that IFNγ specifically activates the upstream section catalyzed by 5-LO/ALOX5 by upregulating 5-LO/ALOX5 mRNA and protein expression. An example of an upstream product is 5-hydroxyeicosatetraenoic acid (5-HETE), which, after conversion to 5-oxo-ETE [35], attracts eosinophils. This process could potentially explain the increased eosinophil infiltration observed in chronic AD lesions which are associated with higher IFNγ expression in the skin compared to acute lesions, as reported by Kiehl et al. [36].

Our findings indicate that IL-13 primarily enhances the downstream production of CysLTs by upregulating LTC4S mRNA expression. This is of special interest because Hsieh et al. [37] demonstrated that upregulation of LTC4S alone, without concurrent increase in 5-LO/ALOX5 protein levels, is sufficient to boost CysLT production in human mast cells [37].

The elevated levels of LTC4S in monocytes from AD skin can also convert LTA4 derived from human eosinophils or neutrophils during inflammation into CysLTs in a process known as transcellular synthesis [38]. LTC4 has been identified as an itch mediator signaling through the CysLT2 receptor expressed on a subset of sensory neurons in dorsal root ganglia in mouse and humans [10, 11]. The elevated baseline expression in monocytes from AD patients and the upregulation of LTC4S mRNA expression by trend in response to IL-13 suggest that the CysLTs synthesized as consequence of this effect could potentially play a role in the severe pruritus associated with IL-13-driven acute AD lesions [39]. A crucial role for LTC4S has also been proposed in skin fibrosis and inflammation. LTC4 stimulates skin fibroblasts to secrete factors that promote keratinocyte proliferation, as well as collagen deposition leading to epidermal thickening [9].

Our study provides a comprehensive view of the regulation of the cysteinyl leukotriene pathway, covering both key enzymes in monocytes and macrophages, and comparing cells from AD patients with those from HVs for the first time. We demonstrated differential regulation of 5-LO/ALOX5 and LTC4S by Th1 and Th2 cytokines and observed notable overexpression in cells from AD patients.

Although our data suggest that modulation of the CysLT pathway might be therapeutically beneficial, especially given the IFNγ–driven upregulation of 5-LO/ALOX5 in macrophages, the use of leukotriene antagonists is currently inconsistent. Existing studies report variable and often modest improvements with leukotriene antagonists [40, 41]. The translation of our findings into effective therapies in clinical applications could be a task for future research.