Artemisia species have been utilized for centuries as Traditional Chinese Medicine and have recently emerged as topical skin care actives in cosmetics. There are more than 300 Artemisia species and thus far Artemisia annua, Artemisia abrotanum, Artemisia absinthium, Artemisia capillaries, Artemisia dracunculus, and Artemisia vulgaris appear to be the most used as raw materials (26). Interest in Artemisia peaked with a Nobel Prize discovery that several species, such as A. annua, abrotanum, and A. vulgaris, contained artemisinin, a sesquiterpenoid lactone proven to be effective for the treatment of malaria (27). This led to phytochemical and biological activity characterization of many of these species and results showed that while there are some shared classes of compounds, the artemisia species slightly differ from each other in their chemical composition (26). Our initial in vitro biological characterization of AN oil extract (from A. annua, artemisinin-free) yielded intriguing results for its potential use to treat inflammatory skin disorders. Specifically, for AD, AN oil extract previously demonstrated the ability to inhibit S. aureus growth as well as S. aureus-induced TSLP, which is linked to AD skin pruritus (10). Moreover, AN oil extract was shown to increase filaggrin, an essential gene in the formation of the epidermal barrier, and reduce Th2 cytokine-induced IL-8 production in normal human epidermal keratinocytes. All the above have been shown to be key contributors to the pathogenesis of AD, providing the rationale to continue investigation into AN oil’s therapeutic potential for AD.
For chronic inflammatory skin disorders like AD and psoriasis, innate and adaptive immunity both play a crucial role. Interestingly, previously published results studying the anti-inflammatory activities of Artemisia predominantly focus on reducing the innate immune response in keratinocytes (28, 29), but not the adaptive response. Having previously studied the activity of AN oil extract in keratinocytes and demonstrating its activity in reducing the innate immune response, we next utilized an hPBMC cell-based assay to confirm for the first time that AN oil extract also acts on lymphocytes, the key mediators of the adaptive immune response. Utilizing T-cell receptor inducer anti-CD3/CD28, AN oil extract not only inhibited IL-4 (a key AD cytokine) and IL-17A (an important psoriasis cytokine) release from hPBMCs, but did so at concentrations lower than the potent topical glucocorticoid clobetasol (Fig. 1A + B). Furthermore, given we utilized primary hPBMCs to better mimic what happens in human skin instead of human leukemia monocytic cell line (THP-1) cells, we observed expected donor to donor variability (30), but nevertheless, AN oil extract effectively reduced cytokine production for all donors.
Having now demonstrated strong cell-based anti-inflammatory activity for cytokines linked to AD and psoriasis, as well as previously showing clinical improvement reducing redness, swelling and itch when applying AN oil extract topically on human subjects with sensitive and acne prone skin (10), we sought to determine AN oil extract’s efficacy in in vivo topical models for skin disease. Previously, Artemisia species have been shown to be effective in several different mouse models including the 2, 4-Dinitrocholrlbenzene (DNCB)-induced AD model, which is the most commonly utilized model (31–34), NC/Nga mice (35, 36), and the oxazolone-induced AD model (37). We instead selected the CPT-induced AD mouse model for four reasons: 1) This model mimics most of the clinical, skin barrier-related, histological, and immunological characteristics observed in AD patients (38); 2) It does not involve hazardous agents like oxazolone or DNCB; 3) It does not affect systemic calcium metabolism in mice, allowing for the CPT to be applied to the ear and dorsal skin at higher concentrations and for a longer time to induce AD inflammatory phenotypes (24); and 4) This would be the first time an Artemisia species would be tested in this specific AD model.
As shown in the results, CPT increased ear thickness (via micrometer measurements and H&E staining), which is a marker for skin inflammation (39), and AN oil extract dose-dependently reduced ear thickness. This reduction in inflammation was also observed visually as mouse ear erythema was greatly reduced as AN oil extract concentration reached 5% (Fig. 2). Moreover, a key protagonist for CPT-induced AD-like skin inflammation is the increase of key pro-inflammatory cytokines, and we demonstrated that AN oil extract significantly inhibited serum levels of IL-1β, IgE, and IL-6 (Table 2), all which have been shown to play a potential role in AD pathogenesis. For example, patients with AD often display elevated levels of total serum IgE, and autoreactive IgE antibodies may elicit an allergic-autoimmune process and contribute to perpetuation of inflammation (40). Also, peripheral blood T cells derived from patients with AD spontaneously produce increased amounts of IL-6 compared to T cells from normal subjects, which reflects the increased activation state of T cells in atopic dermatitis (41). Lastly, IL-1β has been shown to be an early key mediator for the acquisition of an AD phenotype through induction of TSLP and alteration of epidermal homeostasis (42). Altogether, this clearly demonstrates that AN oil extract is efficacious when applied topically to reduce the inflammatory pathways and biomarkers associated with AD in this preclinical model.
Given that AN oil extract inhibited Th-17A response in vitro, which plays an important role in psoriasis (Fig. 1), and previously demonstrating downregulation of TSLP in macrophages (10), also linked to psoriasis and AD, we sought to determine if AN oil extract would be effective when applied topically in vivo. Several animal models mimicking human psoriasis have been developed and utilized. We selected the IMQ-induced psoriasis preclinical model because it allows for the elucidation of underlying mechanisms and the evaluation of new therapies against psoriasis in a rapid and convenient manner (43). Moreover, unlike AD where several different Artemisia species were tested in different models, as of this report only one species, A. capillaris formulated in a topical cream, has been tested for anti-psoriatic activity and it was performed in the IMQ-induced psoriasis model (44). Thus, despite A. capillaris possessing a very different phytochemical profile compared to AN oil extract, we selected the in vivo IMQ model to assess its efficacy. As expected, and previously shown utilizing this model, application of IMQ to the dorsal skin of mice led to a visual increase in psoriatic scaling (Fig. 3), thickening of the epidermis, and infiltration of inflammatory cells (Fig. 4) which were all decreased when AN oil extract was co-applied. To verify these observations quantitatively, we measured PASI, a common psoriasis scoring tool where generally, PASI > 12 is severe, PASI 7 to 12 is moderate, and PASI less than 7 is mild (45). IMQ + vehicle application resulted in a PASI score in the 10–11 range, representing the induction of moderate psoriasis. Topical application of AN oil extract demonstrated a dose-dependent and statistically significant reduction in PASI score with AN oil tested at 3% and 5% producing scores in the 5–6 range, improving the psoriatic condition from moderate to mild (Table 3).
In addition to being characterized by red, scaly lesions formed by the hyperproliferation of epidermal keratinocytes, several inflammatory cytokines have been shown to be elevated in psoriasis lesions, and the serum concentrations of a subset of these also correlate with psoriasis disease severity (46). Two such cytokines are IL-6 and TNF-α, which have both been reported to be potential biomarkers for psoriasis and targets for treatment response (47) with the development of anti-TNF (48) and IL-6 inhibitor therapeutics (49). In skin lesions of psoriasis patients, IL-1β levels have been found to be increased, and effective treatment leads to a significant decrease in epidermal IL-1β expression, suggesting that IL-1β and its family members play a role in the pathogenesis of this disease (50, 51). As shown in Table 4, AN oil extract significantly reduced the levels of all three of these cytokines. Successful development of AN oil extract for treating psoriasis would offer several benefits over the current injectable biologics being utilized, including being more affordable and being a topical therapeutic option. Moreover, being able to downregulate multiple key pro-inflammatory cytokines contributing to the pathogenesis of the disease may be a better long-term approach than targeting a single one.