Cathelicidin LL37 is overexpressed in plaques psoriasis. Microbial ligands are part of the triggers of LL37 over-expression. [Our objectives were] to assess the expression of LL37, of interleukins 17 and 23 in skin ex vivo submitted to a microbial ligand, then to assess the effects of AGH8 (vegetal extract) in the same model. Human skin explants were submitted to a microbial ligand: zymosan A at 1% was applied at the surface of the explants on day 0, D2, D3 and D6. AGH8 was topically applied after removal of the zymosan-disk at same days. Quantitative and immunohistochemistry analysis of LL-37, IL-17 and IL-23 were made (immunostaining of LL37 with anti-LL37 MAB, LL37 immunofluorescence staining was made with anti-mouse IgG. IL-17A and IL23 immunostainings were made using anti-IL-17A or with anti-IL-23 rabbit polyclonal antibodies. On D3 and D8, the basal level of LL37 protein expression in the control explants represented 18.7±7.2% of the epidermis surface. On D8, after zymosan-exposure, the expression of LL-37 was significantly increased by 228% (p<0.001) compared to control. On D3 and D8, AGH8 induced a significant decrease of LL37 expression by 84%, compared to untreated explants. On D8 AGH8 applied on zymosan-exposed explants induced a significant decrease of LL37 expression by 87% (p<0.001), vs zymosan only. In this ex vivo study, the ligand zymosan stimulated inflammatory response in human skin explants involving LL37 and IL-23/IL-17 axis, Topical application of AGH8 induced a significant decrease of the zymosan-induced inflammatory cascade (p<0.001).

**Background** Antimicrobial peptide LL37 is overexpressed in rosacea, in plaques psoriasis and other chronic inflammatory diseases (1-3). Microbial ligands as zymosan are part of the triggers of LL37 over-expression (4, 5).
**Objectives:** To assess the expression of LL37, of interleukins 17 and 23 in skin _ex vivo_ submitted to a microbial ligand, then to assess the effects of AGH8 (vegetal extract) in the same model.
**Materials and methods.** human skin explants from the same donor, maintained in survival (BioEc medium) were submitted to a microbial ligand (from _Candida albicans)_: Zymosan A (Sigma-Aldrich®, ref. Z4250) at 1% in sterile 0.9% NaCl solution was applied on a disk (30 μL/disk) at the surface of the explants for 5 h at 37°C on day 0, day 2, day 3 and day 6. At the same time points, AGH8 was topically applied at a rate of 2 μL/cm² after removal of the zymosan-soaked disk. Quantitative and immunohistochemistry analysis of LL-37, IL-17 and IL-23 were made (_immunostaining of LL37-_ anti-LL37 MAB (Santa Cruz®, Sc-166770, clone D-5), for LL37 Bimmunofluorescence staining was used AlexaFluor® AF488 anti-mouse IgG (Lifetechnologies®, ref: A11001). _IL-17A and IL23 immunostainings_ were made using anti-IL-17A (Abcam®, ref. ab79056) or with anti-IL-23 (Invitrogen®, ref. PA5-20239) rabbit polyclonal antibodies. A negative control without primary antibody incubation has been performed for each immunostaining. The immunostainings of LL37, IL-17A and IL-23 were assessed by microscopical observations (Leica® DMLB microscope, Olympus® DP72 camera,  CellSens® software).
**Results** On D3 and D8, the basal level of LL37 protein expression in the control untreated explants represented 18.7±7.2% and 13.3±4.1% of the epidermis surface respectively (Fig.1A). On D8, after zymosan-exposure, the expression of LL-37 was significantly increased by 228% (p<0.001) compared to control (Fig1B). On day 3 and day 8, the repeated topical application of AGH8 induced a significant decrease of LL37 expression by 84% compared to untreated explants (Fig.1C). On D3 and D8, zymosan induced an increased expression of IL-17 (Fig.2A/B) and of IL-23 (Fig.3A/B). The topical application of AGH8 on zymosan-exposed explants induced a significant decrease of IL-17 (Fig.2C) and respectively IL-23 (Fig.3C) (p<0.001), compared to explants treated with zymosan only.
**Conclusions**: this ex vivo study, the microbial ligand zymosan stimulated inflammatory response in normal human skin explants with the stimulation of antimicrobial peptide cathelicidin LL 37 expression and subsequent upregulation of IL 23 and IL 17 expressions Topical application of AGH 8 induced a significant decrease of the zymosan induced inflammatory cascade The early down regulation of LL 37 overexpression induced by microbial ligands could have a clinical application by focusing the upstream prevention of the activation of pro inflammatory axis IL 17 IL 23 in chronic inflammatory diseases as psoriasis.
**References**
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2. Fuentes-Duculan J, Bonifacio KM, Hawkes JE, et al. Autoantigens ADAMTSL5 and LL37 are significantly upregulated in active Psoriasis and localized with keratinocytes, dendritic cells and other leukocytes. Exp Dermatol, 2017;26:1075-1082.
3. Lande R, Gregorio J, Facchinetti V, et al. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature, 2007;449:564-569.
4. Hurabielle C, Link VM, Bouladoux N, et al. Immunity to commensal skin fungi promotes psoriasiform skin inflammation. Proc Natl Acad Sci U S A, 2020;117:16465-16474.
5. Sparber F, De Gregorio C, Steckholzer S, et al. The Skin Commensal Yeast Malassezia Triggers a Type 17 Response that Coordinates Anti-fungal Immunity and Exacerbates Skin Inflammation. Cell Host Microbe, 2019;25:389-403.e6.

ESDR391 - Cathelicidin LL37 and skin inflammation: ex vivo study

The scientific meeting of the European Society for Dermatological Research (ESDR) is held yearly, usually in September. With an international attendance of more than 1000 skin scientists from around the globe, the four-day event includes short oral presentations from peer-reviewed abstracts, keynote lectures from international experts, sponsored symposia, satellite programs, educational events as well as an important focus on posters, networking and building collaborations amongst scientists. For more information about this year’s meeting visit our website.