424 Cell-based immunofluorescence test applying recombinant laminin 332 for the serological differential diagnosis of pemphigoid S Goletz 1 , C Probst 2 , L Komorowski 2 , W Schlumberger 2 , D Zillikens 3 , W Sto¨ cker 2 and E Schmidt 1,3 1 Lu¨beck Institute of Experimental Dermatology (LIED), University of Lu¨beck, Lu¨beck, Germany, 2 Experimental Immunology, Euroimmun, Lu¨beck, Germany and 3 Department of Dermatology, University of Lu¨beck, Lu¨beck, Germany A subgroup of patients with mucous membrane pemphigoid (MMP) exhibits autoimmune reactions against laminin 332 (Lam332) and can be identified by the presence of the corre- sponding autoantibodies. Nearly 30% of patients with anti-Lam332 MMP develop solid cancers, yet no commercial diagnostic assay for anti-Lam332 autoantibodies is widely available. In this study, recombinant Lam332 or fragments of it were used in a cell-based immunofluorescence (IF) test applying a heterotrimer of alpha3, beta3, and gamma2 as well as the chains separately expressed in HEK293 cells. Subsequently, sera from 93 anti-Lam332 positive MMP patients defined by Western blot reactivity with extracellular matrix of cultured human keratinocytes were tested. Controls included sera from patients with non-anti-Lam332 MMP (n¼153) serologically defined by lack of dermal site reactivity with salt-split skin and/or by reactivity against BP180 and/or BP230. Furthermore, sera from patients with bullous pemphigoid (n¼20), pemphigus vulgaris (n¼20), non-inflammatory dermatoses (n¼22), and from blood donors (n¼100) were included. The IF test with the heterotrimer full length laminin 332 as antigen substrate revealed a sensitivity of 69%, with the alpha3 chain 43%, the beta3 chain 34%, and the gamma2 chain 10% at a specificity of 100% with each of the substrates. The combination did not significantly increase the sensitivity. Of the 93 MMP sera that were reactive with the cell-derived Lam332 by Western blotting, 72 reacted with the dermal site of human salt-split skin. The sensitivity in this clearer defined MMP group with the Lam332 heterotrimer was 86%. The novel IF-based assay with recombinant Lam332 as a substrate is beneficial for the serological diagnosis of anti-Lam332 MMP. 425 Oxysterol function of lymphocyte cell death is differentially defined by types of cholesterol modification H Takahashi, H Nomura, H Iriki and M Amagai Dermatology, Keio University, Tokyo, Japan Crucial roles of cholesterol metabolites in immunity has recently attracted much attention. We previously demonstrated that interleukin-27 (IL-27), an immunoregulatory cytokine, in- duces cholesterol 25-hydroxylase (Ch25h) in CD4 + T cells under antigen stimulation. Ch25h is an enzyme converting cholesterol into 25-hydroxycholesterol (25OHC). Here we show that 25OHC is secreted from IL-27-stimualted T cells and kills activated lymphocytes in vitro. Since 25OHC is further metabolized to 7a, 25-dihydroxycholesterol (7a, 25OHC) by CYP7B1, ability of cell death induction was compared among cholesterol, 25OHC, and 7a, 25OHC, in which structural difference is only one hydroxyl group added to the precursors. When those molecules were supplied to T cell culture, only 25OHC, but not cholesterol nor 7a, 25OHC, induced cell death, suggesting specific function of 25OHC among them. Besides 25OHC, various hydroxyl derivatives of cholesterol were generated in the body and how unique 25OHC function is among these derivatives is still unclear. To clarify that, the function of 25OHC was further compared to other cholesterol derivatives at 1 - 1000 nM that are irrespective of Ch25h but generated in vivo. Among eight derivatives, 27-hydroxycholesterol (27OHC), 24(R/S), 25-epoxycholesterol, 20a-hydroxycholesterol (20aOHC), recapitulated cell death in activated CD4 + T cells, but 7a-hydroxycholesterol, 7b-hydroxycholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, and 7-ketocholesterol did not. Quanti- tatively, 27OHC and 24(R/S), 25-epoxycholesterol could induce cell death at 100 nM similar to 25OHC, but 20aOHC needed higher concentration (1000 nM) to induce cell death. These results suggested that cell death induction is a specific function authorized to limited cholesterol derivatives and 25OHC is one of the most potent molecules to induce cell death in lymphocytes. Since delicate difference of chemical structure significantly impact choles- terol derivative function, this information would be beneficial to find the receptor and syn- thesize super-agonist as a new drug in the future. 426 Dual interleukin-17A and interleukin-17F neutralisation with bimekizumab provides evidence for interleukin-17F contribution to immune-mediated inflammatory skin response A Maroof 1 , T Smallie 1 , S Archer 1 , C Simpson 1 , M Griffiths 1 , D Baeten 2 and S Shaw 1 1 UCB Pharma, Slough, United Kingdom and 2 UCB Pharma, Brussels, Belgium Interleukin (IL)-17A and IL-17F are pro-inflammatory cytokines that share similar biological function and structural homology. Both are known to be upregulated in several human im- mune-mediated inflammatory diseases, however, compared with IL-17A, the role of IL-17F is less clear. We hypothesised that IL-17F also contributes to chronic tissue inflammation in lesional skin from patients with psoriatic arthritis (PsA). To test this, we developed bimeki- zumab, a humanised monoclonal IgG1 antibody that potently and selectively neutralises IL- 17A and IL-17F. Immunostaining was performed on lesional skin from patients with PsA to probe for IL-17F protein. Primary normal human dermal fibroblasts (NHDFs) were stimulated with recombinant IL-17A and IL-17F, with/without TNF, to assess the effect on inflammatory cytokine production. Primary NHDFs were treated with Th17-cell supernatant and blocked with cytokine-specific antibodies including bimekizumab, to assess the role of IL-17A and IL- 17F in inflammatory gene and protein expression. IL-17F protein was detected in lesional skin from patients with PsA. Stimulation of primary NHDFs with recombinant IL-17F, in the presence of TNF, induced production of pro-inflammatory mediators (e.g. IL-8 & IL-6), though to a lesser extent than with recombinant IL-17A. Dual neutralisation of IL-17A and IL-17F with bimekizumab demonstrated greater inhibition of IL-8 (57% lower p<0.0001) and IL-6 (35% lower p<0.0001) production vs IL-17A inhibition alone. Lower levels of expression of 27 inflammation-linked genes including IL-6, IL-8, CXCL1 and CXCL2 were found with bimekizumab vs IL-17A inhibition alone, in Th17-stimulated primary NHDFs. Data indicate that dual neutralisation of IL-17A and IL-17F more profoundly suppresses the inflammatory response of primary NHDFs, than IL-17A blockade alone. Dual IL-17A and IL-17F neutrali- sation may therefore be effective in the treatment of immune-mediated inflammatory dis- eases, including psoriasis and PsA. 427 Finegoldia magna and Corynebacterium kroppenstedtii are significantly enriched in rosacea independent of rosacea subtype: Results of a case-control study BM Rainer 2,1 , E Mongodin 3 , J Bui 3 , A Fischer 1 , H Pasieka 1 , LA Garza 1 , S Kang 1 and A Chien 1 1 Dermatology, Johns Hopkins School of Medicine, Graz, Austria, 2 Dermatology, Medical University of Graz, Graz, Austria and 3 Institute for Genome Sciences, Baltimore, MD Rosacea has long been associated with microorganisms, but the involvement of microbial communities in rosacea’s pathophysiology is unknown. Here, we performed a study to examine the skin microbiota (1) in rosacea patients compared to matched controls, and (2) in erythematous rosacea (ETR) versus papulopustular rosacea (PPR). We used bacterial 16S ri- bosomal RNA gene sequencing on DNA extracted directly from skin samples (nose/cheeks swabs) of participants (21 patients and 21 controls; mean 47.1 years). The V3V4 region of the 16S rRNA gene was targeted, sequenced using Illumina MiSeq, and analysed using the QIIME/Phyloseq software packages. Our final sequence dataset contained 4,036,167 16S rRNA sequences clustered into a total of 1,593 species-level operational taxonomic units. In- depth differential abundance analyses using DESeq2 revealed several statistically significant differences of discrete bacterial taxa between patients and controls, but did not reveal sig- nificant differences between ETR (n¼12) and PPR (n¼9) subtypes. Cheeks microbiota of ro- sacea patients was significantly enriched in bacteria from the genus Finegoldia magna (log2 mean fold change: 4.44; p<0.005), but had a significantly lower relative abundance of Propionibacterium acnes (-3.48; p<0.005) compared to controls. The nose microbiota of patients had significantly higher levels of Corynebacterium kroppenstedtii (4.35; p< 0.005). Our data demonstrate that specific bacterial genera are highly associated with rosacea, and other strains are enriched in healthy skin. Finegoldia magna and C. kroppenstedtii may have important roles in rosacea pathophysiology and could be future targets for therapeutic interventions. 428 CD100-Plexin-B2 promotes the inflammation in psoriasis by activating NF-kB and inflammasome in keratinocytes C Zhang Dermatology, Assistant Researcher, Xi’an, China Psoriasis is an inflammatory skin disease, in which keratinocytes play a crucial role in the pathogenesis. Plexin-B2 (PlxnB2) and its ligand, Semaphorin 4D (Sema4D, CD100), are originally identified as axon-guidance molecules that function during neuronal development; however, studies also showed that CD100-Plexins participate in various immune responses. To explore the function and mechanism of CD100-PlxnB2 in the pathogenesis of psoriasis. Expression and interaction of PlxnB2 and CD100 in psoriasis patients, imiquimod-induced psoriasis-like dermatitis of mice and primary keratinocytes cells were analyzed by immu- nohistochemistry, quantitative RT-PCR, ELISA, Western blot and co-immunoprecipitation. Inflammatory responses, cytokine expression and signaling pathway activation were analyzed by using ELISA, quantitative PCR, Western blot and flow cytometry. We found that the expression of PlxnB2 on keratinocytes was specifically increased in the lesional skin of psoriasis patients and imiquimod (IMQ)-induced psoriatic dermatitis of mice, but not atopic dermatitis patients and MC903-induced AD mouse model. The levels of soluble CD100 (sCD100) and membrane CD100 (mCD100) were elevated in the sera of psoriasis patients and on the keratinocytes of psoriatic skin, respectively. And the expression of mCD100 on T cells, monocytes and platelet, but not B cells, from PBMC of psoriasis patients was lower than that of healthy controls. By binding to PlxnB2, sCD100 promoted the production of CXCL-1, CCL-20, IL-1b and IL-18 by keratinocytes in cell culture, and activated the NLRP3 inflam- masome. Moreover, CD100-PlxnB2 stimulated NF-kB signaling pathway in keratinocytes through the activation of small GTPase RhoA and Rac1. And mCD100 promoted the PlxnB2- mediated effects exerted by sCD100, and functioned as a co-receptor for PlxnB2. Our data uncovered for the first time that cooperation of CD100 and PlxnB2 promoted the inflam- matory responses in keratinocytes by activating NF-kB and NLRP3 inflammasome, and participated in the pathogenesis of psoriasis. And CD100/PlxnB2 might be a potential ther- apeutic target for psoriasis. 429 Prohapten-activation by human cutaneous cytochrome P450 isoenzymes e identified with a modified KeratinoSens assay L Huth 1 , E Moss 2 , S Huth 1 , C Skazik 1 , A Karlberg 3 , J Lepoittevin 2 , JM Baron 1 and HF Merk 1 1 Dept. of Dermatology & Allergology, Uniklinik RWTH Aachen, Aachen, Germany, 2 Institut de Dermatochimie, Universite´ de Strasbourg, Strasbourg, France and 3 Department of Chemistry and Molecular Biology, Dermatochemistry, University of Gothenburg, Gothen- burg, Sweden Skin is a uniquely susceptible target organ for allergic reactions to small molecular weight compounds (haptens). Prohaptens need metabolic bioactivation in order to become anti- genic. Incubation of prohaptens with single cytochrome P450 isoenzyme (CYP) cocktails or CYP-containing microsomes are generally very cytotoxic. The S9 fraction prepared from rat liver has been tried successfully, however a disadvantage is that it contains murine liver CYPs but not human cutaneous CYPs. This led us to redesign our protocol by spiking rat liver S9 with human cutaneous CYPs (e.g. CYP 1B1) and measuring the activation of NRF2 in Kera- tinoSens cells. Carvoxime (Ca) is a potent prohapten known to be a strong sensitizer in the murine LLNA. It is activated by human skin CYP 1B1 that is not expressed in liver and Ca was not reactive in the KeratinoSens assay using rat liver S9. Addition of Ca to the spiked prep- aration as well as CYP 1B1 itself strongly activated NRF2 whereas heat inactivated prepa- rations showed no reactivity. Several fragrances are prohaptens. The cutaneous metabolom of the fragrance creosol (Cr) was analyzed by HR-MAS NMR after topical application to a 3D human skin equivalent. 1,2 benzoquinone-hapten-protein-adduct and another up to now unidentified hapten-protein-adduct were found in the upper epidermis, formaldehyde in the dermis. Since this metabolism requires a demethylation of Cr, we studied the possible involvement of human cutaneous CYPs in the KeratinoSens assay with rat liver S9 after spiking with human cutaneous CYPs. Addition of Cr to the spiked preparation strongly activated NRF2 and heat inactivated preparations as well as preparations only with S9 showed no reactivity. Taken together this modified KeratinoSens assay allows to identify prohaptens which are bioactivated by human cutaneous CYPs. Inflammation, Immunity and Infection | ABSTRACTS www.jidonline.org S265