523 T-cell cytokines (IL-17 and IFN-g) exacerbates innate immune responses in an organotypic human psoriasis tissue model S Ayehunie, T Landry, C Hedin, A Armento, P Hayden and M Klausner MatTek Corporation, Ashland, Massachusetts, United States Psoriasis is a chronic inflammatory immune mediated skin disease marked by hyper- proliferation, abnormal keratinocyte (KC) differentiation, and leukocyte infiltration. Research into the pathogenesis of psoriasis has been hampered by the lack of in vitro models that provides insight into the mechanisms of disease or efficacy of therapeutics. Species differ- ences between existing rodent models and humans raises major translational issues. Since T cells, particularly Th17 cells) are key players in psoriasis, anti-IL-17A antibodies such as Secukinumab have been developed as therapeutics. Based on these scientific developments, we investigated the role of IL-17 alone or in combination with IFN-g (Th1 cytokines) in exacerbating inflammatory innate immune responses using a reconstructed 3D human pso- riatic tissue model. Normal human keratinocytes (KC) and psoriatic fibroblasts from lesional sites were harvested from primary tissues and cultured using defined serum-free medium to form a highly differentiated 3-dimensional tissue model. The reconstructed psoriatic tissue model was exposed to IL-17 / IFN-g for up to 96 hr (4X exposure). The results showed that: 1) IL-17 increases the Th-17 cell and dendritic cells/monocytes chemoattractant (CCL-20), the neutrophil chemoatractant (IL-8), and the anti microbial peptides (HBD2 and elafin (PI3) by 2- 7-fold. 2) IFN-g induces overexpression of the chemoattractant for activated T cells (CXCL11), IL-8, and TNF-a by 2-324-fold. 3). IL-17 and IFN-g exacerbate inflammation synergistically by increasing expression levels of the neutrophil chemoattractants (CXCL5, IL-8, IL-6), HBD2, PI3, and TNF-a by > 3-fold. In conclusion, we show that a self-sustaining inflammatory feedback loop that involves T-cell cytokines (IL-17, IFN- g) and KC/fibroblast derived CCL20, CXCL5, HBD2, PI3, IL-8, and IL-6 is established. Thus, level of innate immune responses such as CCL-20, IL-8, IL-6, and CXCL-5 following treatment can serve as surrogate markers to examine novel biologic therapies. 524 Evaluation of different skin collection methods for assessment of the skin microbiome JA Rock 1 , B Jansen 1 , Z Yao 1 , A Talisha 1 , H Maesa 1 , DM Dinh 2 and J Cope 2 1 DermTech, La Jolla, California, United States and 2 Diversigen, Houston, Texas, United States Background and Objective: The sampling and sequencing of human skin microbiome rep- resents a tremendous opportunity to understand how resident microbes impact common dermatological conditions and skin health. Understanding the differences and limitations in commonly used sample collection methods is paramount to exploiting this up and coming area of research. The objective of this study is to compare the performance of two swab-based collection (SBC) kits (from Genotek and Norgen) and an adhesive patch-based collection (APC) system (from DermTech). Methods and Materials: Following manufacturers’ in- structions, samples were collected from 3 forehead skin locations from 15 subjects using all 3 collection kits. Nucleic acids from each sample were extracted, and the V1V3 hypervariable region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform. Post- sequencing analysis compared the alpha diversity (AD), beta diversity (BD) and taxonomic composition (TC) among sample sites and collection kits. Results: 11 males and 4 females had samples collected from 3 forehead locations. Ten of the subjects refrained from face washing and using any topical products on the day of collection. All sample collection methods, regardless of skin location, had adequate yields for microbiome analysis. Analyses indicated the SBC method yielded similar results overall in terms of AD, BD and TC. The Dermtech APC method resulted in improved richness but similar overall diversity. The APC method also tended to cluster differentially from the SBC methods by Principle Coordinates Analysis using both the weighted and unweighted UniFrac metrics. As the APC enriched more Pelomonas, Bradyrhizobium and Sphingomonas at the genus level compared to SBC. Conclusions: All sampling methods were able to collect sufficient material for microbiome analysis. However, elevated levels of operational taxonomic unit (OTU) richness and differences between the APC and SBC methods suggest that APC method may sample the skin more completely than swab-based methods. 525 Novel peptide from commensal Staphylococcus simulans blocks MRSA quorum sensing and protects host skin from damage MM Brown 1 , D Todd 2 , N Cech 2 and A Horswill 1 1 Immunology & Microbiology, The University of Colorado Anschutz Medical Campus, Aurora, Colorado, United States and 2 Chemistry & Biochemistry, The University of North Carolina Greensboro, Greensboro, North Carolina, United States The human skin is colonized by a diverse array of bacteria, archaea, fungi, and viruses. Recent studies highlight the abundance of commensal coagulase-negative staphylococci (CoNS) as well as the relative absence of methicillin resistant Staphylococcus aureus (MRSA) on healthy skin. A widely recognized mechanism of bacterial competition is the quorum sensing Accessory Gene Regulator (agr) system, ubiquitous among staphylococci. We hypothesize that agr signaling facilitates interspecies cross-talk between CoNS and MRSA, resulting in a colonization advantage for CoNS and protecting the host from MRSA colonization or infection. A screen of CoNS spent media with potential inhibitory activity against MRSA agrsignaling identified Staphylococcus simulans as an attractive candidate to explore this hypothesis. S. simulans is a rare CoNS commensal of human skin, and we found that S. simulans spent media was sufficient to inhibit MRSA agr signaling in vitro. Mass spectrometry analysis of spent media revealed a unique 9 amino acid AIP and an MS confirmed AIP matching the native structure was synthesized for further analysis. The synthetic AIP inhibited MRSA agr signaling in vitro with nanomolar potency. Addition of synthetic AIP reduced MRSA skin damage in a murine model of infection. Finally, mice co-challenged with S. simulans and MRSA were significantly more protected from dermonecrosis than MRSA single challenge. Co-challenged mice also showed accelerated MRSA clearance compared to single challenge. These results suggest that cross-talk between CoNS and MRSA may be important in maintaining healthy skin homeostasis and preventing MRSA skin damage. Current in- vestigations include the characterization of inhibitory AIPs produced by frequently isolated CoNS like Staphylococcus hominis. In vitro and ex vivo models of human skin that better match the native environment are also in development to assess CoNS-MRSA interactions. 526 Gut dysbiosis in alopecia areata patients reveals overabundance of firmicutes and under representation of bacteroides B Sallee 1 , R Perez-Lorenzo 1 , EH Wang 1 , JC Chen 3 , AR Abdelaziz 2 , LA Bordone 1 and A Christiano 1 1 Dermatology, Columbia University, New York, New York, United States, 2 Dermatology, Columbia University Medical Center, New York, New York, United States and 3 Columbia University, New York, New York, United States Alopecia Areata (AA) is one of the most prevalent autoimmune disorders in humans leading to patchy or total loss hair with about a 2% lifetime prevalence. AA has a significant impact on patients’ quality of life, and associations with other autoimmune diseases. The development of AA is influenced by genetic, immunological, and environmental factors, though these are not completely defined. The gut microbiome has an immunomodulatory effect capable of eliciting pathologic immune responses beyond the gut. Our recent studies in the C3H/HeJ mouse model of AA showed that oral broad spectrum antibiotics prevented onset of AA, suggesting the gut microbiota is required for AA onset. Thus, to determine the microbiome composition of patients with AA, we collected skin swabs, hair follicle samples, and stool samples from a cohort of 26 AA patients. Analysis of 16S rRNA sequencing on stool samples revealed significant differential representation of bacterial taxa, between AA patients and healthy subject specifically, members of the firmicutes and bacteroides phyla, similar to our mouse model findings. When AA patients were compared to healthy controls we found under representation the bacteroides phyla and over representation of the firmicutes phyla in AA gut microbiome, similar to changes reported in other autoimmune disorders. Importantly, there was no difference in the skin or hair follicle microbiome in AA patients as compared to healthy controls, underscoring the importance of gut microbiota dysbiosis in AA patients. The presence of gut microbiota dysbiosis in human AA patients provides a rationale for the development of novel therapeutic strategies for AA patients, including Fecal Microbiota Transfer (FMT) and targeted microbial therapy, since restoring the gut microbiota composition to a healthy state has been suggested as an approach to improve the course of autoimmune diseases, such as AA. 527 Uncovering molecular mechanisms involved in the IL-37-mediated tolerogenic dendritic cells PK Vaddi, Y Luo, C Dinarello and M Fujita Dermatology, University of Colorado, Aurora, Colorado, United States The past few years have witnessed a breakthrough in the development of tolerogenic dendritic cell (DC) based therapies for the treatment of autoimmune diseases and solid organ trans- plantation. The interleukin-1 family member IL-37 is a natural suppressor of innate inflam- matory and immune responses. Using mice expressing human IL-37b isoform (IL-37tg), we have previously reported that IL-37 participates in peripheral tolerance through the genera- tion of semi-mature tolerogenic DCs. However, the molecular mechanism(s) and/or regula- tory molecule(s) involved in the IL-37-mediated tolerogenic DCs are yet to be identified. Similar to IL-1a and IL-33, IL-37 has a unique property as an intracrine cytokine, functioning both intracellularlyand extracellularly. To investigate extracellular mechanisms, recombinant IL-37 and neutralizing antibody to IL-37 were used. Despite their effects on IL-1b secretion, neither recombinant IL-37 nor neutralizing antibody affected the ability of DCs to activate T cell proliferation or induce regulatory T cells, suggesting that extracellular IL-37 has limited effects on DC function. Next, to understand molecular pathways altered by IL-37 in DCs, RNAs from wild-type (WT) and IL-37tg DCs were subjected for Illumina microarray analysis. GeneGo pathway analysis identified a key transcription factor NF-kB and 3 pathways including IL-10, CD40, antigen presentation pathway and HMGB1 pathways. Several differentially expressed genes were identified and verified including CXCL1, IL-10 and others. miRNA array analysis using Nanostring miRNA array kit identified immunosuppressive miRNAs. Furthermore, THP-1 human cell line transfected with IL-37 expressed similar mo- lecular profiles to IL-37 mouse DCs and exhibited lower ability to stimulate proliferation of allogeneic naı ¨ve T cells after differentiation into DCs. Together, these findings reveal an immunosuppressive molecular pathways of DCs expressing IL-37. 528 Curcumin-mediated modulation of bacterial communities in inflammatory skin dysbiosis J Tran 1 , A Vaughn 2 , W Burney 2 , R Sivamani 2 and R Crawford 1 1 Biological Sciences, Cali- fornia State University, Sacramento, Elk Grove, California, United States and 2 University of California, Davis, California, United States Dysbiosis of the skin microbiome and underlying immune response to microbial antigens contributes to the pathogenesis of atopic dermatitis and several other disorders. DNA sequencing analysis has shown that bacterial communities of human patients shift to a concurrent reduction of commensal Staphylococcus epidermidis and overabundance of pathogenic species including Staphylococcus aureus and Pseudomonas aeruginosa during bouts of chronic inflammation relapse and a return to greater overall diversity following antibiotic treatment. However, antibiotic resistance and person-to-person variability in mi- crobial communities is rendering currently prescribed therapeutic regimens unsuccessful, thereby highlighting a paucity in novel remediation approaches. To that end, food derivatives including curcumin have been explored for prebiotic intervention as a means of shifting bacterial populations based on antimicrobial and/or metabolic properties, but published studies present conflicting evidence toward efficacy and the underlying mechanisms remain unclear. Here we use a combination of physiological and molecular experiments to show that curcumin differentially modulates phenotypes associated with commensal and pathogenic SA including growth rate and biofilm formation by regulating expression of genes previously shown to be important for adherence and extracellular matrix production. Additionally, curcumin fortified existing biofilms and differentially regulated gene expression profiles in planktonic and biofilm-associated populations. We further show that curcumin regulates production of short and long chain fatty acids by sebocytes grown in cell culture, potentially contributing additive selective effects for specific bacteria species. Collectively, these results suggest the use of curcumin for remodeling the skin milieu and inhabiting microbial com- munities toward a predominance of non-pathogenic organisms. ABSTRACTS | Innate Immunity, Microbiology, and Microbiome S90 Journal of Investigative Dermatology (2019), Volume 139