994 In contrast to myeloid cells, inflammasome activation in keratinocytes is dependent on JNK1 and p38 mitogen-activated protein kinases G Fenini 1 , S Grossi 2 , S Gehrke 3 , H Beer 2 , TK Satoh 1 , E Contassot 4 and LE French 4 1 Dermatology, University of Zu¨rich, Zurich, Switzerland, 2 Dermatology, University of Zurich, Zurich, Switzerland, 3 University of Zurich, Zurich, Switzerland and 4 Dermatology, USZ, Zurich, Switzerland In addition to their barrier functions, keratinocytes are immunologically active cells responding to environmental stressors. Indeed, they express pattern recognition receptors (PRRs) that can sense various stimuli including microbes, chemicals and radiation and release cytokines, chemokines and alarmins. Keratinocytes can secrete the pro-inflammatory cyto- kine IL-1b in response to ultraviolet B (UVB) but the regulation of this process remains incompletely understood. Inflammasomes are key intracellular signaling platforms containing PRRs known as NOD-like receptors that are needed for the processing and release of bio- logically active IL-1b by caspase-1. Inflammasomes have been extensively studied in myeloid cells. Recently, it has been reported that Jun N-terminal kinase-1 (JNK1) is involved in inflammasome activation in myeloid immune cells, an observation that we confirmed in vivo and ex vivo using conditional JNK1 and JNK2 deficient mice. In contrast, little is known about inflammasome activation mechanisms in keratinocytes. Here, we demonstrate that not only JNK but also p38 MAPK are required for inflammasome activation and IL-1b secretion in primary human keratinocytes exposed to UVB or nigericin, a well-known inflammasome activator derived Streptomyces bacteria. Using selective small molecule inhibitors, siRNA gene silencing and CRISPR/Cas9-based deletion, we identified the p38a and p38d isoforms as critical regulators of inflammasome activation as revealed by ASC oligomerization and IL-1b secretion in primary keratinocytes exposed to UVB or nigericin. Altogether, we demonstrate that the activation and regulation of the inflammasome in keratinocytes is not identical to that of myeloid cells. 995 MicroRNA-31 promotes psoriasis by activating macrophages through metabolic reprogramming Y Sun, F Lou and H Wang Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai, China A growing number of evidences highlight the crucial roles of metabolic reprogramming in macrophage activation, which is critical for psoriasis development, while the underlying mechanisms remain largely unknown. Here we report macrophage intrinsic microRNA-31 (miR-31) is markedly induced in the skin of psoriatic patients and imiquimod-induced mouse model of psoriasis. The genetic deficiency of miR-31 in macrophages protects mice from imiquimod-induced psoriasiform lesions, decreases acanthosis and reduces the disease severity. Furthermore, Protein Phosphatase 6 (PP6) is identified as a direct target of miR-31 in macrophages and its dysregulation is responsible for psoriasis severity. We further demon- strate that PP6 interacts with phosphorylates fructose-2,6-biphosphatase isoform 3 (PFKFB3), a major 6-phosphofructo-2-kinase of aerobic glycolysis. Decreased PP6 leads to an impaired kinase function of PFKFB3, eliciting truncated glycolytic capacity and pro-inflammatory cascades in macrophages. Thus, we conclude that macrophage glycolytic metabolism is regulated by miR-31/PP6 axis to adapt to psoriasis proceedings. 996 Atopic dermatitis model on RHE in presence of S.aureus and infiltrating immuno-competent cells M Meloni 1 , E Caviola 1 , S Balzaretti 2 , F Carriero 1 and J Bourland 1 1 VitroScreen SRL, Milan, Italy and 2 VitroScreen SRL, Milano, Italy Atopic dermatitis (AD) is a common skin disease linked to a dysregulation of the immune system and an impaired epidermal barrier. AD lesions show an increased susceptibility to infection by S. aureus, which triggers the production of thymic stromal lymphopoietin (TSLP) by keratinocytes. Based on Reconstructed Human Epidermis (RHE) with a double porosity polycarbonate filter allowing THP-1 monocyte infiltration, a unique in vitro model has been developed to better investigate the cutaneous immune response activated by S. aureus in AD. RHE with impaired barrier were colonized with S. aureus at 2x10 6 CFU/insert. THP-1 infil- trated the RHE for 4 hours. Tissue response was assessed after 4 hours (atopic rash), 16 and 48 hours (delayed inflammation) by qRT-PCR of inflammatory (TNF-a, TSLP, TLR-2), antimi- crobial (human b-defensin 2) response and barrier (filaggrin, CLDN1, ZO-1) markers. Markers of THP-1 differentiation in APC were evaluated (CD86, CD14 and CD11b) as well as Th1/ Th2-associated cytokines (TNF-a, IFN-g, IL-4, IL-5, IL-8, IL-10, IL-13). For selected biomarkers protein expression was confirmed by immunolabelling. THP-1 successfully infiltrated the colonized RHE and differentiated towards a dendritic phenotype. A significant increase of the expression of IL-8, TLR-2 and TNF-a indicated an inflammation in the THP-1-RHE model in presence of S. aureus. TSLP expression presented a 20-fold increase while filaggrin expression decreased when THP-1 were in direct contact with the keratinocytes. S. aureus colonization of the RHE co-cultured with THP-1 also decreased filaggrin expression. Human b-defensin 2 increased by more than 100-fold in the AD model, reflecting keratinocyte reaction to S. aureus. The THP-1-RHE cell migration model seems to fully recapitulate the features of AD in vitro by taking into account the keratinocyte innate and inflammatory response and the im- mune-mediated response in presence of a S. aureus stable colonization. 997 Skin commensal bacteria drive the wound healing response by initiating pDC recruitment and activation in injured skin J Di Domizio 1 , C Belkhodja 1 , P Chenuet 2 , T Murray 1 , O Demaria 1 , C Conrad 1 , D Speiser 1 , B Ryffel 2 and M Gilliet 1 1 University Hospital Lausanne CHUV, Lausanne, Switzerland and 2 University of Orleans, CNRS, Orleans, Centre, France Wound healing of the skin is a complex process that involves the coordinated induction of inflammation and re-epithelialisation. We have previously shown that this process is triggered by pDC infiltration of injured skin and their activation to produce type I IFNs, although the initiating mechanisms are still elusive. Here we used tape stripping of murine skin or skin blister induction of human volunteers as skin injury models to demonstrate that skin commensal bacteria are required to initiate this process. First, we show that the skin commensal bacteria activate neutrophils to express Cxcl10 which drives recruitment of pDC into injured skin. Second, we demonstrate that the presence of the skin microbiota is essential for the ability of Cxcl10 to activate pDC. In fact, Cxcl10 was found to preferentially bind and kill bacteria, leading to the formation of complexes with bacterial DNA. The subsequent release of CXCL10-bacterial DNA complexes into the extracellular environment triggered the activation of pDC via TLR9. By inhibiting pDC infiltration and activation, the depletion of skin microbiota profoundly dampened the inflammatory response in injured skin and delayed the wound closure. Our data uncover a fundamental role of the skin microbiota in the initiation of inflammatory and healing responses in skin wounds and suggest that the host exploits commensalism to efficiently restore barrier integrity. 998 EMT-like phenotype in normal keratinocytes driven by TLR3 activation A Schneider, A Sarfo, Z Cong and AM Nelson Department of Dermatology, Penn State University College of Medicine, Hershey, PA Toll-Like-Receptor 3 (TLR3), a pattern recognition receptor that recognizes dsRNA, activation results in NFkB or interferon-regulatory factor 3 (IRF3) mediated gene transcription of in- flammatory cytokines (e.g. IL-6) and type I and III interferons (e.g. IFN-l). TLR3 also con- tributes to induced-pluripotent stem cell (IPS) programming, wound healing and skin regeneration through Wnt, SHH, and epithelial-mesenchymal transition (EMT) pathways. Our hypothesis is that TLR3 signaling within normal keratinocytes contributes to non-melanoma skin cancer (NMSC) through activation of EMT. In vitro, normal neonatal human embryonic keratinocytes (NHEKs) treated with a TLR3 agonist, polyinosinic:polycytidylic acid (poly IC), exhibit sustained EMT morphology changes that correlate with elevated levels of the inter- mediate filament Vimentin (w2 fold), and transcription factors, ZEB1 (w3-fold), SNAI1 (w2- fold), and TWIST1 (w2-fold), all key EMT markers. Poly IC treatment significantly increased levels of IL-6 (w8-fold) and IFN-l1(w200-fold) in normal keratinocytes. However, treatment of NHEKs with recombinant IL-6 or IFN-l1 proteins do not induce an EMT-like morphology or increase in EMT gene expression. UVB radiation, the major player in NMSC development, also activates TLR3 signaling in keratinocytes. In vitro, NHEKs exposed to UVB (10mJ/cm 2 ) exhibit similar EMT-like morphology and gene expression changes as poly IC treated cells. Chemical inhibition of TLR3 (CU CPT 4a) prior to poly IC or UVB exposure prevents EMT gene expression. We performed RNA-sequencing to investigate common pathways activated by both Poly IC and UVB to uncover additional downstream players responsible for driving the TLR3 induced EMT-phenotype.Taken together, TLR3 activation and the resulting EMT-like phenotype is a result of multiple players, and not likely driven by a single downstream mediator of TLR3 signaling. 999 Prebiotic colloidal oatmeal supports the growth of S. epidermidis and enhances the production of lactic acid F Liu-Walsh 1 , J Hauschild 2 , G Leo 3 , J Masucci 4 , A Tall 5 , NK Tierney 6 and K Capone 1 1 Johnson & Johnson Consumer, Skillman, NJ, 2 Johnson & Johnson Sterility Assurance Industrial Microbiology, Raritan, NJ, 3 Janssen, Springhouse, NJ, 4 Janssen, Springhouse, PA, 5 Johnson & Johnson Consumer EMEA Microbiology, Val de Reuil, Franche-Comte, France and 6 Johnson & Johnson, Skillman, NJ We previously demonstrated that 1% colloidal oatmeal promoted the growth of Staphylo- coccus epidermidis (S. epidermidis), Staphylococcus aureus (S. aureus) and Propionibacte- rium acnes (P. acnes). More importantly, we showed that colloidal oatmeal increased the growth rate of S. epidermidis more than that of S. aureus. We now demonstrate in an in vitro competition assay that 1% colloidal oatmeal selectively promoted the growth of S. epi- dermidis when S. epidermidis and S. aureus were cultured together. These data support a prebiotic mechanism for colloidal oatmeal in balancing the skin microbiome by supporting the growth of a skin health-associated species in the presence of an opportunistic pathogen. Short chain fatty acid (SCFA) analysis revealed that the metabolite profile of S. epidermidis grown in the presence of 1% colloidal oatmeal under aerobic conditions was more similar to S. epidermidis grown under anaerobic conditions in the absence of colloidal oatmeal, indi- cating S. epidermidis utilizes fermentation pathways to metabolize colloidal oatmeal. As a result, S. epidermidis grew faster and generated more lactic acid, a fermentation byproduct. Lactic acid is one of skins natural moisturizing factors (NMF) that provide skin benefits such as moisturization and exfoliation. RNA sequencing of S. epidermidis grown in aerobic and anaerobic conditions confirmed that the glucose metabolic and monosaccharide metabolic processes were down-regulated in S. epidermidis grown under anaerobic conditions and with 1% colloidal oatmeal under aerobic conditions. Together, these data support the conclusion that 1% colloidal oatmeal influences the growth and metabolism of a health-associated skin bacterium, and may play a significant role in the microbiome-diversity improvements seen with a colloidal oat-containing formula in AD subjects. Innate Immunity, Microbiology, Inflammation | ABSTRACTS www.jidonline.org S169