449 In situ mapping of innate lymphoid cells in normal and inflamed human skin M Bru ¨ ggen 2,1 , W Bauer 2 , B Reininger 2 , E Clim 4 , C Captarencu 4 , B Meier 1 , PM Brunner 2,3 , L French 1 and G Stingl 2 1 Dpt of Dermatology, University Hospital Zurich, Zurich, Switzerland, 2 Dpt of Dermatology, DIAID, Medical University of Vienna, Vienna, Austria, 3 Laboratory for Investigative Dermatology, Rockefeller University, New York, NY and 4 Dpt of Application Support and Image Processing, TissueGnostics SRL, Iasi, Romania Innate lymphoid cells (ILCs) have recently been identified also in skin, but their functional role in this organ remains poorly understood. The main aim of our study was to develop a technique to reliably identify ILCs in situ in normal and in inflamed human skin. Most importantly, we wanted to explore the topographical relationship of ILCs to the skin’s various structural and cellular elements. We collected lesional skin biopsies from atopic dermatitis (AD) and psoriasis (Pso) patients (both n¼13) and normal human skin (NHS) samples from healthy controls. An ILC staining panel was established and a computed system coupled to a fluorescence microscope (TissueFAXS) was used for the analysis. Cells were analyzed/gated based on fluorescence intensities of the different channels. A special tool was developed to track and manually validate every cell gated as ILCs. In an additional set of algorithms, we assessed the distance between each validated ILC and different structures/cells. A very scarce ILC population of mostly ILC1s and AHR + ILC3s was observed in NHS. In contrast, AD and Pso skin lesions were infiltrated by clearly visible ILC subsets. We found AD skin to not only harbor ILC2s, but also an AHR + ILC3 population of similar size. Conversely, we encountered almost equal proportions of ILC1s and RORgt + ILC3s, but essentially no ILC2s in Pso skin. The topographic analysis revealed ILCs to reside near the epidermis and in close proximity to T lymphocytes. The latter showed, as previously described, a Th2/Th22-signature in AD and a Th1/Th17 pattern in Pso. Phenotypic and topographic ILC mapping in situ should help us to understand the cross-talk of these cells to their symbionts and, thus, their contribution to skin biology under physiologic and pathologic conditions. 450 The beta2 integrin CD11b regulates the activity of inflammatory monocytes in the regulation of allergic contact dermatitis JT Avery, H Li, DC Bullard and H Xu University of Alabama at Birmingham, Birmingham, AL Allergic contact dermatitis (ACD) is a T lymphocyte-mediated delayed type hypersensitivity immune response in the skin. While most studies have concentrated on mechanisms for the induction of ACD, less attention has been paid to mechanisms for the resolution of inflam- mation in the skin. The current study examines a role of CD11b in the pro-resolving activity of inflammatory monocytes in ACD. CD11b is a member of beta2 integrins. CD11b+ myeloid cells are commonly observed in the inflamed skin of ACD patients and animal models. A common perception is that CD11b is required for the adhesion and migration of myeloid cells. Our data show that ACD is enhanced in CD11b deficient mice compared to wild type animals (P<0.01). Immunohistochemical analysis shows that the number of granulocytes (Gr- 1+) in the allergen challenged skin is higher in CD11b deficient than in wild type mice. In contrast, CD11b deficiency reduces inflammatory monocytes (Ly6C+/Ly6G-) (P<0.01). Further studies show that the inflammatory monocytes sorted from the allergen challenged skin of CD11b deficient mice produce lower levels of IL-10 and TGF-beta than those sorted from wild type mice (P<0.05). IL-10 and TGF-beat are immunosuppressive cytokines and have important roles in monocyte mediated resolution of inflammation. The result suggests that CD11b regulates the pro-resolving activity of inflammatory monocytes in ACD. In sup- porting this, transfer of wild type myeloid cells to CD11b deficient mice prior to challenge inhibits the elicitation of ACD (P<0.05) and reduces neutrophil infiltrations whereas it in- creases inflammatory monocytes in the allergen challenged skin of the CD11b deficient mice (P<0.01). Collectively, the findings reveal a previously unrecognized role of CD11b in the function of inflammatory monocytes in the regulation of ACD. It suggests that targeting CD11b signals may be a new strategy for treatment of the disease. 451 A novel mechanism for long-term protection against CA-MRSA skin reinfection that is not dependent upon antibody responses or CD4+ T cells C Page, DB Lee, B Pinsker, Y Wang, H Liu, R Ortines, J Shahbazian, A Ashbaugh and LS Miller Dermatology, Johns Hopkins University, Baltimore, MD The immune responses that protect against community-acquired methicillin-resistant S. aureus (CA-MRSA) skin reinfections are unclear. We developed a CA-MRSA skin reinfection model in mice (USA300 LAC::lux; 3x10 7 CFU, intradermally) involving a primary infection (1  ) in the lower back (which cleared by 14 days) followed by a secondary infection (2  ) in the upper back on day 28. 1  IL-1b-/- mice developed larger lesions, increased bacterial burden and impaired neutrophil recruitment than wt mice whereas 2  IL-1b-/- mice were completely protected and had no immune impairment. This protection was only seen in the absence of IL-1b since the responses in 1  and 2  wt mice were virtually identical. In addition, the protection was long-lasting since it was still observed in 2  IL-1b-/- mice if we waited 8 weeks between 1  and 2  infections. Interestingly, the protection is not due to antibodies or CD4+ T cells as adoptive transfer of serum containing high levels of CA-MRSA specific antibodies from previously infected 1  IL-1b-/- to naı ¨ve IL-1b-/- did not restore the immune impairment and 2  IL-1b-/- mice were still protected in the absence of CD4+ T cells during 1  and/or 2  infection (GK1.5 mAb treatment). However, 2  IL-1b-/- mice were no longer protected if the egress of lymphocytes from lymph nodes was blocked in 2  IL-1b-/- mice (FTY720 treatment) and the immune impairment in naı ¨ve IL-1b-/- mice could be rescued by adoptive transfer of total lymph nodes cells from previously infected 1  IL-1b-/- mice. Taken together, in the absence of IL-1b, a durable protective immune response against a CA-MRSA infection was generated in lymph nodes that was unexpectedly not due to traditional adaptive immune responses such as antibodies or CD4 + T cells. This response could represent a new immune mechanism to target for future immunotherapies and vaccines against CA-MRSA skin infections. 452 Short chain fatty acids induce regulatory T cells by switching antigen-presenting cells from a stimulatory to a regulatory phenotype T Schwarz, A Bruhs and A Schwarz Department Dermatology, University Kiel, Kiel, Germany It was observed that commensal microbe-derived short chain fatty acids (SCFA) like sodium butyrate induce regulatory T cells (Treg) not only in the colon but in also in the skin. Thus, SCFA produced by commensal skin bacteria under microaerophilic[TS1] conditions may regulate immune responses in the skin via activation or induction of resident cutaneous T cells. Accordingly, application of sodium butyrate inhibited the induction of contact hyper- sensitivity (CHS). Since injection of sodium butyrate-treated and hapten-coupled bone marrow derived dendritic cells (BMDC) rendered recipient mice unresponsive to sensitiza- tion, we postulated that dendritic cells might play an important role in SCFA-mediated in- duction of Treg. Injection of lymph node cells and splenocytes obtained from mice which were injected with butyrate-treated BMDC suppressed the induction of CHS in the recipients, indicating that butyrate-treated BMDC induce Treg. To clarify whether these Treg express the Treg specific transcription factor Foxp3, we utilized DEREG mice (DEpletion of REGulatory T cells) in which Foxp3-positive cells can be depleted by the injection of diphtheria toxin (DT). Lymph node cells obtained from DEREG donors injected with butyrate-treated BMDC significantly suppressed CHS in the recipients. In contrast, sensitization was not suppressed upon transfer of cells obtained from DEREG mice which were treated with DT upon injection of butyrate -treated BMDC. This indicates that Treg induced by butyrate-treated BMDC ex- press Foxp3. FACS analyses revealed down-regulation of the expression of MHC class II and of the costimulatory molecule B7-2 in butyrate-treated BMDC. In addition, butyrate induced the secretion of the immunosuppressive cytokine interleukin-10 by BMDC. Together these data imply that butyrate switches dendritic cells from a stimulatory into a regulatory phenotype which finally induces Treg. 453 Differential regulation of host cutaneous gene expression by skin microbiota J Meisel, G Sfyroera, J Horwinski, J Bugayev, A Tyldsley, B Hodkinson and EA Grice Dermatology, University of Pennsylvania, Philadelphia, PA It is unclear how the skin maintains homeostasis with the colonizing microbiota, and how the host distinguishes commensal or symbiotic microbes from pathogens. We hypothesize that the cutaneous defense response, and in particular innate immune pathways, are differentially regulated in a microbiota-specific manner. We colonized germ free (GF) mice with microbial isolates and/or consortia and used RNA sequencing to identify differentially expressed genes and pathways in the skin. When comparing skin from GF mice to conventionally raised C57BL/6J mice (CONV), 1,017 genes were differentially expressed, and Gene Ontology terms that were significantly enriched included innate immunity genes involved in the complement pathway (i.e. C3) and genes encoding chemokines (i.e. Ccl5) and pattern recognition re- ceptors (i.e. Tlr genes) were up regulated in CONV skin (FDR corrected p-value < 0.05). Keratinocytes provide the first cellular contact with microbiota and are a source of innate immune mediators; thus to determine the cellular contributions to gene expression we interrogated gene expression in CD45- epidermal cells. We are also establishing pathogen- (i.e. Staphylococcus aureus) and commensal- (i.e. S. epidermidis) specific signatures of gene expression which suggest that different microbial consortia elicit distinct host responses. These studies provide novel insights into the regulation of host gene expression and defense responses by the skin microbiota. 454 Washing hands with antibacterial soap may not reduce the risk of contamination VT Gonc ¸alves 2 , DD Rodriguez 1 and LM Vasquez-Pinto 1 1 Natura Innovation and Technol- ogy, Cajamar, Brazil and 2 Biocenter Microbiologica, Sa˜o Paulo, Brazil Skin microbiome has an important role as host guardian, contributing to prevent infections from several microorganisms. Antiseptic soaps are generally made with non-selective anti- bacterial ingredients such as triclosan or triclocarban. Besides killing the skin microbiome, these substances also reach the sewer system and the environment where it can be metab- olized in dangerous substances such as dioxins. Recent studies have questioned the efficacy of antiseptic soaps in preventing infection, especially those caused by virus like flu and common cold. In this study, we compared two hand soap containing different antibacterial ingredients and evaluated the colonization profile of hand microbiome. Our results showed that both the regular soap and the antibacterial one equally remove the bacteria and fungi from skin. However, after 30 minutes, the hands washed with antibacterial soap presented a 9-fold increase in the number of bacteria and a 100-fold increase of funghi when compared with the hands washed with regular soap. These results suggest that washing the hands with antibacterial soap opens an opportunity window for pathogens colonization rather than protect the skin from diseases. S80 Journal of Investigative Dermatology (2016), Volume 136 ABSTRACTS | Innate Immunity, Inflammation & Microbiology