Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types—normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line—upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor α in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor κB (NF-κB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-κB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT).

TGFb is essential the epidermal retention resident memory T cells and Langerhans cells, but the source of TGFb that is important for these functions is unknown. Since the majority of cells in epidermis are keratinocytes (KC), we hypothesized that TGFb from KC might be important for skin homeostasis and the maintenance of skin immune cells. To examine this, we generated K14CreER T2 TGFb fl YFP mice which enables tamoxifen inducible specifically ablation of TGFb in KC. Systemic application of tamoxifen was lethal likely due to esophageal inflammation. Topical application of 4-hydroxytamoxifen (4-OHT) once a day for 2 days was well tolerated. We confirmed the deletion of TGFb in bulge, isthumus and interfollicular keratinocytes based on YFP expression and TGFb mRNA 28 and 84 days post 4-OHT treatment. Mice with KC specific deletion of TGFb developed acanthosis without observable skin inflammation, implicating a TGFb autocrine/paracrine suppression of KC proliferation. We also found that ablation of TGFb in KC has no effect on the numbers of epidermal-resident Langerhans cell, ab T cells and Innate lymphoid. A modest decrease in DETC was observed. In dermis, we did not find any alteration in numbers of DC subsets, ab T cells or dermal gd T cells. Thus, under homeostatic conditions deletion of TGFb in KC enhances epidermal acanthosis but has limited effect on cutaneous residence of most immune cells. 983Pluripotent stem cell model of Nakajo-Nishimura syndrome untangles proinflammatory pathways mediated by oxidative stress Nakajo-Nishimura syndrome (NNS) is an immunoproteasome-associated autoinflammatory disorder caused by a mutation of the PSMB8 gene. Although dysfunction of the immunoproteasome causes various cellular stresses attributed to the overproduction of inflammatory cytokines and chemokines in NNS, the underlying mechanisms of the autoinflammation are still largely unknown. To investigate and understand the mechanisms and signal pathways in NNS, we established a panel of isogenic pluripotent stem cell (PSC) lines with PSMB8 mutation. Activity of the immunoproteasome in PSMB8-mutant PSC-derived myeloid cell lines (MT-MLs) was reduced even without stimulation compared to non-mutant-MLs. Additionally, MT-MLs showed an overproduction of inflammatory cytokines and chemokines, with elevated reactive oxygen species (ROS) and phosphorylated p38 MAPK levels. Treatment with p38 MAPK inhibitor and anti-oxidants decreased the abnormal production of cytokines and chemokines. The current PSC model revealed a specific ROS-mediated inflammatory pathway, providing a platform for the discovery of alternative therapeutic options for NNS and related immunoproteasome disorders. 984Superoxide dismutase 3 controls Th2 cytokine-mediated allergic inflammation through inhibition of periostin S Sah, C Nguyen and T Kim The Catholic University of Korea, Seoul, Republic of Korea Superoxide dismutase 3 (SOD3), an antioxidant enzyme that plays an important role in regulation of inflammation. However, the role of SOD3 and the mechanism(s) b...