406 Abnormal collagen VII expression in non-lesional psoriatic skin

S | Inflammation, Immunity and Infection 406 Abnormal collagen VII expression in non-lesional psoriatic skin B Guban, R Kui, R Bozo, G Groma, I Nemeth, A Bebes, M Szell, L Kemeny and Z Bata-Csorgo 1 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 2 MTA-SZTE Dermatological Research Group, University of Szeged, Szeged, Hungary and 3 Department of Medical Genetics, University of Szeged, Szeged, Hungary Alteration of the extracellular matrix (ECM) is a likely participant in the pathomechanism of psoriasis. ECM defects have been observed in non-lesional (NL) psoriatic skin, including disruption of the laminin, possibly leading to impaired keratinocyte adhesion to the basement membrane. This is associated with aberrant expression of a5b1 integrin in, and extradomainA containing fibronectin (EDAFN) around basal keratinocytes. In our previous work, we found that in NL psoriatic skin, the expression of KGFR and KGF were also increased compared to healthy controls. Because fibronectin can specifically bind to type VII collagen, we aimed to examine the putative connection between EDAFN and collagen VII expression in non-lesional and lesional psoriatic skin. We found that collagen VII expression was decreased to a variable degree in psoriatic NL skin, while in lesional skin its expression was comparable to healthy skin. The antibody clone LH7.2 (Novocastra) was used for visualizing collagen VII. Samples from 6 patients were stained. Collagen VII expression in NL skin seems to correlate with PASI score. High PASI scores (e. g. 19.6) were typically coupled with intensive collagen VII staining in the lesional skin and a weak collagen VII staining in NL skin. In contrast, patients with lower PASI scores (e. g. 9.8) showed no collagen VII expression in NL skin. Collagen VII expression was always present in lesional skin. Also, the further the biopsy was taken from the lesions, the less collagen VII expression was detected in NL skin. This is an unexpected finding and has to be confirmed in larger NL samples. We speculate that increased EDAFN production may be a compensation mechanism for the reduced collagen VII expression in non-lesional skin. S230 Journal of Investigative Dermatology (2016), Volume 136 407 CD34 skin resident precursor cells may contribute to maintain tissue resident macrophages in human skin and are stimulated by substance P and IFNg J Gherardini, Y Uchida, J Cheret, M Alam, M Bertolini and R Paus 1 Dermatoly, Univ Muenster, Muenster, Germany, 2 Monasterium Laboratory, Muenster, Germany and 3 Univ Manchester, Manchester, United Kingdom Besides monocyte-derived macrophages (MACs), self-renewing tissue-resident macrophages (trMACs) are now appreciated to maintain the “physiological” number of intra-cutaneous MACs in murine skin. However, it remains unknown whether such self-renewing trMACs also exist in human skin and how they may be stimulated. We have investigated this possibility in organ-cultured full-thickness human skin, using the pro-inflammatory neuropeptide, substance P (SP), to imitate a “neurogenic skin inflammation” signaling milieu. In the absence of perfused vasculature or bone marrow or intraluminal CD14 monocytes, SP significantly increased the number of CD68 MACs in human dermis ex vivo. Moreover, SP did not suppress MAC apoptosis, as shown by CD68/TUNEL double-immunostaining, and almost no proliferative CD68 cells were detectable in test or control skin. This not only suggested that new MACs can arise from resident cells within human skin, but also that proliferation of trMACs is very unlikely to generate the new CD68 MAC. Interestingly CD34 cells, MAC precursor cells (but not c-Kit cells), were often found to be in direct cell-cell-contact with CD68 cells in human skin, especially in SP-treated samples. Therefore, we are currently probing the hypothesis that human skin trMACs arise from tissue-resident CD34 cells stimulating them to differentiate with M-CSF and IL-34. Similar phenomena were also seen in cytokine-mediated inflammation, triggered by IFNg in our full thickness skin organ culture, suggesting that resident MAC progenitor cells exist in human skin and are able to respond to different inflammatory [and ECM] stimuli to satisfy the demand of MACs, notably under conditions of skin stress (such as skin organ culture). 408 Neutrophil mediated proteolysis of IL-36 members has both regulatory and inflammatory consequences T Macleod, R Doble, M Wittmann, M Stacey and D McGonagle University of Leeds, Leeds, United Kingdom The IL-36 cytokines, IL-36a, b, g and the receptor antagonist, are members of the IL-1 superfamily that have a strong link to psoriatic inflammation. High levels of IL-36a and IL-36g are a prominent characteristic in psoriatic plaques, whilst mutations in the receptor antagonist are highly linked to pustular psoriasis subtypes. As observed with other IL-1 superfamily proteins, the IL-36 members require precise N-terminal cleavage for full biological activity. Using different blood leukocyte and skin resident preparations, and recombinant proteins, we have identified several neutrophil proteases that cleave all members of the IL-36 family generating both active and inactive truncations. Incubation of IL-36 agonists with neutrophil proteases generated biologically inactive truncations (IL-36a I7, IL-36g Y16 and Q17) when tested on primary fibroblasts and keratinocytes, whilst incubation of IL-36Ra generated a biologically active antagonist IL-36Ra V2. However, prolonged incubation of IL-36Ra resulted in further truncation producing IL-36Ra S4, deactivating the antagonist. IL-8, CCL20 and hBD2 were used as outcome measures for biological activity. These findings suggest neutrophils are an important element when considering the dynamics of IL-36 mediated inflammation. Whilst traditionally thought to act in an inflammatory capacity, these findings indicate a regulatory role by generating inactive agonist truncations and activating IL-36Ra. Clearly, neutrophils are capable of influencing IL-36 mediated inflammation to produce both proand anti-inflammatory outcomes, yet whether one outcome is favoured over the other and what factors might sway the balance is yet to be clarified. Given their abundance in psoriatic lesions, and the importance of IL-36 in psoriatic inflammation, a clear understanding of how neutrophils influence IL-36 mediated inflammation is of great importance. 409 Immunological properties of adipose tissue derived mesenchymal stem cells B Guban, J Varga, A Facsko, Z Bata-Csorgo, L Kemeny and Z Vereb 1 Department of Dermatology and AllergologyAllergology, University of Szeged, Szeged, Hungary and 2 2Stem Cells and Eye Research Laboratory, Department of Ophthalmology, University of Szeged, Szeged, Hungary Mesenchymal stem cells (MSC) are the stromal cells of bone marrow, but they can also be found in other tissues including fat as well. Our goals were to isolate and cultivate adipose tissue derived MSCs (ADMSC) and study their role in inflammation. ADMSCs were isolated by the manual enzymatic method from liposuctions. The expression of surface markers and highend glycosylation products were measured by multicolor FACS. Osteogenic, adipogenic and chondrogenic differentiation potential were tested in vitro. ADMSCs were activated by TLR ligands (LPS, Poly:IC) and pro-inflammatory cytokines (TNFa, IL1b, IFNg) and the secreted cytokines measured by ELISA. Cells isolated from adipose tissue showed fibroblastoid morphology and grew as monolayers in vitro and could be maintained in culture for more than 10 passages. ADMSCs expressed the important MSC markers (CD29, CD44, CD73, CD90 and CD105) with absence of endothelial (CD31, VEGFR2) or hematopoietic cell markers (CD34, CD45, CXCR4) respectively. CD49a/Integrin a1, CD51/Integrin aV, CD146/ MCAM were present and CD18/Integrin b2, CD54/ICAM1 were absent in the surface of the in vitro cultured ADMSCs. Majority of the cells showed ConA, WGA, RCA 120, DBA and PHA-E lectin positivity resulting in a special carbohydrate pattern. ADMSCs were able to differentiate into bone, fat and cartilage tissue respectively. Furthermore ADMSCs secreted increased amount of IL-6 and IL-8 after 12h, 24h treatment of LPS, Poly:IC, TNFa and IL1b compared to untreated controls respectively. CXCL-10 could be detected in the supernatants of ADMSCs just due upon Poly:IC treatment. Adipose tissue is an abundant and accessible source of several types of stem and progenitor cells found within the stromal vascular fraction such as ADMSCs. Based upon our results these cells fulfilled the ISCT criteria and could be useful for regenerative therapeutic applications and in daily surgical practice as well. 410 The role of NK cells in WASp related cancer and eczema: Studies on the pathogenesis and possible novel therapies H Brauner, J Kritikou, C Dahlberg, M Baptista, A Wagner, K Karre, J Orange and L Westerberg 1 Dept of Microbiology, Tumor and Cellbiology, Karolinska Insitutet, Stockholm, Sweden and 2 Center for Human Immunobiology, Baylor College of Medicine,