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474 Deciphering immune mechanisms of calcipotriol-induced skin inflammation
Inflammation, Immunity and Infection | ABSTRACTS 472
473
Inducing resolution of inflammation to improve skin defence against external aggressions G Chene, V Baillif, E Van Goethem and M Dubourdeau Ambiotis, Toulouse, France Many skin disorders are linked to inflammation, rending its control a key to treat redness, swelling and other many types of skin discomfort. However, inflammation is also a natural process for host defence. Controlling inflammation has then to be very precise to decrease clinical signs without paralyzing the defence capacity of the skin. It is now well described that inflammation is controlled by specialized pro-resolving mediators (SPMs). SPMs represent the stopping circuits for inflammation. Those mediators are actively involved in return to homeostasis and if they are not produced, the good ending of the inflammatory process will not occur. Improving the production of resolution molecules has numerous beneficial properties in the skin: SPM induce the healing and avoid fibrosis, they also play a positive role in tissue regeneration or in soothing in patients with eczema. They protect before inflammation, as they are naturally present in tissues. Among SPMs, we have studied lipoxin A4 (LxA4) production under inflammatory conditions in a skin-simplified model. We have also further evaluated if elevated amount of LxA4 was able to boost skin defence. In these experiments, we initially showed that a model of simplified skin was able to produce LxA4 during inflammation. Stimulated human immune inflammatory cells with LxA4 were able to engulf more Propionibacterium acnes than unstimulated cells. We have shown that stimulated monocytes with LxA4 were able to produce more anion superoxide than unstimulated cells. Oxygen species are used by cells to destroy engulfed bacteria. Those preliminary results suggest that during inflammation, the skin is able to produce SPMs to control inflammation. Those SPMs allow the defence of the skin by inducing the phagocytosis of P. acnes and certainly its killing by improving the production of oxygen species. Those results underline that a compound that would increase the production of SPMs in the skin would lead to a better control of inflammation, an improvement of cells to destroy the causative agent of inflammation, leading then to a come back to cutaneous homeostasis.
A comparative study of bullous pemphigoid-180 and 230 antibodies in serum and blister fluid samples K Drenovska, M Shahid and S Vassileva Department of Dermatology and Venereology, Medical University, Sofia, Bulgaria The diagnosis of bullous pemphigoid (BP) relies on the detection of tissue bound or circulating in patients’ sera IgG anti-basement membrane (BMZ) autoantibodies. Despite the reported comparable titers of anti-BMZ antibodies in both serum and blister fluid of BP patients using indirect immunofluorescence on salt-split skin substrate, only scarce data exist on the detection of antibodies to the BP antigens, BP180 and BP230, using the more sensitive and specific ELISA assays. In the present study, BP180NC16A and BP230 ELISA kits were used to compare the levels of the corresponding autoantibodies in pairs serum-blister fluid collected simultaneously in 38 newly diagnosed untreated BP patients. Our aim was to investigate whether blister fluid can be an alternative to sera for detecting anti-BP180 and anti-BP230 in the routine diagnosis of BP. Positive results for anti-BP180 antibodies (cut-off 20 RU/ml) were detected in 32 and 30 sera and blister fluid samples, respectively (n ¼ 38, Cohen’s k ¼ 0.826) which represents a very good correlation of the results. Similarly, anti-BP230 antibodies were positive (cut-off 20 RU/ml) in the serum and blister fluid of 13 and 12 patients, respectively (n ¼ 38, Cohen’s k ¼ 0.821). In addition, in 17 out of 38 patients (44.7%) there was full agreement between the ELISA BP180 and BP230 results in the serum-blister fluid pairs, all positive in 12 or all negative in 5 patients, respectively. In the remaining 21 patients the blister fluid tested positive only for anti-BP180 known to be of major pathogenic importance in BP. Only 3 out of 38 blister fluid samples showed negative results for both autoantibodies, whereas the corresponding serum samples were positive either for BP180 or BP230 antibodies. Our results demonstrate a relatively high agreement between the anti-BP180 and antiBP230 values in the pairs serum-blister fluid, the latter being less invasive and easier to obtain in the routine diagnosis of BP, especially in elderly patients or children.
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Deciphering immune mechanisms of calcipotriol-induced skin inflammation V Patra3,1, S Renaudineau1, V Lenief1, S Vuillier1, R Terreux2, P Wolf3, J Nicolas1 and M Vocanson1 1 INSERM U1111-CIRI, Lyon, France, 2 IBCP, Lyon, France and 3 Medical University of Gratz, Gratz, Austria On complexing with nuclear vitamin D receptors (VDRs), the low calcemic vitamin D analog, Calcipotriol (MC903), regulates the transcriptional activity of numerous immune cells. Calcipotriol is thus used for years as an anti-inflammatory agent in the treatment of plaque psoriasis. However, several studies reported that, despite its anti-inflammatory properties, calcipotriol can be pro-inflammatory and induces atopic or contact dermatitis-like symptoms in patients or in mice upon repeated applications. The underlying mechanisms of this phenomenon remain largely unknown. Here, we have developed a mouse model to better characterize the nature and the immune mechanisms of the MC903-induced skin inflammation. When naı¨ve mice received graded doses of topical MC903, they developed a unique, dose-dependent, two-wave, skin inflammatory response. The inflammation was mediated successively by innate (from D3 to D6) and adaptive immunity (from D7 to D12). Combining cellular (using FACS, mAb-depletion and adoptive transfer experiments) and molecular (qRT-PCR) approaches, we demonstrated that both CD4+ and CD8+ T cells, producing IL-4, IL-17A and IFNg, mediate the late phase of MC903-induced inflammation. Interestingly, in vitro restimulation assays using cells collected from draining lymph nodes at D6 post application, showed that the late phase of MC903-induced inflammation is antigendependant and not the mere result of MC903 stimulation of VDRs. Such observations were next confirmed by the potent and local CD8+ (bot not CD4+) T cell-mediated memory reaction triggered to the site of previously affected skin, one day after MC903 re-test. We are currently exploring the nature of the antigens responsible for MC903-induced inflammation, but preliminary in silico investigations suggested that two calcipotriol metabolites, but not calcipotriol itself, are endowed with hapten properties. This study should open important avenues to better understand the MC903 adverse effects observed in humans treated by topical calcipotriol.
Abnormal basal membran protein expression in non-lesional psoriatic skin B Guba´n1, R Kui1, I Ne´meth1, A Bebes1, M Sze´ll3,2, L Keme´ny1,2 and Z Bata-Cso¨rgo¨1,2 1 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 2 MTA-SZTE Dermatological Research Group, Szeged, Hungary and 3 Department of Medical Genetics, University of Szeged, Szeged, Hungary Most studies on psoriasis focus on lesional skin, however healthy looking non-lesional skin already carries alterations that could be a basis of the manifestation of the disease. These include extracellular matrix defects that have been observed in psoriatic non-lesional skin, such as the disruption of laminin networks, which may lead to impaired keratinocyte adhesion to the basement membrane. This process is also associated with aberrant expression of the a5b1 integrin complex and the extradomain-A-containing fibronectin (EDA+FN) around basal keratinocytes. We set out to examine further the basement membrane alterations by focusing on type VII collagen and laminin 332 (LAMA3) proteins in non-lesional skin of psoriatic patients. We compared protein expression from samples derived near and far from lesions using immunfluorescent staining. Type VII collagen and LAMA3 are uniformly expressed in psoriatic lesional skin, as well as in healthy skin, but not in non-lesional skin. We found that the type VII collagen and LAMA3 expression in non-lesional skin corresponded with the PASI score of the patients and the distance of the sample from the lesion. High PASI scores were typically coupled with intensive type VII collagen staining in lesional and in nonlesional skin. In contrast, patients with low PASI score showed nearly no detectable type VII collagen protein in the NL skin. Also, NL samples taken further from lesions expressed less type VII collagen and LAMA3. In psoriatic NL skin changes in basal membrane proteins create a damaged, unstable dermal-epidermal junction zone that could manifest in a chronic inflammatory phenotype (lesional skin).
WITHDRAWN
Biofilms-producing Propionibacterium acnes attend at both inflamed and noninflamed acne lesions E Di Domenico1, I Cavallo1, L Toma2, G Prignano1, M Gallo1, F Pimpinelli1, V Bordignon1, C Cavallotti3, B Capitanio3 and F Ensoli1 1 Clinical Pathology and Microbiology, San Gallicano, Rome, Italy, 2 Infectious Disease Consultant, Regina Elena National Cancer Institute, Rome, Italy and 3 Department of Dermatology, San Gallicano, Rome, Italy Acne vulgaris is a common inflammatory disorder of the sebaceous follicles, affecting more than 80% of young adolescents that can also persist into adulthood. However, the mechanisms by which inflammatory lesions arise are poorly understood. Although, considerable evidences suggest that Propionibacterium acnes participates in the pathogenesis of acne vulgaris, the possible role of biofilm formation by P. acnes remains, as yet, to be elucidated. In the present study, we investigated the putative role of biofilm production by P. acnes in comedogenesis and in the progression towards inflamed acne lesions. The biofilm-forming ability of P. acnes strains was tested by a specific in vitro method, based on the clinical biofilm ring test (c-BRT). For each patient, we collected samples from healthy skin and from different type of acne lesions including microcomedon, comedon, papule and pustule in order to assess the presence of P. acnes. Microbial identification was performed using the VITEKÒII automated system and by sequencing 16S rRNA gene. Our results revealed that P. acnes strains were present in all the stages of acne progression, namely from the non-inflamed microcomedon/comedon condition to the inflammatory phase characterized by papule/ pustule. Nevertheless, a significantly higher number of P. acnes were isolated at both inflamed (papule and pustule) and noninflamed (comedon) acne lesions compared with microcomedon and healthy skin. Notably, all the strains analyzed were able to produce biofilm. This study revealed that biofilm production by P. acnes is likely to participate in the development of both noninflamed and inflamed acne lesions by promoting bacterial persistence and thus contributing to the worsening and chronicity of the inflammatory disease.
www.jidonline.org S273
473
Inducing resolution of inflammation to improve skin defence against external aggressions G Chene, V Baillif, E Van Goethem and M Dubourdeau Ambiotis, Toulouse, France Many skin disorders are linked to inflammation, rending its control a key to treat redness, swelling and other many types of skin discomfort. However, inflammation is also a natural process for host defence. Controlling inflammation has then to be very precise to decrease clinical signs without paralyzing the defence capacity of the skin. It is now well described that inflammation is controlled by specialized pro-resolving mediators (SPMs). SPMs represent the stopping circuits for inflammation. Those mediators are actively involved in return to homeostasis and if they are not produced, the good ending of the inflammatory process will not occur. Improving the production of resolution molecules has numerous beneficial properties in the skin: SPM induce the healing and avoid fibrosis, they also play a positive role in tissue regeneration or in soothing in patients with eczema. They protect before inflammation, as they are naturally present in tissues. Among SPMs, we have studied lipoxin A4 (LxA4) production under inflammatory conditions in a skin-simplified model. We have also further evaluated if elevated amount of LxA4 was able to boost skin defence. In these experiments, we initially showed that a model of simplified skin was able to produce LxA4 during inflammation. Stimulated human immune inflammatory cells with LxA4 were able to engulf more Propionibacterium acnes than unstimulated cells. We have shown that stimulated monocytes with LxA4 were able to produce more anion superoxide than unstimulated cells. Oxygen species are used by cells to destroy engulfed bacteria. Those preliminary results suggest that during inflammation, the skin is able to produce SPMs to control inflammation. Those SPMs allow the defence of the skin by inducing the phagocytosis of P. acnes and certainly its killing by improving the production of oxygen species. Those results underline that a compound that would increase the production of SPMs in the skin would lead to a better control of inflammation, an improvement of cells to destroy the causative agent of inflammation, leading then to a come back to cutaneous homeostasis.
A comparative study of bullous pemphigoid-180 and 230 antibodies in serum and blister fluid samples K Drenovska, M Shahid and S Vassileva Department of Dermatology and Venereology, Medical University, Sofia, Bulgaria The diagnosis of bullous pemphigoid (BP) relies on the detection of tissue bound or circulating in patients’ sera IgG anti-basement membrane (BMZ) autoantibodies. Despite the reported comparable titers of anti-BMZ antibodies in both serum and blister fluid of BP patients using indirect immunofluorescence on salt-split skin substrate, only scarce data exist on the detection of antibodies to the BP antigens, BP180 and BP230, using the more sensitive and specific ELISA assays. In the present study, BP180NC16A and BP230 ELISA kits were used to compare the levels of the corresponding autoantibodies in pairs serum-blister fluid collected simultaneously in 38 newly diagnosed untreated BP patients. Our aim was to investigate whether blister fluid can be an alternative to sera for detecting anti-BP180 and anti-BP230 in the routine diagnosis of BP. Positive results for anti-BP180 antibodies (cut-off 20 RU/ml) were detected in 32 and 30 sera and blister fluid samples, respectively (n ¼ 38, Cohen’s k ¼ 0.826) which represents a very good correlation of the results. Similarly, anti-BP230 antibodies were positive (cut-off 20 RU/ml) in the serum and blister fluid of 13 and 12 patients, respectively (n ¼ 38, Cohen’s k ¼ 0.821). In addition, in 17 out of 38 patients (44.7%) there was full agreement between the ELISA BP180 and BP230 results in the serum-blister fluid pairs, all positive in 12 or all negative in 5 patients, respectively. In the remaining 21 patients the blister fluid tested positive only for anti-BP180 known to be of major pathogenic importance in BP. Only 3 out of 38 blister fluid samples showed negative results for both autoantibodies, whereas the corresponding serum samples were positive either for BP180 or BP230 antibodies. Our results demonstrate a relatively high agreement between the anti-BP180 and antiBP230 values in the pairs serum-blister fluid, the latter being less invasive and easier to obtain in the routine diagnosis of BP, especially in elderly patients or children.
474
475
476
477
Deciphering immune mechanisms of calcipotriol-induced skin inflammation V Patra3,1, S Renaudineau1, V Lenief1, S Vuillier1, R Terreux2, P Wolf3, J Nicolas1 and M Vocanson1 1 INSERM U1111-CIRI, Lyon, France, 2 IBCP, Lyon, France and 3 Medical University of Gratz, Gratz, Austria On complexing with nuclear vitamin D receptors (VDRs), the low calcemic vitamin D analog, Calcipotriol (MC903), regulates the transcriptional activity of numerous immune cells. Calcipotriol is thus used for years as an anti-inflammatory agent in the treatment of plaque psoriasis. However, several studies reported that, despite its anti-inflammatory properties, calcipotriol can be pro-inflammatory and induces atopic or contact dermatitis-like symptoms in patients or in mice upon repeated applications. The underlying mechanisms of this phenomenon remain largely unknown. Here, we have developed a mouse model to better characterize the nature and the immune mechanisms of the MC903-induced skin inflammation. When naı¨ve mice received graded doses of topical MC903, they developed a unique, dose-dependent, two-wave, skin inflammatory response. The inflammation was mediated successively by innate (from D3 to D6) and adaptive immunity (from D7 to D12). Combining cellular (using FACS, mAb-depletion and adoptive transfer experiments) and molecular (qRT-PCR) approaches, we demonstrated that both CD4+ and CD8+ T cells, producing IL-4, IL-17A and IFNg, mediate the late phase of MC903-induced inflammation. Interestingly, in vitro restimulation assays using cells collected from draining lymph nodes at D6 post application, showed that the late phase of MC903-induced inflammation is antigendependant and not the mere result of MC903 stimulation of VDRs. Such observations were next confirmed by the potent and local CD8+ (bot not CD4+) T cell-mediated memory reaction triggered to the site of previously affected skin, one day after MC903 re-test. We are currently exploring the nature of the antigens responsible for MC903-induced inflammation, but preliminary in silico investigations suggested that two calcipotriol metabolites, but not calcipotriol itself, are endowed with hapten properties. This study should open important avenues to better understand the MC903 adverse effects observed in humans treated by topical calcipotriol.
Abnormal basal membran protein expression in non-lesional psoriatic skin B Guba´n1, R Kui1, I Ne´meth1, A Bebes1, M Sze´ll3,2, L Keme´ny1,2 and Z Bata-Cso¨rgo¨1,2 1 Department of Dermatology and Allergology, University of Szeged, Szeged, Hungary, 2 MTA-SZTE Dermatological Research Group, Szeged, Hungary and 3 Department of Medical Genetics, University of Szeged, Szeged, Hungary Most studies on psoriasis focus on lesional skin, however healthy looking non-lesional skin already carries alterations that could be a basis of the manifestation of the disease. These include extracellular matrix defects that have been observed in psoriatic non-lesional skin, such as the disruption of laminin networks, which may lead to impaired keratinocyte adhesion to the basement membrane. This process is also associated with aberrant expression of the a5b1 integrin complex and the extradomain-A-containing fibronectin (EDA+FN) around basal keratinocytes. We set out to examine further the basement membrane alterations by focusing on type VII collagen and laminin 332 (LAMA3) proteins in non-lesional skin of psoriatic patients. We compared protein expression from samples derived near and far from lesions using immunfluorescent staining. Type VII collagen and LAMA3 are uniformly expressed in psoriatic lesional skin, as well as in healthy skin, but not in non-lesional skin. We found that the type VII collagen and LAMA3 expression in non-lesional skin corresponded with the PASI score of the patients and the distance of the sample from the lesion. High PASI scores were typically coupled with intensive type VII collagen staining in lesional and in nonlesional skin. In contrast, patients with low PASI score showed nearly no detectable type VII collagen protein in the NL skin. Also, NL samples taken further from lesions expressed less type VII collagen and LAMA3. In psoriatic NL skin changes in basal membrane proteins create a damaged, unstable dermal-epidermal junction zone that could manifest in a chronic inflammatory phenotype (lesional skin).
WITHDRAWN
Biofilms-producing Propionibacterium acnes attend at both inflamed and noninflamed acne lesions E Di Domenico1, I Cavallo1, L Toma2, G Prignano1, M Gallo1, F Pimpinelli1, V Bordignon1, C Cavallotti3, B Capitanio3 and F Ensoli1 1 Clinical Pathology and Microbiology, San Gallicano, Rome, Italy, 2 Infectious Disease Consultant, Regina Elena National Cancer Institute, Rome, Italy and 3 Department of Dermatology, San Gallicano, Rome, Italy Acne vulgaris is a common inflammatory disorder of the sebaceous follicles, affecting more than 80% of young adolescents that can also persist into adulthood. However, the mechanisms by which inflammatory lesions arise are poorly understood. Although, considerable evidences suggest that Propionibacterium acnes participates in the pathogenesis of acne vulgaris, the possible role of biofilm formation by P. acnes remains, as yet, to be elucidated. In the present study, we investigated the putative role of biofilm production by P. acnes in comedogenesis and in the progression towards inflamed acne lesions. The biofilm-forming ability of P. acnes strains was tested by a specific in vitro method, based on the clinical biofilm ring test (c-BRT). For each patient, we collected samples from healthy skin and from different type of acne lesions including microcomedon, comedon, papule and pustule in order to assess the presence of P. acnes. Microbial identification was performed using the VITEKÒII automated system and by sequencing 16S rRNA gene. Our results revealed that P. acnes strains were present in all the stages of acne progression, namely from the non-inflamed microcomedon/comedon condition to the inflammatory phase characterized by papule/ pustule. Nevertheless, a significantly higher number of P. acnes were isolated at both inflamed (papule and pustule) and noninflamed (comedon) acne lesions compared with microcomedon and healthy skin. Notably, all the strains analyzed were able to produce biofilm. This study revealed that biofilm production by P. acnes is likely to participate in the development of both noninflamed and inflamed acne lesions by promoting bacterial persistence and thus contributing to the worsening and chronicity of the inflammatory disease.
www.jidonline.org S273