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Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity
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Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
P10-06[C01-03] Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity Catharina Sagita Moniaga ∗ , Mariko Hara-Chikuma Center for Innovation in Immunoregulative Technology and Therapeutics Graduate School of Medicine Kyoto University, Japan Aquaporins (AQPs, AQP0-12) are membrane water/glycerol channel proteins that participate in various cellular functions in many cell types. AQP9 is expressed in several immune cells including neutrophils; however, its role in cutaneous immune response remains unknown. Here we show the involvement of AQP9 in skin allergic inflammation using AQP9 knockout (AQP9−/−) mice and the contact hypersensitivity (CHS) model. First, AQP9−/− mice showed impairment of CHS responses to 2,4-dinitrofluorobenzene (DNFB) compared with wild-type (WT) mice. The sensitization phase of CHS was examined by evaluating the immune cells accumulated in skin draining lymph nodes (LNs) after DNFB application. Adoptive transfer of AQP9−/− LNs cells into the ears of WT recipients resulted in impaired CHS response, indicating the defect of sensitization in AQP9−/−. IL-17 production was decreased in AQP9−/− compared with WT LNs cells, though the T cell proliferation and IFN-gamma production levels were comparable. WT mice showed high neutrophil accumulation in LNs at 18 h after DNFB application, but it was diminished in AQP9−/− mice. In accordance, we found the attenuation of AQP9−/− neutrophil migration to LNs after DNFB application. Furthermore, the neutrophil migration in response to CCR7 ligands was suppressed in AQP9−/− using chemotaxis chambers. Finally, we found that the administration of Gr-1 (neutrophil) antibody during the sensitization phase suppressed IL-17 production from LNs cells as well as CHS response in WT mice, suggesting the implication of neutrophils for IL-17 production during the development of CHS. These data suggest that AQP9-expressing neutrophils play an important role in the sensitization phase of CHS through their migration function. http://dx.doi.org/10.1016/j.jdermsci.2016.08.191 P10-07[C01-04] The possible mechanisms of the recruitment of Tregs in the lesional skin of extramammary Paget’s disease by RANKL/RANK pathways Taku Fujimura ∗ , Yumi Kambayashi, Sadanori Furudate, Masayuki Asano, Aya Kakizaki, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan It is reported that receptor of activated nuclear factor kappa ligand (RANKL) and that the interaction of RANK and RANKL promotes mammary tumorgenesis and bone metastasis of breast cancer (BC). Since extramammary Paget’s disease (EMPD) is a skin adenocarcinoma of apocrine gland origin and highly metastatic to bones, we hypothesized Paget cells might express RANKL. To prove our hypothesis, we first examined the expression of RANKL, RANK, and MMP7, which can proteolytically cleave sRANKL from its membrane-bound form. The immunohistochemical studies demonstrated that most of the Paget cells expressed RANKL and MMP7, but not RANK, while stromal cells with the expression of CD163 and Arginase 1, possibly M2 macrophages, expressed RANK. RANKL and MMP7 expression by Paget cells was confirmed at the
mRNA level. Moreover, EMPD tissue culture revealed that sRANKL was actually released from tumor tissues. To clarify the consequence of the interaction of sRANKL and RANK on M2 macrophages, we induced M2 macrophages expressing RANK in vitro and stimulated them with sRANKL, which demonstrated their significant induction of several cancer-related chemokines including CCLl7, CXCL1, CXCL2, and CXCL8. Based on this observation, we examined CCL17 expression in the lesional skin of EMPD and found immunohistologically that M2 macrophages expressed CCL17 and that the lesional skin showed increased CCL17 mRNA expression. In addition, we also demonstrated that these M2 macrophages were surrounded by regulatory T cells (Tregs), which are well known to express CCR4. These studies suggest that the RANKL/RANK pathway might contribute the recruitment of Tregs and maintain the immunosuppressive tumor microenvironment in EMPD. http://dx.doi.org/10.1016/j.jdermsci.2016.08.192 P10-08[C01-05] Interaction of adhesion molecules of keratinocytes and Langerhans cells in the epidermis in contact hypersensitivity Akiko Nishibu ∗ , Takashi Mochizuki The Department of Dermatology, Kanazawa Medical University, Japan In both induction phase and elicitation phase of contact hypersensitivity (CHS), keratinocytes (KC) and Langerhans cells (LC) play critical roles. It is presumed that LC should migrate within or out of the epidermis in the CHS process. However, details have not been made clear yet. In order to clarify cell kinetics of LC and adhesion molecules of KC in the epidermis, we used the genetically engineered mouse produced by crossing the desmoglein 3-EGFP mouse and the CD11c-EYFP mouse. CHS was induced by hapten application and time-lapse in vivo imaging by confocal microscopy was performed. In terms of results, in the stationary state, LC remained in the same position and a movement of dendrites of LC was slightly observed. An expression of desmoglein of KC, in contact with LC, had decreased or disappeared. In the elicitation phase of CHS, an extension and retraction motion of dendrites of LC was obviously enhanced, and approximately 1% of LC moved with an amoeboid motion in the epidermis. When LC moved in the epidermis, the surrounding KC changed shape flexibly. The expression of desmoglein decreased along with the LC movement and recovered promptly within several minutes after the LC migration. http://dx.doi.org/10.1016/j.jdermsci.2016.08.193
Abstracts / Journal of Dermatological Science 84 (2016) e1–e88
P10-06[C01-03] Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity Catharina Sagita Moniaga ∗ , Mariko Hara-Chikuma Center for Innovation in Immunoregulative Technology and Therapeutics Graduate School of Medicine Kyoto University, Japan Aquaporins (AQPs, AQP0-12) are membrane water/glycerol channel proteins that participate in various cellular functions in many cell types. AQP9 is expressed in several immune cells including neutrophils; however, its role in cutaneous immune response remains unknown. Here we show the involvement of AQP9 in skin allergic inflammation using AQP9 knockout (AQP9−/−) mice and the contact hypersensitivity (CHS) model. First, AQP9−/− mice showed impairment of CHS responses to 2,4-dinitrofluorobenzene (DNFB) compared with wild-type (WT) mice. The sensitization phase of CHS was examined by evaluating the immune cells accumulated in skin draining lymph nodes (LNs) after DNFB application. Adoptive transfer of AQP9−/− LNs cells into the ears of WT recipients resulted in impaired CHS response, indicating the defect of sensitization in AQP9−/−. IL-17 production was decreased in AQP9−/− compared with WT LNs cells, though the T cell proliferation and IFN-gamma production levels were comparable. WT mice showed high neutrophil accumulation in LNs at 18 h after DNFB application, but it was diminished in AQP9−/− mice. In accordance, we found the attenuation of AQP9−/− neutrophil migration to LNs after DNFB application. Furthermore, the neutrophil migration in response to CCR7 ligands was suppressed in AQP9−/− using chemotaxis chambers. Finally, we found that the administration of Gr-1 (neutrophil) antibody during the sensitization phase suppressed IL-17 production from LNs cells as well as CHS response in WT mice, suggesting the implication of neutrophils for IL-17 production during the development of CHS. These data suggest that AQP9-expressing neutrophils play an important role in the sensitization phase of CHS through their migration function. http://dx.doi.org/10.1016/j.jdermsci.2016.08.191 P10-07[C01-04] The possible mechanisms of the recruitment of Tregs in the lesional skin of extramammary Paget’s disease by RANKL/RANK pathways Taku Fujimura ∗ , Yumi Kambayashi, Sadanori Furudate, Masayuki Asano, Aya Kakizaki, Setsuya Aiba Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Japan It is reported that receptor of activated nuclear factor kappa ligand (RANKL) and that the interaction of RANK and RANKL promotes mammary tumorgenesis and bone metastasis of breast cancer (BC). Since extramammary Paget’s disease (EMPD) is a skin adenocarcinoma of apocrine gland origin and highly metastatic to bones, we hypothesized Paget cells might express RANKL. To prove our hypothesis, we first examined the expression of RANKL, RANK, and MMP7, which can proteolytically cleave sRANKL from its membrane-bound form. The immunohistochemical studies demonstrated that most of the Paget cells expressed RANKL and MMP7, but not RANK, while stromal cells with the expression of CD163 and Arginase 1, possibly M2 macrophages, expressed RANK. RANKL and MMP7 expression by Paget cells was confirmed at the
mRNA level. Moreover, EMPD tissue culture revealed that sRANKL was actually released from tumor tissues. To clarify the consequence of the interaction of sRANKL and RANK on M2 macrophages, we induced M2 macrophages expressing RANK in vitro and stimulated them with sRANKL, which demonstrated their significant induction of several cancer-related chemokines including CCLl7, CXCL1, CXCL2, and CXCL8. Based on this observation, we examined CCL17 expression in the lesional skin of EMPD and found immunohistologically that M2 macrophages expressed CCL17 and that the lesional skin showed increased CCL17 mRNA expression. In addition, we also demonstrated that these M2 macrophages were surrounded by regulatory T cells (Tregs), which are well known to express CCR4. These studies suggest that the RANKL/RANK pathway might contribute the recruitment of Tregs and maintain the immunosuppressive tumor microenvironment in EMPD. http://dx.doi.org/10.1016/j.jdermsci.2016.08.192 P10-08[C01-05] Interaction of adhesion molecules of keratinocytes and Langerhans cells in the epidermis in contact hypersensitivity Akiko Nishibu ∗ , Takashi Mochizuki The Department of Dermatology, Kanazawa Medical University, Japan In both induction phase and elicitation phase of contact hypersensitivity (CHS), keratinocytes (KC) and Langerhans cells (LC) play critical roles. It is presumed that LC should migrate within or out of the epidermis in the CHS process. However, details have not been made clear yet. In order to clarify cell kinetics of LC and adhesion molecules of KC in the epidermis, we used the genetically engineered mouse produced by crossing the desmoglein 3-EGFP mouse and the CD11c-EYFP mouse. CHS was induced by hapten application and time-lapse in vivo imaging by confocal microscopy was performed. In terms of results, in the stationary state, LC remained in the same position and a movement of dendrites of LC was slightly observed. An expression of desmoglein of KC, in contact with LC, had decreased or disappeared. In the elicitation phase of CHS, an extension and retraction motion of dendrites of LC was obviously enhanced, and approximately 1% of LC moved with an amoeboid motion in the epidermis. When LC moved in the epidermis, the surrounding KC changed shape flexibly. The expression of desmoglein decreased along with the LC movement and recovered promptly within several minutes after the LC migration. http://dx.doi.org/10.1016/j.jdermsci.2016.08.193