Rui Aoki

Anti-NXP2 autoantibodies in adult patients with idiopathic inflammatory myopathies: possible association with malignancy

Objectives Myositis-specific autoantibodies (MSAs) are useful tools for identifying clinically homogeneous subsets and predicting prognosis of patients with idiopathic inflammatory myopathies (IIM) including polymyositis (PM) and dermatomyositis (DM). Recent studies have shown that anti-NXP2 antibody (Ab) is a major MSA in juvenile dermatomyositis (JDM). In this study the frequencies and clinical associations of anti-NXP2 Ab were evaluated in adult patients with IIM. Methods Clinical data and serum samples were collected from 507 adult Japanese patients with IIM (445 with DM and 62 with PM). Eleven patients with JDM, 108 with systemic lupus erythematosus, 433 with systemic sclerosis and 124 with idiopathic pulmonary fibrosis were assessed as disease controls. Serum was examined for anti-NXP2 Ab by immunoprecipitation and western blotting using polyclonal anti-NXP2 Ab. Results Seven patients (1.6%) with adult DM and one (1.6%) with adult PM were positive for anti-NXP2 Ab. Except for two patients with JDM, none of the disease controls were positive for this autoantibody. Among eight adult patients with IIM, three had internal malignancies within 3 years of diagnosis of IIM. Another patient with DM also had a metastatic cancer at the diagnosis. All of the carcinomas were at an advanced stage (stage IIIb–IV). Conclusions While less common than in juvenile IIM, anti-NXP2 Ab was found in adult IIM. Anti-NXP2 Ab may be associated with adult IIM with malignancy.

Gap Junction-Mediated Intercellular Communication between Dendritic Cells (DCs) Is Required for Effective Activation of DCs

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-γ or TNF-α plus IFN-γ. LPS- plus IFN-γ-induced dye-coupling was mediated by DC-derived TNF-α. In addition, CpG plus IFN-γ induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-α. LPS- plus IFN-γ-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-γ-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.

Osteopontin is produced by mast cells and affects IgE-mediated degranulation and migration of mast cells.

Osteopontin (OPN), originally discovered in bone as an extracellular matrix protein, was identified in many cell types in the immune system, presumably being involved in many aspects of pathogenesis of inflammatory and immune diseases. Mast cells are also involved in such pathological aspects by secreting multiple mediators. However, it has not been determined whether mast cells produce OPN and whether it affects their function. To test this, we used murine fetal skin‐derived cultured mast cells (FSMC) and bone marrow‐derived cultured mast cells. We found that OPN was spontaneously produced by FSMC and inducible by ionomycin and FcϵRI aggregation in bone marrow‐derived cultured mast cells. In the presence of mast cell growth factors, FSMC were similarly generated from both OPN‐deficient (OPN–/–) and ‐sufficient (OPN+/+) mice without significant differences in yield, purity, granularity, and viability. Using OPN–/– FSMC, we found that recombinant OPN augmented IgE‐mediated degranulation and induced FSMC chemotaxis. Both effects were mediated by OPN receptors (i.e. CD44 and integrin αv). IgE‐mediated passive cutaneous anaphylaxis was significantly reduced in OPN–/– mice compared with OPN+/+ mice, indicating physiological relevance of OPN. These results indicate that OPN is a mast cell mediator, enhances mast cell responses to antigen, and thus may influence mast cell‐related pathological conditions.

Mast Cells Play a Key Role in Host Defense against Herpes Simplex Virus Infection through TNF-α and IL-6 Production

The essential contribution of mast cells (MCs) to bacterial host defense has been well established; however, little is known about their role in viral infections in vivo. Here, we found that intradermal injection with herpes simplex virus 2 (HSV-2) into MC-deficient Kit(W/Wv) mice led to increased clinical severity and mortality with elevated virus titers in HSV-infected skins. Ex vivo HSV-specific tetramer staining assay demonstrated that MC deficiency did not affect the frequency of HSV-specific cytotoxic T lymphocytes (CTLs) in draining lymph nodes. Moreover, the high mortality in Kit(W/W-v) mice was completely reversed by intradermal reconstitution with bone marrow-derived MCs (BMMCs) from wild-type, but not TNF(-/-) or IL-6(-/-) mice, indicating that MCs or, more specifically, MC-derived tumor necrosis factor (TNF) and IL-6 can protect mice from HSV-induced mortality. However, HSV did not directly induce TNF-α or IL-6 production by BMMCs; supernatants from HSV-infected keratinocytes induced the production of these cytokines by BMMCs without degranulation. Furthermore, IL-33 expression was induced in HSV-infected keratinocytes, and blocking the IL-33 receptor T1/ST2 on BMMCs significantly reduced TNF-α and IL-6 production by BMMCs. These results indicate the involvement of MCs in host defense at HSV-infected sites through TNF-α and IL-6 production, which is induced by keratinocyte-derived IL-33.

Antimicrobial Peptide LL-37 Produced by HSV-2-Infected Keratinocytes Enhances HIV Infection of Langerhans Cells

Herpes simplex virus (HSV)-2 shedding is associated with increased risk for sexually acquiring HIV. Because Langerhans cells (LCs), the mucosal epithelium resident dendritic cells, are suspected to be one of the initial target cell types infected by HIV following sexual exposure, we examined whether and how HSV-2 affects HIV infection of LCs. Although relatively few HSV-2/HIV-coinfected LCs were detected, HSV-2 dramatically enhanced the HIV susceptibility of LCs within skin explants. HSV-2 stimulated epithelial cell production of antimicrobial peptides (AMPs), including human β defensins and LL-37. LL-37 strongly upregulated the expression of HIV receptors in monocyte-derived LCs (mLCs), thereby enhancing their HIV susceptibility. Culture supernatants of epithelial cells infected with HSV-2 enhanced HIV susceptibility in mLCs, and this effect was abrogated by blocking LL-37 production. These data suggest that HSV-2 enhances sexual transmission of HIV by increasing HIV susceptibility of LCs via epithelial cell production of LL-37.

A case of lymphoepithelioma-like carcinoma of the skin associated with Epstein-Barr virus infection.

Lymphoepithelioma-like carcinoma of the skin (LELCS) is a rare cutaneous neoplasm with histopathologic similarities to nasopharyngeal carcinoma. The association of nasopharyngeal carcinoma with Epstein-Barr virus (EBV) is well documented. EBV has also been reported to be associated with LELC in only four sites (the stomach, salivary glands, lung, and thymus), but not in the skin. We report herein a case of EBV-positive LELCS. An 82-year-old female presented with a red nodule on the right cheek. Histologically, the entire dermis was occupied by atypical tumor cell nests with dense lymphocytic infiltration. Neoplastic cells were strongly positive for cytokeratin 14 but were negative for cytokeratins 19 and 20. EBV genomes in neoplastic cells were detected by polymerase chain reaction analysis and in situ hybridization for EBV-encoded RNA, suggesting an association with EBV.

Circadian Gene Clock Regulates Psoriasis-Like Skin Inflammation in Mice

There are several reports suggesting that the pathophysiology of psoriasis may be associated with aberrant circadian rhythms. However, the mechanistic link between psoriasis and the circadian time-keeping system, “the circadian clock,” remains unclear. This study determined whether the core circadian gene, Clock, had a regulatory role in the development of psoriasis. For this purpose, we compared the development of psoriasis-like skin inflammation induced by the Toll-like receptor 7 ligand imiquimod (IMQ) between wild-type mice and mice with a loss-of-function mutation of Clock. We also compared the development of IMQ-induced dermatitis between wild-type mice and mice with a loss-of-function mutation of Period2 (Per2), another key circadian gene that inhibits CLOCK activity. We found that Clock mutation ameliorated IMQ-induced dermatitis, whereas the Per2 mutation exaggerated IMQ-induced dermatitis, when compared with wild-type mice associated with decreased or increased IL-23 receptor (IL-23R) expression in γ/δ+ T cells, respectively. In addition, CLOCK directly bound to the promoter of IL-23R in γ/δ+ T cells, and IL-23R expression in the mouse skin was under circadian control. These findings suggest that Clock is a novel regulator of psoriasis-like skin inflammation in mice via direct modulation of IL-23R expression in γ/δ+ T cells, establishing a mechanistic link between psoriasis and the circadian clock.

Oral Administration of the CCR5 Inhibitor, Maraviroc, Blocks HIV Ex Vivo Infection of Langerhans Cells within the Epithelium

TO THE EDITOR Preexposure prophylaxis (PrEP) with oral administration of an antiretroviral is a potential method for preventing acquisition of HIV. A controlled trial in men who have sex with men (the iPrEx trial) showed that daily oral use of tenofovir disoproxil fumarateemtricitabine (TDF-FTC; Truvada) reduced transmission rates by 44% (Grant et al., 2010). In addition, the HIV Prevention Trial Network (HPTN) 052 trial recently confirmed that antiretroviral treatment leads to 96% reduction in transmission among HIV-negative heterosexual partners of HIV-positive individuals (Cohen et al., 2011). Similar trials, however, with TDF-FTC (the FEM-PrEP trial) or TDF alone (the VOICE trial) were stopped because of poor outcomes (van der Straten et al., 2012). Different results among various trials, which used identical antiretroviral regimens, could be explained by varying compliance with drug use and/or varying drug concentration and activity within the exposed tissue (Patterson et al., 2011). Langerhans cells (LCs) are CCR5þ dendritic cells located within genital skin and mucosal epithelium (Lederman et al., 2006). In female rhesus macaques exposed intravaginally to simian immunodeficiency virus, up to 90% of initially infected target cells were LCs (Hu et al., 2000). Ex vivo experiments with human foreskin explants show that epidermal LCs are target cells for HIV, providing a likely explanation for why circumcision greatly reduces the probability of acquiring HIV (Ganor et al., 2010). LCs also express CD4 and CCR5, but not CXCR4, within the tissue and demonstrate the distinctive characteristics of emigrating from tissue to draining lymph nodes in order to interact with T cells after contact with pathogens (Kawamura et al., 2000). Indeed, epidermal LCs are readily infected ex vivo with R5 HIV, but not with X4 HIV, and promote high levels of infection upon interaction with cocultured CD4þ T cells (Kawamura et al., 2000; Ogawa et al., 2013). Thus, LCs probably have an important role in disseminating HIV soon after exposure to virus. Epidemiologic observations have found that the majority of HIV strains isolated from patients soon after initial infection are R5 HIV strains (i.e., they utilize CCR5; Lederman et al., 2006). Not surprisingly, individuals with homozygous defects in CCR5 are largely protected from sexually acquiring HIV (Lederman et al., 2006). In addition, three different CCR5-binding topically applied compounds protected female macaques from sexually acquiring simian/human immunodeficiency virus: the N-terminally modified chemokine analog PSC-RANTES, the small-molecule inhibitor CMPD167, and maraviroc (MVC) (Lederman et al., 2006; Veazey et al., 2010). In addition to topical application to vaginal mucosa, oral delivery of CMPD167 protected macaques from vaginal simian/human immunodeficiency virus challenge (Veazey et al., 2005). Given these data, orally administered MVC may prove to be particularly important in PrEP regimens, although its ability to prevent HIV acquisition is unknown. In the current study, 20 healthy volunteers were randomly divided into four equal groups; they received 300 mg of MVC orally twice daily for 1, 2, 3, or 14 days. To obtain epidermal tissues, all subjects underwent suction blistering of the skin before and 2 hours after the last MVC dose. All subjects had plasma and semen collected 2 hours after their last dose. MVC concentrations in serum, semen, and epidermal tissues were determined by using the liquid chromatography– mass spectometry method, as described previously (Takahashi et al., 2010). Mean concentration±SD in the epidermis was 21.91±13.80, 23.36± 13.28, and 31.54±20.61 nM for individuals taking drug for 1, 2, or 3 days (n1⁄45 for each), respectively. MVC concentrations tended to be higher with a longer dosing period. Consistent with recent data showing high levels of MVC in genital tissue (Dumond et al., 2009), these results indicate that MVC rapidly distributes into the skin at high concentrations. In addition, MVC was detected in semen of all subjects (Supplementary Figure S1 online). To understand how HIV traverses skin and genital mucosa, an ex vivo model was developed whereby resident LCs within epithelial tissue explants are exposed to HIV and then allowed to emigrate from tissue, thus mimicking conditions that occur after mucosal exposure to HIV (Kawamura et al., 2000; Ogawa et al., 2013). In this model, although relatively few productively infected LCs are identified, these cells induce high levels of HIV infection when cocultured with resting autologous CD4þ T cells (Kawamura et al., 2000). In preliminary experiments, HIV infection of LCs, as well as subsequent virus transmission from emigrated LCs to cocultured CD4þ T cells, was decreased in a dose-dependent manner when skin explants were pretreated with various concentrations of MVC before See related commentary on pg 2662

Recruitment of plasmacytoid dendritic cells to skin regulates treatment responsiveness of actinic keratosis to imiquimod

Fig. 1. pDCs are recruited to IMQ-treated AK lesion and the number of pDCs recruited to clinical course. Left panel shows the duration of healing by tIMQ in total AK patients an pictures of AK treated by tIMQ in groups without or with inflammation. Representative im microscopic field (c) in AK lesions before and after IMQ treatment. Results are shown as m (d) Correlation between duration of AK clearance and number of BDCA-2+ pDCs in AK les