pDC Maturation Research Articles

Differential long noncoding RNA signatures in activated plasmacytoid dendritic cells (IRM6P.659)

Abstract Long noncoding RNAs (lncRNAs) represent a novel class of RNA which act through diverse mechanisms to exert control over a wide range of cellular processes. Through control of transcription, post-transcriptional regulation, or cellular signaling events, lncRNAs can function as potent modulators of innate immune responses, however very few lncRNAs have been investigated. We utilized whole exome sequencing of plasmacytoid dendritic cells (pDC) treated with IFN (interferon)-β, TLR7 agonist (toll like receptor, R848), and R848 with type I IFN blockade to identify 910 noncoding transcripts which are differentially regulated by IFN signaling and TLR7 activation. Through dissection of the stimulatory events controlling lncRNA expression, we have identified subsets of type I IFN- and TLR7-induced or suppressed lncRNAs. We hypothesize that many of these transcripts participate in the orchestration of innate and adaptive antiviral immunity through their modulation of pDC function. Dysregulation of these lncRNAs could significantly alter pDC maturation, IFN production, antigen presentation, turnover or localization, events which would be consequential during viral infection or IFN-driven autoimmune disease.

IFN-β Therapy Regulates TLR7-Mediated Response in Plasmacytoid Dendritic Cells of Multiple Sclerosis Patients Influencing an Anti-Inflammatory Status.

Plasmacytoid dendritic cells (pDCs) display altered immune-phenotype in multiple sclerosis (MS) patients and are found actively recruited in postmortem MS brain lesions, implying that their immune regulation may represent an important aspect of MS pathogenesis. Because of the reported Toll-like receptor 7 (TLR7) implication in autoimmunity, in this study we characterized how IFN-β therapy impacts on pDC activation to TLR7 triggering in MS patients, aspect only poorly investigated so far. In vivo IFN-β administration regulates pDC functions in TLR7-treated peripheral blood mononuclear cell (PBMC) cultures differently from what is observed in isolated cells, suggesting that IFN-β may activate inhibitory mechanisms in MS peripheral blood involved in turning off pDC response to dampen the ongoing inflammation. Indeed, IL-10, a key regulatory cytokine found increased upon TLR7 stimulation in in vivo IFN-β-exposed PBMCs, directly reduced pDC-mediated IFN-α production. IFN-β therapy also shaped T-cell responses by decreasing TLR7-induced pDC maturation and inducing T-cell inhibitory molecules. Accordingly, raised pDC-induced IL-27 and decreased IL-23 expression, together with high IL-10 level, contribute to inhibit Th17 cell differentiation. Our study uncovered a role for IFN-β in the regulation of TLR7-mediated pDC responses in MS toward an anti-inflammatory phenotype opening new opportunities to better understand mechanisms of action of this drug in controlling MS immunopathogenesis.

HMGB1 secretion during cervical carcinogenesis promotes the acquisition of a tolerogenic functionality by plasmacytoid dendritic cells.

Acquisition of an impaired functionality by plasmacytoid dendritic cells (pDCs) contributing to cancer progression has been documented in different types of cancers. In the present study, we postulate that molecules secreted by (pre)neoplastic epithelial cells of the genital tract (cervix/vulva) might attract pDCs but also modify their proper functionality, allowing these cells to initiate a tolerogenic response interfering with antitumor immunity. We demonstrated that pDCs are recruited during the cervical metaplasia-dysplasia-cancer sequence, through the action of their chemoattractant, chemerin. We showed that stimulated-pDCs exposed to cervical/vulvar tumor microenvironment display an altered phenotype. We also demonstrated that cervical/vulvar neoplastic keratinocytes inhibit the proper function of pDCs by decreasing their IFNα secretion in response to CpG oligonucleotides. In parallel, we observed that (pre)neoplastic areas of the cervix are infiltrated by FoxP3(+) Treg cells which colocalize with pDCs. Accordingly, pDCs cocultured with cervical/vulvar neoplastic keratinocytes have the capacity to induce a Treg cell differentiation from naïve CD4(+) T cells, which is in agreement with the development of a tolerogenic response. We identified HMGB1 as a soluble factor produced by neoplastic keratinocytes from the genital tract involved in pDCs functional alteration. Indeed, this molecule inhibited pDC maturation, decreased IFNα secretion following TLR9 stimulation and forced these cells to become tolerogenic. In contrast, inhibition of HMGB1 restored pDC phenotype. Our findings indicate that the use of inhibitory molecules notably directed against HMGB1 in cervical/vulvar (pre)neoplastic lesions might prevent alterations of pDCs functionality and represent an attractive therapeutic strategy to overcome immune tolerance in cancers.

Sensing of immature particles produced by dengue virus infected cells induces an antiviral response by plasmacytoid dendritic cells.

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.

HIV-1 and HIV-2 differentially mature plasmacytoid dendritic cells into IFN-producing cells or APCs.

HIV-1 causes a progressive impairment of immune function. HIV-2 is a naturally attenuated form of HIV, and HIV-2 patients display a slow-progressing disease. The leading hypothesis for the difference in disease phenotype between HIV-1 and HIV-2 is that more efficient T cell-mediated immunity allows for immune-mediated control of HIV-2 infection, similar to that observed in the minority of HIV-1-infected long-term nonprogressors. Understanding how HIV-1 and HIV-2 differentially influence the immune function may highlight critical mechanisms determining disease outcome. We investigated the effects of exposing primary human peripheral blood cells to HIV-1 or HIV-2 in vitro. HIV-2 induced a gene expression profile distinct from HIV-1, characterized by reduced type I IFN, despite similar upregulation of IFN-stimulated genes and viral restriction factors. HIV-2 favored plasmacytoid dendritic cell (pDC) differentiation into cells with an APC phenotype rather than IFN-α-producing cells. HIV-2, but not HIV-1, inhibited IFN-α production in response to CpG-A. The balance between pDC maturation into IFN-α-producing cells or development of an APC phenotype differentiates the early response against HIV-1 and HIV-2. We propose that divergent paths of pDC differentiation driven by HIV-1 and HIV-2 cause the observed differences in pathogenicity between the two viruses.

Broadly neutralizing antibody VRC01 prevents HIV-1 transmission from plasmacytoid dendritic cells to CD4 T lymphocytes.

Plasmacytoid dendritic cells (pDC) poorly replicate human immunodeficiency virus type 1 (HIV-1) but efficiently transfer HIV-1 to adjacent CD4 T lymphocytes. We found that coculture with T lymphocytes downregulates SAMHD1 expression, enhances HIV-1 replication, and increases pDC maturation and alpha interferon (IFN-α) secretion. HIV-1 transfer to T lymphocytes is inhibited by broadly neutralizing antibody VRC01 with efficiency similar to that of cell-free infection of T lymphocytes. Interestingly, prevention of HIV-1 transmission by VRC01 retains IFN-α secretion. These results emphasize the multiple functions of VRC01 in protection against HIV-1 acquisition.

LPS and HIV-1 induce a mature phenotype in human plasmacytoid dendritic cells and enhance their programmed death ligand-1 expression (VIR1P.970)

Abstract Aberrant immune activation and immune exhaustion are features characteristic of chronic HIV infection. Elevated plasma LPS levels resulting from disruption of gut mucosal integrity during HIV infection have been implicated as a major factor contributing to immune activation. In chronic HIV-1 infection immune exhaustion molecules PD-1 and its ligand PD-L1 negatively regulate the function of exhausted CD8 T cells. The pDC constitute only a minor portion of the PBMC population, but are crucial contributors to the innate as well as adaptive immune response. We have confirmed our previous results that pDC express low levels of TLR4 and respond to LPS. We undertook this study to examine the influence of LPS on virally stimulated pDC maturation and PD-L1 expression and mechanisms thereof. In order to do so we primed pDC with LPS prior to stimulation with HIV. We observed that LPS enhanced HIV-1-induced pDC maturation (CD83 and CD40 expression) and PD-L1 expression. PD-L1 was up-regulated on mature pDC specifically by LPS and HIV combination treatment versus individual treatment with LPS or HIV. The combination treatment specifically induced co-expression of PD-L1 with positive co-stimulatory molecule CD86 but not CD80. Additionally, LPS enhanced CD4 expression on pDC but depressed HIV-1 induced CXCR4 and CCR5 expression. Together, these results indicate that in response to HIV stimulation, pDC adopt a tolerogenic phenotype (i.e. PD-L1-positive) and LPS helps drive this.

Functional limitations of plasmacytoid dendritic cells limit type I interferon, T cell responses and virus control in early life.

Infant mortality from viral infection remains a major global health concern: viruses causing acute infections in immunologically mature hosts often follow a more severe course in early life, with prolonged or persistent viral replication. Similarly, the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) causes acute self-limiting infection in adult mice but follows a protracted course in infant animals, in which LCMV-specific CD8+ T cells fail to expand and control infection. By disrupting type I IFNs signaling in adult mice or providing IFN-α supplementation to infant mice, we show here that the impaired early life T cell responses and viral control result from limited early type I IFN responses. We postulated that plasmacytoid dendritic cells (pDC), which have been identified as one major source of immediate-early IFN-I, may not exert adult-like function in vivo in the early life microenvironment. We tested this hypothesis by studying pDC functions in vivo during LCMV infection and identified a coordinated downregulation of infant pDC maturation, activation and function: despite an adult-like in vitro activation capacity of infant pDCs, the expression of the E2-2 pDC master regulator (and of critical downstream antiviral genes such as MyD88, TLR7/TLR9, NF-κB, IRF7 and IRF8) is downregulated in vivo at baseline and during LCMV infection. A similar pattern was observed in response to ssRNA polyU, a model ligand of the TLR7 viral sensor. This suggests that the limited T cell-mediated defense against early life viral infections is largely attributable to / regulated by infant pDC responses and provides incentives for novel strategies to supplement or stimulate immediate-early IFN-α responses.

Cross-linking of CD81 by HCV-E2 protein inhibits human intrahepatic plasmacytoid dendritic cells response to CpG-ODN

Plasmacytoid dendritic cells (pDCs) are reported to be defective in HCV-infected patients, the mechanisms of which remain poorly understood. We isolated liver derived mononuclear cells (LMNCs) and pDCs from normal liver tissues of benign tumor dissections and liver transplant donors. Isolated pDCs and LMNCs were cultured with precoated HCV envelop protein E2 (HCV-E2) or anti-CD81 mAb in the presence of CpG-ODN. Our results show that cross-linking of CD81 by either HCV-E2 or anti-CD81 mAb inhibits IFN-α secretion in CpG-induced pDCs; down-regulates HLA-DR, CD80 and CD86 expression in pDCs; and suppresses CpG-ODN induced proliferation and survival of pDCs. The blockade of CD81 by soluble anti-CD81 antibody restores pDCs response to CpG-ODN. These results suggest that HCV E2 protein interacts with CD81 to inhibit pDC maturation, activation, and IFN-α production, and may thereby contribute to the impaired innate anti-viral immune response in HCV infection.

Plasmacytoid, conventional, and monocyte‐derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation

DCs express receptors sensing microbial, danger or cytokine signals, which when triggered in combination drive DC maturation and functional polarization. Maturation was proposed to result from a discrete number of modifications in conventional DCs (cDCs), in contrast to a cell-fate conversion in plasmacytoid DCs (pDCs). cDC maturation is generally assessed by measuring cytokine production and membrane expression of MHC class II and co-stimulation molecules. pDC maturation complexity was demonstrated by functional genomics. Here, pDCs and cDCs were shown to undergo profound and convergent changes in their gene expression programs in vivo during viral infection. This observation was generalized to other stimulation conditions and DC subsets, by public microarray data analyses, PCR confirmation of selected gene expression profiles, and gene regulatory sequence bioinformatics analyses. Thus, maturation is a complex process similarly reshaping all DC subsets, including through the induction of a core set of NF-κB- or IFN-stimulated genes irrespective of stimuli.

Identification of novel oligonucleotides from mitochondrial DNA that spontaneously induce plasmacytoid dendritic cell activation

This study tested the hypothesis that mtDNA fragments carry immunostimulatory motifs that naturally induce immune activation by PDC. Genomic and mtDNA induced similar IFN-α production after transfection into PBMCs using the liposomal transfection reagent DOTAP. Shortening of mtDNA to CpG islands enhanced the immunostimulatory activity, based on the presence of unmethylated CpG DNA. Further fragmentation into mtODN, which exhibited similarities to published CpG ODN, resulted in a strong immunostimulatory activity in addition to PDC maturation and migration. The addition of the human cathelicidin LL-37 to CpG islands induced spontaneous PDC IFN-α production. Notably, one phosphodiester mtODN with a double-palindromic structure induced PDC IFN-α production in the absence of DOTAP. Flow cytometry, life-cell, and confocal imaging revealed attachment and spontaneous uptake into PDC, colocalizing, in part, with TLR9 in early endosomal vesicles. This process was accompanied by a moderate but significant PDC maturation in addition to B cell and NK cell activation (P<0.05). Altogether, our data indicate that fragmented mtDNA, which may be released as a consequence of apoptotic, necrotic, and necroptotic cell death, can act as a DAMP. For the first time, our study provides a mechanism how longer and shorter mtDNA fragments can be taken up naturally by the PDC and thus, may contribute to acute and chronic immune activation.

Measles Virus Vaccine–Infected Tumor Cells Induce Tumor Antigen Cross-Presentation by Human Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDC) are antigen-presenting cells specialized in antiviral response. The measles virus vaccine is proposed as an antitumor agent to target and specifically kill tumor cells without infecting healthy cells. Here, we investigated, in vitro, the effects of measles virus vaccine-infected tumor cells on the phenotype and functions of human pDC. We studied maturation and tumor antigen cross-presentation by pDC, exposed either to the virus alone, or to measles virus vaccine-infected or UV-irradiated tumor cells. We found that only measles virus vaccine-infected cells induced pDC maturation with a strong production of IFN-α, whereas UV-irradiated tumor cells were unable to activate pDC. This IFN-α production was triggered by the interaction of measles virus vaccine single-stranded RNA (ssRNA) with TLR7. We observed that measles virus vaccine-infected tumor cells were phagocytosed by pDC. Interestingly, we showed cross-presentation of the tumor antigen NYESO-1 to a specific CD8(+) T-cell clone when pDC were cocultured with measles virus vaccine-infected tumor cells, whereas pDC were unable to cross-present NYESO-1 after coculture with UV-irradiated tumor cells. Altogether, our results suggest that the use of measles virus vaccine in antitumor virotherapy induces immunogenic tumor cell death, allowing pDC to mature, produce high amounts of IFN-α, and cross-present tumor antigen, thus representing a mode of recruiting these antigen-presenting cells in the immune response. Clin Cancer Res; 19(5); 1147-58. ©2012 AACR.

EBV stimulates TLR‐ and autophagy‐dependent pathways and impairs maturation in plasmacytoid dendritic cells: Implications for viral immune escape

Plasmacytoid DCs (pDCs) are crucial mediators in the establishment of immunity against most viruses, given their extraordinary capacity to produce a massive quantity of type I IFN. In this study we investigate the response of pDCs to infection with EBV, a γ-herpes virus that persists with an asymptomatic infection in immunocompetent hosts, although in certain conditions it can promote development of cancers or autoimmune diseases. We show that high amounts of type I IFNs were released from isolated pDCs after exposure to EBV by a mechanism requiring TLRs and a functional autophagic machinery. We next demonstrate that EBV can infect pDCs via viral binding to MHC class II molecule HLA-DR and that pDCs express EBV-induced latency genes. Furthermore, we observe that EBV is able to induce activation but not maturation of pDCs, which correlates with an impaired TNF-α release. Accordingly, EBV-infected pDCs are unable to mount a full T-cell response, suggesting that impaired pDC maturation, combined with a concomitant EBV-mediated upregulation of the T-cell inhibitory molecules B7-H1 and ICOS-L, could represent an immune-evasion strategy promoted by the virus. These mechanisms might lead to persistence in immunocompetent hosts or to dysregulated immune responses linked to EBV-associated diseases.

TLR-activated conventional DCs promote γ-secretase-mediated conditioning of plasmacytoid DCs

Cooperative events between DC subsets involve cell contact and soluble factors. Upon viral challenge, murine pDCs induce cDC cooperation through CD40-CD40L interactions and IL-15 secretion, whereas in humans, the same effect is mediated by IFN-α. Conversely, during bacterial infections, pDC maturation may be induced by activated cDCs, although no mechanisms had been described so far. Here, we investigate how human pDCs are "conditioned" by cDCs. Blood-borne DC subsets (cDCs and pDCs) were sorted from healthy donors. IL-3-maintained pDCs were cocultured with LPS-activated, poly (I:C)-activated, or control cDCs [cDC(LPS), cDC(P(I:C)), cDC(CTRL)]. Coculture experiments showed that cDC(LPS)-conditioned pDCs up-regulated maturation markers, such as CD25 and CD86, whereas SNs contained higher amounts of IL-6 and CCL19 compared with control conditions. Gene-expression analyses on sorted cDC(LPS) or cDC(P(I:C)) conditioned pDCs confirmed the induction of several genes, including IL-6 and CCL19 and remarkably, several Notch target genes. Further studies using the γ-secretase/Notch inhibitor DAPT and soluble Notch ligands resulted in a significantly reduced expression of canonical Notch target genes in conditioned pDCs. DAPT treatment also hampered the secretion of CCL19 (but not of IL-6) by cDC(LPS) conditioned pDCs. These results reveal the involvement of γ-secretase-mediated mechanisms, including the Notch pathway, in the cell contact-dependent communication between human DC subsets. The resulting partial activation of pDCs after encountering with mature cDCs endows pDCs with an accessory function that may contribute to T cell recruitment and activation.

Defects in CD54 and CD86 Up-regulation by Plasmacytoid Dendritic Cells During Pregnancy

Physiological modulation of the immune system is required for foetal tolerance during pregnancy. However, this immune regulation might lead to impaired self-defence against pathogens. Indeed, pregnant women are more susceptible to newly encountered viruses comparing to non-pregnant women, as exemplified by the prevalence of severe complications in pregnant women infected with the pandemic influenza virus in 2009. Plasmacytoid dendritic cells (pDCs) are specialized dendritic cells that recognise viral antigens and initiate both innate and adaptive immune responses. We therefore sought to determine whether the number and/or the functions of peripheral blood pDCs are regulated during pregnancy. pDC maturation and interferon (IFN)-α production were analysed in response to Toll-like receptor (TLR) stimulation of peripheral blood mononuclear cells from pregnant and non-pregnant women. Our results reveal that pDC frequency is slightly decreased, while the IFN-α production in response to TLR stimulation increases during pregnancy. Interestingly, the up-regulation of the co-stimulatory receptors CD54 (ICAM1) and CD86 is significantly decreased in pDCs from pregnant women as compared to controls, suggesting a possible impact on T-cell responses. In conclusion, we propose that the modulation of CD54 and CD86 expression on peripheral blood pDCs during pregnancy might decrease the initiation of adaptive antiviral immune responses.

Capture of cell-derived microvesicles (exosomes and apoptotic bodies) by human plasmacytoid dendritic cells

cDCs and pDCs differ in multiple aspects. Among those, antigen capture is a recognized feature of cDCs, whereas pDCs display poor capacity to capture cell-derived antigens. However, animal models of organ transplantation suggested a role for pDCs in tolerance induction via phagocytosis of donor antigens. In a transplantation setting, microvesicles, such as apoptotic bodies and exosomes secreted by the graft, may be potential sources of alloantigen. Here, we tested the capacity of human pDCs to capture exosomes and apoptotic bodies from Jurkat T cells. Exosomes and apoptotic bodies were indeed captured by pDCs, although required longer times of incubation when compared with the highly endocytic cDCs. In cDCs and pDCs, exosome capture was more efficient than apoptotic bodies. Endocytosis inhibitors clearly impaired exosome capture by cDCs, although this could not be verified in pDCs as a result of cellular toxicity. Functionally, capture of Jurkat-derived exosomes did not induce nor prevent pDC maturation, and exosome-loaded pDCs induced T cell proliferation, suggesting a link between capture and presentation. Thus, exosomes and apoptotic bodies may be sources of antigen for human pDCs.

LPS enhances HIV-1-induced maturation and programmed death ligand-1 expression on human plasmacytoid dendritic cells (168.27)

Abstract Elevated plasma levels of lipopolysaccharide (LPS) resulting from disruption of gut mucosal integrity during HIV infection has been implicated as a major factor contributing to immune activation that is a hallmark of HIV infection. The negative costimulatory molecule, Programmed Death-1 (PD-1), and its ligand, Programmed Death Ligand -1(PD-L1), negatively regulate the function of exhausted CD8 T cells in chronic HIV-1 infection leading to suppression of specific anti-viral T cell responses. In HIV-infected individuals, increased PD-L1 expression on APCs correlates directly with viral load, indicating a potential effect of the virus on PD-L1 expression. Since the mechanisms of PD-L1 up-regulation in these settings are not understood, we investigated how PD-L1 expression is regulated in plasmacytoid dendritic cells (pDC). We also investigated the influence of LPS on virally stimulated pDC maturation and PD-L1 expression. By kinetic studies, we observed that LPS enhanced HIV-1 induced PD-L1, CD40 and CD83 expression on pDC, and that PD-L1 expression was up-regulated on matured pDC. IFN-α expression preceded PD-L1 expression on pDC following HIV-1 stimulation, but IFN-a by itself only modestly increased PD-L1 expression, indicating that up-regulation of PD-L1 on pDC was specifically a primary response to HIV stimulation. Together, these results suggest that pDC adopt a tolerogenic phenotype following maturation during HIV-1 infection and LPS helps mediate this process.

Positive RAR/RXR and Negative STAT3 Regulation of FOXP3 Transcription is Mediated by Control of SMAD3 Access and Binding to an Enhancer

Some clinical trials and epidemiologic studies revealed improvements in symptoms of ulcerative colitis after smoking and nicotine patch treatment. However, mechanisms underlying the ameliorating effect of nicotine remain poorly understood. We have previously reported that nicotine administration ameliorates murine oxazolone (OXZ)-induced colitis through alpha7 nicotine acetylcholine receptors (α7nAChRs). In addition, we have shown that mRNA of α7nAChRs was expressed in plasmacytoid dendritic cells (pDCs) of OXZ colitis mouse colon. The aim of this study was to investigate a pathophysiological role of pDCs and immunological effects of nicotine on pDCs in ulcerative colitis. Method: OXZ-induced colitis was developed in BALB/c mice. Lamina propria mononuclear cells (LPMCs) of the colon and mesenteric lymph node (MLN) were collected in OXZ colitis mice. pDCs were isolated from LPMCs by using magnetic cell separation system (BD biosciences). In addition, mouse bone marrow (BM) cells were differentiated to pDC-like cells (BMpDC) by Flt3 ligand. Furthermore, immunological effects of nicotine on BMpDCwere examined by flow cytometry and BMpDC migration was examined by chemotaxis assay using EZ-TAXIScan. Moreover, the effect of nicotine administration on pDC subset proportion was investigated in the MLN of OXZ colitis mice. Result: α7nAChRs mRNA was expressed in pDC of OXZ colitis mouse colon and BMpDC, but not BM conventional dendritic cells (cDC). Flow cytometry analysis revealed that neither CD80, CD86 nor MHC class II expression in BMpDC was affected with nicotine in the induction of pDC maturation by CpG-DNA. On the other hand, CCL21induced migration of mature BMpDC was inhibited by nicotine and α7nAChRs selective agonist GTS-21, and α7nAChRs selective antagonist methyllycaconitine (MLA) blocked the inhibitory effect of nicotine and GTS-21. Furthermore, nicotine administration downregulated pDC subset proportion in the MLN of OXZ colitis mice. Conclusion: These data suggest that the activation of α7nAChRs reduces pDC-mediated antigen presentation to naive CD4 T cells by inhibiting pDC migration from colon to MLN, thereby alleviating the symptoms in OXZ colitis. Our results may contribute to the development of therapeutic medicines for ulcerative colitis by activating the cholinergic anti-inflammatory pathway.

Nicotine Improves Symptoms in Murine Oxazolone-Induced Ulcerative Colitis by Inhibiting Migration of Plasmacytoid Dendritic Cells Through Alpha7 Nicotinic Acetylcholine Receptors

Some clinical trials and epidemiologic studies revealed improvements in symptoms of ulcerative colitis after smoking and nicotine patch treatment. However, mechanisms underlying the ameliorating effect of nicotine remain poorly understood. We have previously reported that nicotine administration ameliorates murine oxazolone (OXZ)-induced colitis through alpha7 nicotine acetylcholine receptors (α7nAChRs). In addition, we have shown that mRNA of α7nAChRs was expressed in plasmacytoid dendritic cells (pDCs) of OXZ colitis mouse colon. The aim of this study was to investigate a pathophysiological role of pDCs and immunological effects of nicotine on pDCs in ulcerative colitis. Method: OXZ-induced colitis was developed in BALB/c mice. Lamina propria mononuclear cells (LPMCs) of the colon and mesenteric lymph node (MLN) were collected in OXZ colitis mice. pDCs were isolated from LPMCs by using magnetic cell separation system (BD biosciences). In addition, mouse bone marrow (BM) cells were differentiated to pDC-like cells (BMpDC) by Flt3 ligand. Furthermore, immunological effects of nicotine on BMpDCwere examined by flow cytometry and BMpDC migration was examined by chemotaxis assay using EZ-TAXIScan. Moreover, the effect of nicotine administration on pDC subset proportion was investigated in the MLN of OXZ colitis mice. Result: α7nAChRs mRNA was expressed in pDC of OXZ colitis mouse colon and BMpDC, but not BM conventional dendritic cells (cDC). Flow cytometry analysis revealed that neither CD80, CD86 nor MHC class II expression in BMpDC was affected with nicotine in the induction of pDC maturation by CpG-DNA. On the other hand, CCL21induced migration of mature BMpDC was inhibited by nicotine and α7nAChRs selective agonist GTS-21, and α7nAChRs selective antagonist methyllycaconitine (MLA) blocked the inhibitory effect of nicotine and GTS-21. Furthermore, nicotine administration downregulated pDC subset proportion in the MLN of OXZ colitis mice. Conclusion: These data suggest that the activation of α7nAChRs reduces pDC-mediated antigen presentation to naive CD4 T cells by inhibiting pDC migration from colon to MLN, thereby alleviating the symptoms in OXZ colitis. Our results may contribute to the development of therapeutic medicines for ulcerative colitis by activating the cholinergic anti-inflammatory pathway.

Spatiotemporal Regulation of Heat Shock Protein 90-Chaperoned Self-DNA and CpG-Oligodeoxynucleotide for Type I IFN Induction via Targeting to Static Early Endosome

Recent studies have suggested that TLR9 signaling in early endosomes leads to IFN-alpha production by plasmacytoid dendritic cells (pDCs), whereas TLR9 signaling in late endosomes induces pDC maturation, IL-6, and TNF-alpha secretion. In this study, we show that human DNA as well as CpG-oligodeoxynucleotides (ODNs) in complex with heat shock protein 90 (Hsp90) stimulate pDCs to produce large quantities of IFN-alpha. The Hsp90-CpG-A complexes are targeted into the Rab5+, early endosomal Ag 1+-static early endosome postinternalization by DCs, suggesting that preferential sorting of Hsp90-chaperoned self-DNA/CpG-ODNs to the static endosome is required for signaling through TLR9 for IFN-alpha production. Interestingly, Hsp90-mediated preferential static early endosomal translocation of CpG-ODNs triggers robust IFN-alpha production from murine conventional DCs. Thus, extracellular Hsp90 converts inert self-DNA/CpG-ODNs into a potent trigger of IFN-alpha production via spatiotemporal regulation.