pDC Maturation Research Articles

Siglec-H ablation in plasmacytoid dendritic cells alleviates acute liver injury by promoting IL-21+ CD4 T cells

Abstract Siglec-H is an immune receptor specifically expressed in plasmacytoid dendritic cells (pDCs) that inhibits IFN-α production and regulates pDC maturation. Although, the role of pDCs in autoimmune disorders is well-known, the effect of Siglec-H expression in pDCs in acute liver injury (ALI) remains elusive. To investigate Siglec-H/pDCs axis in ALI, BDCA2 transgenic and Siglec-H knockout (SH KO) mice were administrated with concanavalin A (ConA) to induce a T cell-mediated ALI in combination of either pDCs depletion with anti-BDCA2 antibody or anti-IL-21R and Stat3 blockade. Here, we report that specific depletion of pDCs in BDCA2 transgenic mice exacerbated ALI. Surprisingly, SH KO mice were resistant to ALI (lower ALT, hyaluronic acid, IL-6 and TNF-α). Th1/IFN-γ response and Stat1 signaling were both inhibited in the liver of SH KO mice while intrahepatic IL-21 and Stat3 signaling pathways were elevated. Mechanistically, we identified that Siglec-H-null pDCs exhibited immunosuppressive phenotypes (CD40LowCCR9Low), resulting in a defective CD4 T cell activation and a promotion of IL-21-producing CD4 T cells in the liver. Finally, IL-21R and Stat3 inhibitions in Siglec-H deficient mice restored ConA-induced ALI. We conclude that, Siglec-H-null pDCs mediate hepatic tolerance via induction of IL-21+CD4 T cells in the liver during ALI. These findings indicate that targeting Siglec-H/pDCs axis may provide a novel approach to modulate CD4 T cell polarization and liver disease.

Siglec-H-/- Plasmacytoid Dendritic Cells Protect Against Acute Liver Injury by Suppressing IFN-γ/Th1 Response and Promoting IL-21+ CD4 T Cells

Siglec-H is a receptor specifically expressed in mouse plasmacytoid dendritic cells (pDCs), which functions as a negative regulator of interferon-α production and plays a critical role in pDC maturation to become antigen-presenting cells. The function of pDCs in autoimmune and inflammatory diseases has been reported. However, the effect of Siglec-H expression in pDCs in liver inflammation and diseases remains unclear. Using the model of concanavalin A-induced acute liver injury (ALI), we investigated the Siglec-H/pDCs axis during ALI in BDCA2 transgenic mice and Siglec-H-/- mice. Anti-BDCA2 antibody, anti-interleukin (IL)-21R antibody, and Stat3 inhibitor were used to specifically deplete pDCs, block IL21 receptor, and inhibit Stat3 signaling, respectively. Splenocytes and purified naive CD4 T cells and bone marrow FLT3L-derived pDCs were cocultured and stimulated with phorbol myristate acetate/ionomycin and CD3/CD28 beads, respectively. Data showed that specific depletion of pDCs aggravated concanavalin A-induced ALI. Remarkably, alanine aminotransferase, hyaluronic acid, and proinflammatory cytokines IL6 and tumor necrosis factor-α levels were lower in the blood and liver of Siglec-H knockout mice. This was associated with attenuation of both interferon-γ/Th1 response and Stat1 signaling in the liver of Siglec-H knockout mice while intrahepatic IL21 and Stat3 signaling pathways were upregulated. Blocking IL21R or Stat3 signaling in Siglec-H knockout mice restored concanavalin A-induced ALI. Finally, we observed that the Siglec-H-null pDCs exhibited immature and immunosuppressive phenotypes (CCR9LowCD40Low), resulting in reduction of CD4 T-cell activation and promotion of IL21+CD4 T cells in the liver. During T-cell-mediated ALI, Siglec-H-null pDCs enhance immune tolerance and promote IL21+CD4 T cells in the liver. Targeting Siglec-H/pDC axis may provide a novel approach to modulate liver inflammation and disease.

Dark pigmentation and related low FMOD expression increase IL-3 and facilitateplasmacytoid dendritic cell maturation

According to epidemiological research, skin autoimmune diseases are more prevalent among black Americans. We postulated that pigment-producing melanocytes may contribute to local immune regulation in the microenvironment. We examined murine epidermal melanocytes in vitro to determine the role of pigment production in immune responses mediated by dendritic cell (DC) activation. Our study revealed that darkly pigmented melanocytes produce more IL-3 and the pro-inflammatory cytokines, IL-6 and TNF-α, and consequently induce plasmacytoid DC (pDC) maturation. Additionally, we demonstrate that low pigment-associated fibromodulin (FMOD) interferes with cytokine secretion and subsequent pDC maturation.

Human Plasmacytoid Dendritic Cells Express C-Type Lectin Receptors and Attach and Respond to Aspergillus fumigatus.

Plasmacytoid dendritic cells (pDCs) have been implicated as having a role in antifungal immunity, but mechanisms of their interaction with fungi and the resulting cellular responses are not well understood. In this study, we identify the direct and indirect biological response of human pDCs to the fungal pathogen Aspergillus fumigatus and characterize the expression and regulation of antifungal receptors on the pDC surface. Results indicate pDCs do not phagocytose Aspergillus conidia, but instead bind hyphal surfaces and undergo activation and maturation via the upregulation of costimulatory and maturation markers. Measuring the expression of C-type lectin receptors dectin-1, dectin-2, dectin-3, and mannose receptor on human pDCs revealed intermediate expression of each receptor compared with monocytes. The specific dectin-1 agonist curdlan induced pDC activation and maturation in a cell-intrinsic and cell-extrinsic manner. The indirect activation of pDCs by curdlan was much stronger than direct stimulation and was mediated through cytokine production by other PBMCs. Overall, our data indicate pDCs express various C-type lectin receptors, recognize and respond to Aspergillus hyphal Ag, and serve as immune enhancers or modulators in the overarching fungal immune response.

Low Density Lipoprotein Exposure of Plasmacytoid Dendritic Cells Blunts Toll-like Receptor 7/9 Signaling via NUR77.

Background: Pathogens or trauma-derived danger signals induced maturation and activation of plasmacytoid dendritic cells (pDCs) is a pivotal step in pDC-dependent host defense. Exposure of pDC to cardiometabolic disease-associated lipids and proteins may well influence critical signaling pathways, thereby compromising immune responses against endogenous, bacterial and viral pathogens. In this study, we have addressed if hyperlipidemia impacts human pDC activation, cytokine response and capacity to prime CD4+ T cells. METHODS AND RESULTS: We show that exposure to pro-atherogenic oxidized low-density lipoproteins (oxLDL) led to pDC lipid accumulation, which in turn ablated a Toll-like receptor (TLR) 7 and 9 dependent up-regulation of pDC maturation markers CD40, CD83, CD86 and HLA-DR. Moreover, oxLDL dampened TLR9 activation induced the production of pro-inflammatory cytokines in a NUR77/IRF7 dependent manner and impaired the capacity of pDCs to prime and polarize CD4+ T helper (Th) cells. CONCLUSION: Our findings reveal profound effects of dyslipidemia on pDC responses to pathogen-derived signals.

TNF-α Regulates Human Plasmacytoid Dendritic Cells by Suppressing IFN-α Production and Enhancing T Cell Activation.

Human plasmacytoid dendritic cells (pDCs) play a vital role in modulating immune responses. They can produce massive amounts of type I IFNs in response to nucleic acids via TLRs, but they are also known to possess weak Ag-presenting properties inducing CD4+ T cell activation. Previous studies showed a cross-regulation between TNF-α and IFN-α, but many questions remain about the effect of TNF-α in regulating human pDCs. In this study, we showed that TNF-α significantly inhibited the secretion of IFN-α and TNF-α of TLR-stimulated pDCs. Instead, exogenous TNF-α promoted pDC maturation by upregulating costimulatory molecules and chemokine receptors such as CD80, CD86, HLA-DR, and CCR7. Additionally, RNA sequencing analysis showed that TNF-α inhibited IFN-α and TNF-α production by downregulating IRF7 and NF-κB pathways, while it promoted Ag processing and presentation pathways as well as T cell activation and differentiation. Indeed, TNF-α-treated pDCs induced in vitro higher CD4+ T cell proliferation and activation, enhancing the production of Th1 and Th17 cytokines. In conclusion, TNF-α favors pDC maturation by switching their main role as IFN-α-producing cells to a more conventional dendritic cell phenotype. The functional status of pDCs might therefore be strongly influenced by their overall inflammatory environment, and TNF-α might regulate IFN-α-mediated aspects of a range of autoimmune and inflammatory diseases.

Graft-versus-host disease depletes plasmacytoid dendritic cell progenitors to impair tolerance induction.

Graft-versus-host disease (GVHD) causes failed reconstitution of donor plasmacytoid dendritic cells (pDCs) that are critical for immune protection and tolerance. We used both murine and human systems to uncover the mechanisms whereby GVHD induces donor pDC defects. GVHD depleted Flt3-expressing donor multipotent progenitors (MPPs) that sustained pDCs, leading to impaired generation of pDCs. MPP loss was associated with decreased amounts of MPP-producing hematopoietic stem cells (HSCs) and oxidative stress-induced death of proliferating MPPs. Additionally, alloreactive T cells produced GM-CSF to inhibit MPP expression of Tcf4, the transcription factor essential for pDC development, subverting MPP production of pDCs. GM-CSF did not affect the maturation of pDC precursors. Notably, enhanced recovery of donor pDCs upon adoptive transfer early after allogeneic HSC transplantation repressed GVHD and restored the de novo generation of donor pDCs in recipient mice. pDCs suppressed the proliferation and expansion of activated autologous T cells via a type I IFN signaling-dependent mechanism. They also produced PD-L1 and LILRB4 to inhibit T cell production of IFN-γ. We thus demonstrate that GVHD impairs the reconstitution of tolerogenic donor pDCs by depleting DC progenitors rather than by preventing pDC maturation. MPPs are an important target to effectively bolster pDC reconstitution for controlling GVHD.

Blockade of Deubiquitylating Enzyme USP7 in Plasmacytoid Dendritic Cells Stimulates Anti-Myeloma Immunity

Introduction Proteasome inhibitors (PIs) are a backbone standard of care in the treatment of relapsed/refractory and newly diagnosed multiple myeloma (MM). Although PIs represent a major advance, not all MM responds, and relapse develops due to the development of drug-resistance. Besides the 26S proteasome, the ubiquitin proteasome pathway harbors many other sites for therapeutic intervention, with the potential to overcome PI-resistance. We showed that inhibition of deubiquitylating enzyme USP7, upstream of the 20S proteasome, induces cell death even in MM cells resistant to PI therapies. To date, however, the effect of USP7 inhibition on the immunosuppressive and tumor-promoting MM-host bone marrow (BM) accessory cells such as plasmacytoid dendritic cells (pDC) remains unclear. Here, we utilized our co-culture models of pDCs, T cells, and NK cells with autologous patient MM cells to examine whether USP7 inhibition also stimulates anti-MM immunity. Methods MM cell lines were validated by fingerprinting, and MM patient BM/PB samples (n = 9) were obtained after informed consent. We used minimally cytotoxic concentrations of a novel USP7 inhibitor XL177A (0.1-0.5 µM). Activation/maturation assays MM-pDCs were treated with XL177A (0.2 uM; 24h), followed by evaluation of CD86/HLA-DR markers by FACS. USP7-HA-UbVME Labeling: pDC representative Cal-1 cells were treated with DMSO or XL177A for 6h; proteins were labeled with ubiquitin (Ub) active site probe Ub-vinyl methyl ester (HAUbVME) for 30 mins, followed by immunoblot analysis using anti-USP7 antibodies. CTL/NK activity assays MM CD8+ T- or NK-cells cells were cultured with autologous pDCs (1:10 pDC:T/NK ratio) with XL177A for 3 days; after washing to remove drug, cells were cultured with pre-stained (CellTrace Violet) target MM cells (10:1 E/T ratio; T/NK:MM) for 24h, followed by FACS quantification of viable MM cells. Statistical parameters were calculated using GraphPad Prism. Results 1) Treatment of MM cell lines and patient MM cells with XL177A for 24h significantly decreases their viability (IC50 range 0.6 uM to 5 uM) (mean ± SD; p < 0.05; n=4). 2) Blockade of USP7 by XL177A activates MM patient BM-pDCs, evidenced by an increase in pDC maturation markers CD86 and HLA-DR. As a negative control, treatment of pDCs with XL177B, an inactive (R) enantiomer of XL177A, had no effect on CD86 or HLA-DR expression on pDCs. 3) XL177A specificity against USP7 DUB activity was assessed using competition assays between XL177A and Ub-active site labeling probe HAUbVME: treatment of pDC-Cal-1 cells with XL177A blocked the labeling HAUbME probe, confirming its specificity. Similar results were noted using another USP7 inhibitor P5091. 4) Functional studies showed that XL177A stimulates MM-specific CD8+ CTL activity, evidenced by a decrease in the viability of patient MM cells (p = 0.0004; n= 9). 5) XL177A triggered pDC-induced NK cell-mediated MM-specific cytolytic activity (p<0.05). Finally, 6) XL177A decreased Treg (CD4+/CD25+/Foxp3+) populations in MM. Conclusion Targeting USP7 DUB enzyme restores both innate and adaptive immune responses in MM. These findings, coupled with our prior findings showing that inhibition of USP7 overcomes PI-resistance in MM cells, suggest that targeting USP7 is a novel therapeutic strategy to overcome both PI-resistance and immune suppression in MM. Disclosures Buhrlage: Adenoid Cystic Carcinoma Foundation: Other: Scientific Advisory Board. Chauhan:consultant to Stemline Therapeutics, Inc., and Equity owner in C4 Therapeutics.: Consultancy, Other: Equity owner in C4 Therapeutics.; Oncopeptide AB: Consultancy. Anderson:Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

Regulation of Id2 gene expression in plasmacytoid dendritic cells

Abstract Plasmacytoid dendritic cells (pDCs) are specialized type I interferon (IFN-I) producing cells that mediate anti-viral responses, anti-tumor immunity, and autoimmunity. When exposed to Toll-like receptor 7 (TLR7)- and TLR9-ligands, pDCs mature and produce IFN-Is; pDCs also acquire conventional DC (cDC)-like morphology and features, including the cell surface expression of antigen presentation and co-stimulatory molecules, secretion of additional cytokines, and ability to activate adaptive T cell responses. Yet, the transcriptional pathways regulating TLR-induced pDC maturation are poorly characterized. We found the mRNA and protein expression of the inhibitor of DNA binding protein 2 (Id2) was induced in TLR7/9-stimulated pDCs, and Id2 expression correlated with greater cDC-like features. Id2 normally blocks pDC development by antagonizing the pDC-master regulator, E2-2, while E2-2 suppresses Id2 transcription. Thus, we hypothesize TLR-activated signaling pathways in pDCs control the abundance of transcriptional regulators (e.g., E2-2) at the Id2 promoter as well as the promoter chromatin state, leading to induction of Id2 transcription during pDC maturation. Using genetic knockout mouse models, we found Id2 induction was independent of interferon alpha receptor and nuclear factor kappa beta signaling pathways. Chromatin-immunoprecipitation assays suggested E2-2 abundance at the Id2 promoter decreased following TLR stimulation, while activating histone modifications increased. These data are consistent with transcriptional induction of Id2 upon TLR stimulation. Future work will investigate the effect of additional regulators and TLR-regulated pathways on Id2 induction in TLR7/9-stimulated pDCs.

Human Miscarriage Is Associated With Dysregulations in Peripheral Blood-Derived Myeloid Dendritic Cell Subsets.

Dendritic cells (DC) are critically involved in decisions related to the acceptance or rejection of the foreign fetal antigens by the maternal immune system. However, particularly for human peripheral blood DCs (PBDC), available literature is rather inconsistent and the factors regulating these cells are ill-defined. Here, we investigated the phenotype and functionality of different human PBDC subsets during normal and pathologic pregnancies and studied an involvement of human chorionic gonadotropin (hCG) in PBDC regulation. Peripheral blood samples were obtained from normal pregnant women in all three trimesters, from first trimester miscarriage patients and from healthy non-pregnant women. Samples were analyzed for plasma hCG levels, for regulatory T (Treg) cell numbers, for frequencies of total and mature plasmacytoid (PDC) and myeloid (MDC1 and MDC2) PBDC subsets and for their cytokine secretion. In vitro assays, culturing PDC, MDC1 or MDC2 in the presence of two trophoblast cell lines, placenta explant supernatants or two hCG preparations were performed. The Treg-inducing capability of hCG- or non-hCG-treated stimulated MDC1 was assessed. Total and mature MDC1 and MDC2 frequencies increased during the first and second trimester of normal pregnancy, respectively. Miscarriage was associated with a reduced MDC1 and an increased MDC2 activation profile. PDC were not altered neither during normal pregnancy progression nor during miscarriage. In vitro, the culture of isolated PBDC subsets in the presence of placenta-derived factors impaired the maturation of MDC1 and differentially affected PDC maturation. An inhibitory effect on MDC1 and PDC maturation was also proven for the urine-derived hCG preparation. Finally, we observed a Treg cell elevation during early normal pregnancy that was not present in miscarriages. Stimulated MDC1 induced Treg cells in vitro, however, hCG was not involved in this process. Our findings suggest that during normal pregnancy PBDC subsets are differentially regulated dependent on gestational age. Miscarriage seems to be associated with dysregulations in the myeloid PBDC subsets and with disturbances in Treg cell frequencies. Moreover, our results propose an interdependency between MDC1 and Treg cells during early pregnancy. hCG, although shown to impair MDC1 maturation, does not seem to be a key regulator of PBDC alterations during pregnancy.

Regulation of NF-κB- and STAT1-mediated plasmacytoid dendritic cell functions by A20.

Dendritic cells (DCs) are professional antigen presenting cells involved in the induction of T cell-mediated adaptive immunity. Plasmacytoid DCs (pDCs) originate from lymphoid precursors and produce type I interferons (IFNs) in response to pathogens. A20 is considered as a negative regulator of toll-like receptor (TLR) signaling pathways, in which Toxoplasma gondii- derived profilin (TgPRF) is a TLR11/12 ligand recognised by DCs to stimulate their maturation/activation. Little is known about contributions of A20 to changes in biological properties of pDCs. The present study, therefore, explored whether pDC functions are influenced by A20. To this end, bone marrow cells were isolated and cultured with Flt3L to attain CD8DCs, CD11bDCs and pDCs and followed by challenge with TgPRP in the presence or absence of A20 siRNA. Expression of maturation markers were analysed by flow cytometry, and secretion of inflammatory cytokines by ELISA, cell migration by a transwell migration assay and expression of signalling molecules by western blotting. As a result, treatment with A20 siRNA enhanced activations of IκB-α and STAT-1, leading to increases in expressions of maturation markers and cytokine productions as well as migration of TgPRP-treated pDCs, while mature CD11bDCs produced at higher levels of TNF-α and IL-6 only. In addition, functions of CD8DCs remained unaltered following A20 silencing. The effects of A20 on pDC maturation and activation were completely abolished by IKK inhibitor and partially blunted by fludarabine. In conclusion, the inhibitory effects of A20 on pDC functions are expected to affect the immune response in T. gondii infection.

Differential effects of glycerophospholipids versus glycosphingolipids on dendritic cell subsets

Abstract Self-glycerophospholipids, such as phosphatidic acid (PA), bind CD1d, and activate phospholipid-reactive T cells (PLT) (Eur J Immunol 2018). Glycosphingolipids, such as α-GalCer (αGC), activate iNKT cells and exhibit many immune effects, including dendritic cell (DC) maturation. Here, we investigated the effect of glycerophospholipids on DC. Immunization with PA led to a rapid and persistent increase in the proportions and total numbers of plasmacytoid DC (pDC, CD11c+PDCA1+B220+ cells). In contrast, pDCs were initially reduced after αGC injection, but the total numbers recovered later. PA also caused an increase, albeit delayed as compared to increase in pDC, in myeloid DC (mDC)/non-pDC (CD11c+PDCA1− cells). mDC were increased at later timepoints with αGC. Further, PA increased expression of MHCII, CD86 and CD40 on pDC, but only a minimal increase in these on mDC. In contrast, αGC caused a minimal increase in these on pDC, but increased CD80 and CD86 expression on mDC. Finally, only pDC, but not mDC, produced IFNα after αGC or PA. Intriguingly, αGC that caused a less profound pDC maturation than PA induced more IFNα by pDC than PA. αGC, but not PA, also increased TNFα production by pDC. Overall, the two lipid Ag elicited different, complex effects on DC subsets: PA led to rapid expansion and maturation of pDC but did not increase their production of pro-inflammatory cytokines. In contrast, αGC that had little effects on pDC maturation and expansion increased IFNα and TNFα production by pDC. mDC were increased with both PA and αGC, but their costimulatory molecules’ expression increased more with αGC than with PA. Such differential effects of different lipids on DC subsets may have important immune and pathogenetic implications.

Toll-Like Receptor 7 (TLR-7) and TLR-9 Agonists Improve Hepatitis C Virus Replication and Infectivity Inhibition by Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) are innate immune cells with high antiviral activity triggered by Toll-like receptor 7 (TLR-7) and TLR-9 stimulation. Moreover, they are important mediators between innate and adaptive immunity. Although nowadays there is available an effective therapeutic arsenal against hepatitis C virus (HCV), a protective vaccine is not available. We have analyzed the pDCs' response to HCV infection in a hepatitis C virus (HCV)-Huh7.5 virus-cell system, which allows completion of the virus infectious cycle. pDCs were cocultured following human immunodeficiency virus (HIV) aldrithiol-2 (AT-2 [TLR-7 agonist]) inactivation and CpG (TLR-9 agonist) stimulation. We employed three virus derivatives-wild-type Jc1, interferon (IFN)-resistant virus IR, and high-replicative-fitness virus P100-in order to explore additional IFN-α-related virus inhibition mechanisms. pDCs inhibited HCV infectivity and replication and produced IFN-α. After TLR-7 and TLR-9 stimulation, inhibition of infectivity and IFN-α production by pDCs were enhanced. TLR-7 stimulation drove higher TNF-related apoptosis-inducing ligand (TRAIL) expression in pDCs. Additionally, TLR-7- and TLR-9-stimulated pDCs exhibited a mature phenotype, improving the antigen presentation and lymph node homing-related markers. In conclusion, pDCs could serve as a drug target against HCV in order to improve antiviral activity and as an enhancer of viral immunization.IMPORTANCE We implemented a coculture system of pDCs with HCV-infected hepatoma cell line, Huh7.5. We used three HCV derivatives in order to gain insight into pDCs' behavior against HCV and associated antiviral mechanisms. The results with this cell coculture system support the capacity of pDCs to inhibit HCV replication and infectivity mainly via IFN-α, but also through additional mechanisms associated with pDC maturation. We provided evidence that TLR agonists can enhance antiviral pDCs' function and can induce phenotypic changes that may facilitate the interplay with other immune cells. These findings suggest the possibility of including TLR agonists in the strategies of HCV vaccine development.

Long Noncoding RNA Signatures Induced by Toll-Like Receptor 7 and Type I Interferon Signaling in Activated Human Plasmacytoid Dendritic Cells.

Long noncoding RNAs (lncRNAs) exhibit highly lineage-specific expression and act through diverse mechanisms to exert control over a wide range of cellular processes. lncRNAs can function as potent modulators of innate immune responses through control of transcriptional and posttranscriptional regulation of mRNA expression and processing. Recent studies have demonstrated that lncRNAs participate in the regulation of antiviral responses and autoimmune disease. Despite their emerging role as immune mediators, the mechanisms that govern lncRNA expression and function have only begun to be characterized. In this study, we explore the role of lncRNAs in human plasmacytoid dendritic cells (pDCs), which are critical sentinel sensors of viral infection and contribute to the development of autoimmune disease. Using genome-wide sequencing approaches, we dissect the contributions of Toll-like receptor 7 (TLR7) and type I interferon (IFN-I) in the regulation of coding and noncoding RNA expression in CAL-1 pDCs treated with R848 or IFNβ. Functional enrichment analysis reveals both the unique and synergistic roles of TLR7 and IFN-I signaling in the orchestration of pDC function. These observations were consistent with primary cell immune responses elicited by detection of viral infection. We identified and characterized the conditional TLR7- and IFN-I-dependent regulation of 588 lncRNAs. Dysregulation of these lncRNAs could significantly alter pDC maturation, IFN-I and inflammatory cytokine production, antigen presentation, costimulation or tolerance cues, turnover, or localization, all consequential events during viral infection or IFN-I-driven autoimmune diseases such as systemic lupus erythematosus. These findings demonstrate the differential responsiveness of lncRNAs to unique immune stimuli, uncover regulatory mechanisms of lncRNA expression, and reveal a novel and tractable platform for the study of lncRNA expression and function.

TLR7 polymorphism, sex and chronic HBV infection influence plasmacytoid DC maturation by TLR7 ligands

TLR7 agonists are of high interest for the treatment of cancer, auto-immunity and chronic viral infections. They are known to activate plasmacytoid dendritic cells (pDCs) to produce high amounts of Type I Interferon (IFN) and to facilitate T and B cell responses, the latter with the help of maturation markers such as CD40, CD80 and CD86. The TLR7 single nucleotide polymorphism (SNP) rs179008 (GLn11Leu), sex and chronic viral infection have all been reported to influence pDC IFN production. It is unknown, however, whether these factors also influence pDC phenotypic maturation and thereby IFN-independent pDC functions. Furthermore, it is unclear whether SNP rs179008 influences HBV susceptibility and/or clearance.Here we investigated whether the SNP rs179008, sex and HBV infection affected phenotypic maturation of pDCs from 38 healthy individuals and 28 chronic HBV patients. In addition, we assessed SNP prevalence in a large cohort of healthy individuals (n = 231) and chronic HBV patients (n = 1054).Consistent with previous reports, the rs179008 variant allele was largely absent in Asians and more prevalent in Caucasians. Among Caucasians, the SNP was equally prevalent in healthy and chronically infected males. The SNP was, however, significantly more prevalent in healthy females than in those with chronic HBV infection (42 versus 28%), suggesting that in females it may offer protection from chronic infection. Ex vivo experiments demonstrated that induction of the co-stimulatory molecules CD40 and CD86 by TLR7 ligands, but not TLR9 ligands, was augmented in pDCs from healthy SNP-carrying females. Furthermore, CD80 and CD86 upregulation was more pronounced in females independent of the SNP. Lastly, our data suggested that chronic HBV infection impairs pDC maturation. These findings provide insight into factors determining TLR7 responses, which is important for further clinical development of TLR7-based therapies.

Myeloid and plasmacytoid dendritic cell subsets are differentially regulated by the pregnancy hormone human Chorionic Gonadotropin

Abstract Dendritic cells (DCs) are critically involved in the establishment and maintenance of fetal tolerance. However, the precise mechanisms underlying their regulation during pregnancy are still not defined. The placenta-derived hormone human Chorionic Gonadotropin (hCG) possesses immune modulatory properties and is commonly used in artificial reproductive techniques (ART). Here, we analyzed the effect of placenta-derived hCG and two hCG preparations on different DC subsets. After isolation from peripheral blood of non pregnant women, DCs were stimulated with LPS to induce maturation. In parallel, DCs were co-cultured with placenta-derived hCG (JEG-3 cells), with urine-derived hCG or recombinant hCG. The total number and maturation state of two myeloid DC (MDC1; MDC2) and one plasmacytoid DC subset (PDC) was evaluated. Additionally, the levels of IL-6, IL-8, IL-10, IL-12 and TNF were determined in co-culture supernatants. In all experiments, unstimulated DCs and stimulated DCs cultured alone served as controls. JEG-3 impaired the LPS-induced increase of total MDC1 while both hCG preparations hindered the increment of total MDC1 and MDC2. Moreover, JEG-3 interfered with PDC and MDC1 maturation while both hCG preparations hampered maturation of all DC subsets. However, significant effects could only be observed on the MDC1 subset. Cytokine assays revealed that JEG-3 and both hCG preparations impaired LPS-induced secretion of all analyzed cytokines. Our results suggest that hCG from distinct sources regulates DC subsets in a different manner. This information does not only contribute to our understanding of a differential regulation of various DC subsets during pregnancy but is also relevant for the use of hCG preparations in ART.

84. Alpha 1 Antitrypsin Protein & Gene Therapies for the Treatment of Lupus in Animal Models

Systemic lupus erythematosus (SLE) is an autoimmune rheumatic disorder. Although the pathogenesis of SLE remains elusive, over-reactive dendritic cells (DCs), which promote B cell activation and autoantibodies secretion, play critical roles in the disease development. Alpha-1 antitrypsin (AAT) is a multifunctional protein with anti-inflammatory, cytoprotective and immunoregulatory properties. We has shown that AAT has protective effect in autoimmune disease models including type 1 diabetes and rheumatoid arthritis. In this study, we tested therapeutic potentials of AAT in controlling DC and B cells function and the development of SLE in a spontaneous lupus mouse model. Using bone marrow cells from normal (C57BL/6) and lupus prone (B6. TC, MRL/lpr) mice, we showed that AAT treatment significantly inhibited lipopolysaccharide (LPS) (TLR4 agonist) or CpG (TLR9 agonist) induced DC (cDC and pDC) maturation. In this in vitro system, AAT significantly inhibited the production of IFN-I, IL-1β, IL-6 and TNF-α from DCs. AAT treated DC also have significantly lower potential in stimulating B cell proliferation and functions. Based on these results, performed following experiments using spontaneous lupus mouse models. In order to test this therapeutic potential, we treated female MRL/lpr mice (at 7-weeks of age, n=10) with clinical grade of AAT (2 mg/mouse, every 3 days) or PBS for 11 weeks. Consistent with our in vitro observations, results from this experiment showed that AAT treatment significantly inhibited DC maturation and reduced DC susceptibility to LPS stimulation. Importantly, AAT treatment significantly lowered serum antibody (anti-dsDNA IgG and anti-nuclear antibodies, ANAs) levels and urine albumin levels. Detailed pathological examinations showed that AAT treatment prevented kidney disease development. To confirm this observations and test the long-term effect of AAT treatment, we performed second experiment using both AAT protein and gene therapies. In this experiment, we treated adult female NZM2410 (another commonly used spontaneous lupus model) mice with AAT protein and rAAV8-CB-AAT vector (1×1011 vg/mouse, single IP injection). While 100% mice in control group develop lupus and died at 46 week of age, 50% mice in AAT protein treatment group remain lupus free at 55 weeks of age. Intriguingly, AAT gene therapy (rAAV8-CB-AAT) significantly prevent lupus development (70% mice remain lupus free at 63 weeks of age, P=0.0059). These results clearly demonstrated the therapeutic effect of AAT protein and gene therapy in lupus mouse model. Our results also indicated the advantage of rAAV8 mediated gene therapy for the chronic autoimmune diseases. In summary, we showed AAT inhibited the activation and function of DCs and B cells in vitro and in vivo. We also showed AAT treatment (protein therapy and gene therapy) prevent lupus development in spontaneous lupus mouse models. Considering the safety profile of AAT and rAAV, our results may be translated into clinical application and lead to a novel (safe and effective) therapy for SLE in humans. This work was supported by grants from University of Florida and Grifols, Inc.

CD28 Deficiency Enhances Type I IFN Production by Murine Plasmacytoid Dendritic Cells.

Type I IFNs (IFN-I) are key innate mediators that create a profound antiviral state and orchestrate the activation of almost all immune cells. Plasmacytoid dendritic cells (pDCs) are the most powerful IFN-I-producing cells and play important roles during viral infections, cancer, and autoimmune diseases. By comparing gene expression profiles of murine pDCs and conventional DCs, we found that CD28, a prototypic T cell stimulatory receptor, was highly expressed in pDCs. Strikingly, CD28 acted as a negative regulator of pDC IFN-I production upon TLR stimulation but did not affect pDC survival or maturation. Importantly, cell-intrinsic CD28 expression restrained pDC (and systemic) IFN-I production during in vivo RNA and DNA viral infections, limiting antiviral responses and enhancing viral growth early after exposure. Finally, CD28 also downregulated IFN-I response upon skin injury. Our study identified a new pDC regulatory mechanism by which the same CD28 molecule that promotes stimulation in most cells that express it is co-opted to negatively regulate pDC IFN-I production and limit innate responses.

Estrogen Receptor α Deficiency Modulates TLR Ligand-Mediated PDC-TREM Expression in Plasmacytoid Dendritic Cells in Lupus-Prone Mice.

Female lupus-prone NZM2410 estrogen receptor α (ERα)-deficient mice are protected from renal disease and have prolonged survival compared with wild-type littermates; however, the mechanism of protection is unknown. Plasmacytoid dendritic cells (pDCs) and type I IFN drive lupus pathogenesis. Estrogen acting via ERα enhances both pDC development and IFN production. The objectives for this study were to determine if ERα modulates pDC function and IFN activity in predisease NZM2410 mice as a possible protective mechanism of ERα deficiency in lupus-prone mice. We measured the effect of ERα deficiency on spleen pDC frequency, number, maturation, and activation state. ERα deficiency reduced type I IFN activity and the frequency of MHC class II(+) pDCs in the spleen without altering overall pDC frequency, number, or maturation state. Additionally, ERα-deficient NZM2410 mice had a significantly decreased frequency of pDCs expressing PDC-TREM, a modulator of TLR-mediated IFN production. After in vitro TLR9 stimulation, ERα deficiency significantly reduced the expression of PDC-TREM on pDCs from both NZM2410 and C57BL/6 mice. Thus, we have identified a significant effect of ERα deficiency on pDCs in predisease NZM2410 mice, which may represent a mechanism by which ERα deficiency protects NZM2410 mice from lupuslike disease.

Activated T cells enhance interferon-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes

ObjectivesPatients with systemic lupus erythematosus (SLE) have an ongoing interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs). We investigated whether T cells can promote IFN-α production by pDCs.MethodsHuman pDCs were...