Hydroxychloroquine is associated with impaired interferon-alpha and tumor necrosis factor-alpha production by plasmacytoid dendritic cells in systemic lupus erythematosus

Osteoarthritis is a prevalent and disabling disease affecting an increasingly large swathe of the world population. While clinical osteoarthritis is a late-stage condition for which disease-modifying opportunities are limited, osteoarthritis typically develops over decades, offering a long window of time to potentially alter its course. The etiology of osteoarthritis is multifactorial, showing strong associations with highly modifiable risk factors of mechanical overload, obesity and joint injury. As such, characterization of pre-osteoarthritic disease states will be critical to support a paradigm shift from palliation of late disease towards prevention, through early diagnosis and early treatment of joint injury and degeneration to reduce osteoarthritis risk. Joint trauma accelerates development of osteoarthritis from a known point in time. Human joint injury cohorts therefore provide a unique opportunity for evaluation of pre-osteoarthritic conditions and potential interventions from the earliest stages of degeneration. This review focuses on recent advances in imaging and biochemical biomarkers suitable for characterization of the pre-osteoarthritic joint as well as implications for development of effective early treatment strategies.     

Activation with CpG-A and CpG-B oligonucleotides reveals two distinct regulatory pathways of type I IFN synthesis in human plasmacytoid dendritic cells

Two different CpG oligonucleotides (ODN) were used to study the regulation of type I IFN in human plasmacytoid dendritic cells (PDC): ODN 2216, a CpG-A ODN, known to induce high amounts of IFN-alpha in PDC, and ODN 2006, a CpG-B ODN, which is potent at stimulating B cells. CpG-A ODN showed higher and prolonged kinetics of type I IFN production compared with that of CpG-B ODN. In contrast, CpG-B ODN was more active than CpG-A ODN in stimulating IL-8 production and increasing costimulatory and Ag-presenting molecules, suggesting that CpG-A and CpG-B trigger distinct regulatory pathways in PDC. Indeed, CpG-A ODN, but not CpG-B ODN, activated the type I IFNR-mediated autocrine feedback loop. PDC were found to express high constitutive levels of IFN regulatory factor (IRF)7. IRF7 and STAT1, but not IRF3, were equally up-regulated by both CpG-A and CpG-B. CD40 ligand synergistically increased CpG-B-induced IFN-alpha independent of the IFNR but did not affect CpG-B-induced IFN-beta. In conclusion, our studies provide evidence for the existence of two distinct regulatory pathways of type I IFN synthesis in human PDC, one dependent on and one independent of the IFNR-mediated feedback loop. The alternate use of these pathways is based on the type of stimulus rather than the quantity of IFN-alphabeta available to trigger the IFNR. Constitutive expression of IRF7 and the ability to produce considerable amounts of IFN-alpha independent of the IFNR seem to represent characteristic features of PDC.     

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).     

Activation of plasmacytoid dendritic cells by Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with multiple human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Following primary infection, KSHV typically goes through a brief period of lytic replication prior to the establishment of latency. Plasmacytoid dendritic cells (pDCs) are the major producers of type 1 interferon (IFN), primarily in response to virus infection. Toll-like receptors (TLRs) are key components of the innate immune system, and they serve as pathogen recognition receptors that stimulate the host antiviral response. pDCs express exclusively TLR7 and TLR9, and it is through these TLRs that the type 1 interferon response is activated in pDCs. Currently, it is not known whether KSHV is recognized by pDCs and whether activation of pDCs occurs in response to KSHV infection. We now report evidence that KSHV can infect human pDCs and that pDCs are activated upon KSHV infection, as measured by upregulation of CD83 and CD86 and by IFN-α secretion. We further show that induction of IFN-α occurs through activation of TLR9 signaling and that a TLR9 inhibitor diminishes the production and secretion of IFN-α by KSHV-infected pDCs.     

Role for plasmacytoid dendritic cells in anti-HIV innate immunity

The role of plasmacytoid dendritic cells (pDC) in anti-HIV immunity is mostly represented by the production of type I IFN in response to HIV infection in vitro and in vivo. This production is decreased in HIV-1 infected patients at the time of primary infection and during chronic disease in association with progression of disease. Circulating pDC counts are decreased concomitantly with type I IFN, and both factors correlate inversely overall with viral loads and positively with CD4+ T-cell counts. These parameters might be used in clinical immunology to monitor treatment and as predictive factors of immune control of HIV-1 replication to help decide whether to interrupt antiretroviral treatment. They may be related to control of HIV replication as well as to pathogenesis of infection, perhaps in setting the balance between immunity or tolerance to the virus. A better understanding of these parameters is required while attempts to use IFN-alpha or ligands of Toll-like receptors found on pDC are being made.     

Decreased interferon-alpha production and impaired regulatory function of plasmacytoid dendritic cells induced by the hepatitis C virus NS 5 protein

pDC are known to produce large amount of IFN-alpha/beta in response to viruses, and act as a major link between the innate and adaptive immune response. This study concentrated on the interaction of human peripheral blood derived pDC with HCV NS3, NS4, and NS5 proteins, and their maturation, cytokine secretion and functional properties. It was shown that HCV NS5 interferes with CD40L induced maturation of pDC as indicated by decreased expression of CD83 and CD86 markers. CpG ODN stimulated HCV NS3 and NS5 treated pDC showed decreased production of IFN-alpha. In the case of NS3, IFN-alpha production was reduced to 126 pg/ml as compared to 245 pg/ml in controls (P < 0.01), and with NS5, IFN-alpha production was reduced to 92 pg/ml as compared to 238 pg/ml in controls (P < 0.05). In the presence of HCV NS5, the T cell stimulatory capacity of pDC was impaired, as indicated by decreased proliferation of T cells, and decreased production by the T cells of IFN-gamma, which were down to 86 pg/ml as compared to 260 pg/ml in controls (P < 0.05). These results suggest that HCV NS5 impairs pDC function and is in agreement with several other in vivo studies indicating decreased numbers of, and dysfunctional pDC, in chronic HCV infected patients.     

Profound depletion of host conventional dendritic cells, plasmacytoid dendritic cells, and B cells does not prevent graft-versus-host disease induction

The efficacy of allogeneic hematopoietic stem cell transplantation is limited by graft-versus-host disease (GVHD). Host hematopoietic APCs are important initiators of GVHD, making them logical targets for GVHD prevention. Conventional dendritic cells (DCs) are key APCs for T cell responses in other models of T cell immunity, and they are sufficient for GVHD induction. However, we report in this article that in two polyclonal GVHD models in which host hematopoietic APCs are essential, GVHD was not decreased when recipient conventional DCs were inducibly or constitutively deleted. Additional profound depletion of plasmacytoid DCs and B cells, with or without partial depletion of CD11b(+) cells, also did not ameliorate GVHD. These data indicate that, in contrast with pathogen models, there is a surprising redundancy as to which host cells can initiate GVHD. Alternatively, very low numbers of targeted APCs were sufficient. We hypothesize the difference in APC requirements in pathogen and GVHD models relates to the availability of target Ags. In antipathogen responses, specialized APCs are uniquely equipped to acquire and present exogenous Ags, whereas in GVHD, all host cells directly present alloantigens. These studies make it unlikely that reagent-based host APC depletion will prevent GVHD in the clinic.     

Chlamydia pneumoniae infection induced allergic airway sensitization is controlled by regulatory T-cells and plasmacytoid dendritic cells

Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2-/-, and TLR4-/- mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2-/- mice, but not in TLR4-/- mice, due to differential Treg responses in these genotypes. TLR2-/- mice had reduced numbers of Tregs in the lung during CP infection while TLR4-/- mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.     

TLR ligand-dependent activation of naive CD4 T cells by plasmacytoid dendritic cells is impaired in hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling, we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C), R-848, or CpG), in the presence or absence of cytokine (TNF-alpha or IL-3), and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons, the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast, PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation, and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection.     

Epstein-Barr virus induction by a serum factor. Purification of a high molecular weight protein that is responsible for induction

Serum contains a factor that induces Epstein-Barr virus antigens in latently infected human lymphoblastoid cell lines and that cooperates with chemical inducers. Here we report the purification of a protein that is responsible for the effect. Using ammonium sulphate precipitation, chromatography on DEAE-agarose and cellulose, molecular sieve chromatography on Bio-Gel A-5, and glycerol gradient centrifugation, the factor was enriched about 300,000-fold compared to calf serum. Under neutral conditions, the protein chromatographed as a high molecular weight protein of about 5.5 X 10(5) both in its active and inactive form. Both forms of the molecule sedimented at about 7 s. The factor is an acidic protein with a pI of 5. It seems to be composed of high molecular and low molecular weight subunits. Nanogram amounts of purified factor are sufficient for measurable inducing effects.     

Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy

Recent evidence suggests that the previously enigmatic cell type designated plasmacytoid monocytes can function as dendritic cells and contribute substantially to both innate and adaptive immunity. This cell type has previously been described only in bone marrow, blood, and organized lymphoid tissue, but not at effector sites with direct Ag exposure such as the mucosae. Plasmacytoid dendritic cells (P-DCs) matured in vitro can induce T cells to produce allergy-promoting Th2 cytokines; therefore, their possible occurrence in nasal mucosa during experimentally elicited allergic rhinitis was examined. Patients with silent nasal allergy were challenged topically with relevant allergen daily for 7 days. Biopsy specimens as well as blood samples were obtained before and during such provocation, and P-DCs were identified by their high expression of CD123 (IL-3R alpha-chain), together with CD45RA. Our results showed that P-DCs were present in low and variable numbers in normal nasal mucosa but increased dramatically during the allergic reaction. This accumulation concurred with the expression of the L-selectin ligand peripheral lymph node addressin on the mucosal vascular endothelium. The latter observation was particularly interesting in view of the high levels of L-selectin on circulating P-DC precursors and of previous reports suggesting that these cells can enter organized lymphoid tissue via high endothelial venules (which express peripheral lymph node addressin constitutively). Together, our findings suggested that P-DCs are involved in the triggering of airway allergy and that they are directed to allergic lesions by adhesion molecules that normally mediate leukocyte extravasation in organized lymphoid tissue.     

Recruitment of dendritic cells is responsible for intestinal epithelial damage in the pathogenesis of necrotizing enterocolitis by Cronobacter sakazakii

Cronobacter sakazakii is a Gram-negative pathogen associated with the cases of necrotizing enterocolitis (NEC) that result from formula contamination. In a mouse model of NEC, we demonstrate that C. sakazakii infection results in epithelial damage by recruiting greater numbers of dendritic cells (DCs) than macrophages and neutrophils in the gut and suppresses DC maturation, which requires outer membrane protein A (OmpA) expression in C. sakazakii. Pretreatment of intestinal epithelial cell monolayers with supernatant from OmpA(+) C. sakazakii/DC culture markedly enhanced membrane permeability and enterocyte apoptosis, whereas OmpA(-) C. sakazakii/DC culture supernatant had no effect. Analysis of OmpA(+) C. sakazakii/DC coculture supernatant revealed significantly greater TGF-β production compared with the levels produced by OmpA(-) C. sakazakii infection. TGF-β levels were elevated in the intestinal tissue of mice infected with OmpA(+) C. sakazakii. Cocultures of CaCo-2 cells and DCs in a "double-layer" model followed by infection with OmpA(+) C. sakazakii significantly enhanced monolayer leakage by increasing TGF-β production. Elevated levels of inducible NO synthase (iNOS) were also observed in the double-layer infection model, and abrogation of iNOS expression prevented the C. sakazakii-induced CaCo-2 cell monolayer permeability despite the presence of DCs or OmpA(+) C. sakazakii/DC supernatant. Blocking TGF-β activity using a neutralizing Ab suppressed iNOS production and prevented apoptosis and monolayer leakage. Depletion of DCs in newborn mice protected against C. sakazakii-induced NEC, whereas adoptive transfer of DCs rendered the animals susceptible to infection. Therefore, C. sakazakii interaction with DCs in intestine enhances the destruction of the intestinal epithelium and the onset of NEC due to increased TGF-β production.     

Cutting edge: selective usage of chemokine receptors by plasmacytoid dendritic cells

The existence of dendritic cell (DC) subsets is firmly established, but their trafficking properties are virtually unknown. In this study, we show that myeloid (M-DCs) and plasmacytoid (P-DCs) DCs isolated from human blood differ widely in the capacity to migrate to chemotactic stimuli. The pattern of chemokine receptors expressed by blood M-DCs and P-DCs, with the exception of CCR7, is similar. However, most chemokine receptors of P-DCs, in particular those specific for inflammatory chemokines and classical chemotactic agonists, are not functional in circulating cells. Following maturation induced by CD40 ligation, the receptors for inflammatory chemokines are down-regulated, and CCR7 on P-DCs becomes coupled to migration. The drastically impaired capacity of blood P-DCs to migrate in response to inflammatory chemotactic signals contrasts with the response to lymph node-homing chemokines, indicating a propensity to migrate to secondary lymphoid organs rather than to sites of inflammation.     

Characterization of myeloid and plasmacytoid dendritic cells in human lung

Dendritic cells (DCs) are bone marrow-derived mononuclear cells that play a central role in the initiation of immune responses. Because human lung DCs have been incompletely characterized, we enumerated and phenotyped mononuclear cell populations from excess lung tissue obtained at surgery. Myeloid DCs (MDCs) were identified as CD1c(+)CD11c(+)CD14(-)HLA-DR(+) cells and comprised approximately 2% of low autofluorescent (LAF) mononuclear cells. Plasmacytoid DCs (PDCs) were characterized as CD123(+)CD11c(-)CD14(-)HLA-DR(+) cells and comprised approximately 1.0% of the LAF mononuclear cells. Cells enriched in MDCs expressed CD86, moderate CD80, and little CD40, but cells enriched in PDCs had little to no expression of these three costimulatory molecules. CD11c(+)CD14(-) lineage-negative (MDC-enriched) LAF cells were isolated and shown to be much more potent in stimulating an alloreaction than CD11c(+)CD14(+) lineage-negative (monocyte-enriched) LAF cells. PDC-enriched cells were more capable of responding to a TLR-7 agonist by secreting IFN-alpha than MDC-enriched cells. MDC-enriched cells were either CD123(+) or CD123(-), but both subsets secreted cytokines and chemokines typical of MDC upon stimulation with a TLR-4 agonist and both subsets failed to secrete IFN-alpha upon stimulation with a TLR-7 agonist. By immunohistochemistry, we identified MDCs throughout different anatomical locations of the lung. However, our method did not allow the localization of PDCs with certainty. In conclusion, in the human lung MDCs were twice as numerous and expressed higher levels of costimulatory molecules than PDCs. Our data suggest that both lung DC subsets exert distinct immune modulatory functions.     

Productive infection of plasmacytoid dendritic cells with human immunodeficiency virus type 1 is triggered by CD40 ligation

Immature plasmacytoid dendritic cells are the principal alpha interferon-producing cells (IPC), responsible for primary antiviral immunity. IPC express surface molecules CD4, CCR5, and CXCR4, which are known coreceptors required for human immunodeficiency virus (HIV) infection. Here we show that IPC are susceptible to and replicate HIV type 1 (HIV-1). Importantly, viral replication is triggered upon activation of IPC with CD40 ligand, a signal physiologically delivered by CD4 T cells. Immunohistochemical staining of tonsil from HIV-infected individuals reveals HIV p24(+) IPC, consistent with in vivo infection of these cells. IPC exposed in vitro to HIV produce alpha interferon, which partially inhibits viral replication. Nevertheless, IPC efficiently transmit HIV-1 to CD4 T-cells, and such transmission is also augmented by CD40 ligand activation. IPC produce RANTES/CCL5 and MIP-1alpha/CCL3 when exposed to HIV in vitro. IPC also induce na?ve CD4 T cells to proliferate and would therefore preferentially infect these cells. These results indicate that IPC may play an important role in the dissemination of HIV.     

Systemic control of plasmacytoid dendritic cells by CD8+ T cells and commensal microbiota

The composition of the intestinal microbial community is a distinctive individual trait that may divergently influence host biology. Because dendritic cells (DC) regulate the quality of the host response to microbiota, we evaluated DC in mice bearing distinct enteric microbial communities divergent for colitis susceptibility. Surprisingly, a selective, systemic reduction of plasmacytoid dendritic cells (pDC) was observed in isogenic mice with different microbiota: restricted flora (RF) vs specific pathogen free (SPF). This reduction was not observed in germfree mice, suggesting that the pDC deficiency was not simply due to a lack of intestinal microbial products. The microbial action was linked to cytotoxic CD8(+) T cells, since pDC in RF mice were preserved in the CD8(-/-) and perforin(-/-) genotypes, partially restored by anti-CD8beta Ab, and augmented in SPF mice bearing the TAP(-/-) genotype. Direct evidence for pDC cytolysis was obtained by rapid and selective pDC depletion in SPF mice transferred with RF CD8(+) T cells. These data indicate that commensal microbiota, via CTL activation, functionally shape systemic immune regulation that may modify risk of inflammatory disease.     

Propaedeutic study for the delivery of nucleic acid-based molecules from PLGA microparticles and stearic acid nanoparticles

We studied the mechanism governing the delivery of nucleic acid-based drugs (NABD) from microparticles and nanoparticles in zero shear conditions, a situation occurring in applications such as in situ delivery to organ parenchyma. The delivery of a NABD molecule from poly(DL-lactide-co-glycolide) (PLGA) microparticles and stearic acid (SA) nanoparticles was studied using an experimental apparatus comprising a donor chamber separated from the receiver chamber by a synthetic membrane. A possible toxic effect on cell biology, as evaluated by studying cell proliferation, was also conducted forjust PLGA microparticles. A mathematical model based on the hypothesis that NABD release from particles is due to particle erosion was used to interpret experimental release data. Despite zero shear conditions imposed in the donor chamber, particle erosion was the leading mechanism for NABD release from both PLGA microparticles and SA nanoparticles. PLGA microparticle erosion speed is one order of magnitude higher than that of competing SA nanoparticles. Finally, no deleterious effects of PLGA microparticles on cell proliferation were detected. Thus, the data here reported can help optimize the delivery systems aimed at release of NABD from micro- and nanoparticles.     

Type I interferon is required for T helper (Th) 2 induction by dendritic cells

Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN‐I) in enabling this process. An IFN‐I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN‐I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1^?/? mice were incapable of initiating Th2 responses in vivo. These data demonstrate for the first time that the influence of IFN‐I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses.