Induction of HLA-B27 heavy chain homodimer formation after activation in dendritic cells

Introduction

Ankylosing spondylitis (AS) is a severe, chronic inflammatory arthritis, with a strong association to the human major histocompatibilty complex (MHC) class I allele human leucocyte antigen (HLA) B27. Disulfide-linked HLA-B27 heavy-chain homodimers have been implicated as novel structures involved in the aetiology of AS. We have studied the formation of HLA-B27 heavy-chain homodimers in human dendritic cells, which are key antigen-presenting cells and regulators of mammalian immune responses.

Method

Both an in vitro dendritic-like cell line and monocyte-derived dendritic cells from peripheral blood were studied. The KG-1 dendritic-like cell line was transfected with HLA-B27 cDNA constructs, and the cellular distribution, intracellular assembly and ability of HLA-B27 to form heavy-chain homodimers was compared with human monocyte-derived dendritic cells after stimulation with bacterial lipopolysaccharide (LPS).

Results

Immature KG-1 cells expressing HLA-B27 display an intracellular source of MHC class I heavy-chain homodimers partially overlapping with the Golgi bodies, but not the endoplasmic reticulum, which is lost at cell maturation with phorbyl-12-myristate-13-acetate (PMA) and ionomycin. Significantly, the formation of HLA-B27 homodimers in transfected KG-1 cells is induced by maturation, with a transient induction also seen in LPS-stimulated human monocyte-derived dendritic cells expressing HLA-B27. The weak association of wildtype HLA-B*2705 with the transporter associated with antigen processing could also be enhanced by mutation of residues at position 114 and 116 in the peptide-binding groove to those present in the HLA-B*2706 allele.

Conclusion

We have demonstrated that HLA-B27 heavy-chain homodimer formation can be induced by dendritic cell activation, implying that these novel structures may not be displayed to the immune system at all times. Our data suggests that the behaviour of HLA-B27 on dendritic cells may be important in the study of inflammatory arthritis.     

Lnc-DC regulates cellular turnover and the HBV-induced immune response by TLR9/STAT3 signaling in dendritic cells

Background

Lnc-DC is a specific group of long non-coding (Lnc) RNAs in dendritic cells (DCs). Its function has been previously studied, and includes roles in dendritic cell differentiation and the progression of some diseases. In this study, we observed the critical role of Lnc-DC in regulating the differentiation, growth, and apoptosis of dendritic cells.

Methods

We first isolated peripheral blood mononuclear cells to culture and induce into DCs, which were then co-cultured with hepatitis B virus (HBV)-secreting HepG2.2.15 cells for the detection of changes in Lnc-DC. The expression levels of TLR9, p-STAT3, and SOCS3 were tested with qPCR and western blot. MTT assays were used to analyze the cell proliferation, cell cycle, and apoptosis. We used ELISA to test the expression of TNF-α, IL-1β, IL-6, IL-12p40, and IFN-γ.

Results

Co-culture with HBV-secreting HepG2.2.15 cells increased the level of Lnc-DC and activated TLR9/STAT3 signaling. The HBV DNA level (IU/ml) was positively correlated with levels of Lnc-DC and TLR9, further demonstrating that Lnc-DC was associated with the immune response of HBV. Lnc-DC was shown to regulate TLR9/STAT3 signaling in dendritic cells. More interestingly, the regulation of Lnc-DC controlled the immune response by reducing the concentration of secreted TNF-α, IL-6, IL-12, and IFN-γ, as well as increasing the IL-1β concentration in dendritic cells.

Conclusion

Lnc-DC is important in regulating the growth, apoptosis, and immune response of dendritic cells mediated by TLR9/STAT3 signaling, and was also activated by HBV. This study provides a previously unidentified mechanism underlying the immune response in dendritic cells.

     

Investigation of the geometry of the dendritic tree of retinal ganglion cells

Two parameters are proposed for classifying the structure of the dendritic trees of retinal ganglion cells: the mean distance between the ends of the terminal segments of the dendrites from the soma ( ) and the ratio between the number of horizontal and vertical dendritic segments (a). By using these parameters the dendritic trees of ganglion cells were divided into four types. To describe the two-dimensional structure of dendritic trees the following parameters are used: the distribution of intermediate and terminal dendritic segments in each series of branches depending on the angle of their orientation relative to the boundaries of the inner plexiform layer — p_Z (), q_Z (), and depending on their length — P_Z (1), q_Z (1). Histograms of these functions are given. By means of these histograms the two-dimensional structure of the dendritic tree can be drawn in such a way as to reproduce the structural features of actual dendritic trees with the accuracy of the parameters defining their appearance. The structural and functional significance of the parameters used is discussed.Kaunas Medical Institute. Translated from Neirofiziologiya, Vol. 5, No. 3, pp. 307–314, May–June, 1973.     

Phenotypic and functional abnormalities of bone marrow-derived dendritic cells in systemic lupus erythematosus

Introduction  

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoreactive T and B cells, which are believed to be secondary to deficient dendritic cells (DCs). However, whether DC abnormalities occur during their development in the bone marrow (BM) or in the periphery is not known.     

Uterine epithelial cell regulation of DC-SIGN expression inhibits transmitted/founder HIV-1 trans infection by immature dendritic cells

Background

Sexual transmission accounts for the majority of HIV-1 infections. In over 75% of cases, infection is initiated by a single variant (transmitted/founder virus). However, the determinants of virus selection during transmission are unknown. Host cell-cell interactions in the mucosa may be critical in regulating susceptibility to infection. We hypothesized in this study that specific immune modulators secreted by uterine epithelial cells modulate susceptibility of dendritic cells (DC) to infection with HIV-1.

Methodology/Principal Findings

Here we report that uterine epithelial cell secretions (i.e. conditioned medium, CM) decreased DC-SIGN expression on immature dendritic cells via a transforming growth factor beta (TGF-β) mechanism. Further, CM inhibited dendritic cell-mediated trans infection of HIV-1 expressing envelope proteins of prototypic reference. Similarly, CM inhibited trans infection of HIV-1 constructs expressing envelopes of transmitted/founder viruses, variants that are selected during sexual transmission. In contrast, whereas recombinant TGF- β1 inhibited trans infection of prototypic reference HIV-1 by dendritic cells, TGF-β1 had a minimal effect on trans infection of transmitted/founder variants irrespective of the reporter system used to measure trans infection.

Conclusions/Significance

Our results provide the first direct evidence for uterine epithelial cell regulation of dendritic cell transmission of infection with reference and transmitted/founder HIV-1 variants. These findings have immediate implications for designing strategies to prevent sexual transmission of HIV-1.     

Th17 cells and activated dendritic cells are increased in vitiligo lesions

Background

Vitiligo is a common skin disorder, characterized by progressive skin de-pigmentation due to the loss of cutaneous melanocytes. The exact cause of melanocyte loss remains unclear, but a large number of observations have pointed to the important role of cellular immunity in vitiligo pathogenesis.

Methodology/Principal Findings

In this study, we characterized T cell and inflammation-related dermal dendritic cell (DC) subsets in pigmented non-lesional, leading edge and depigmented lesional vitiligo skin. By immunohistochemistry staining, we observed enhanced populations of CD11c+ myeloid dermal DCs and CD207+ Langerhans cells in leading edge vitiligo biopsies. DC-LAMP+ and CD1c+ sub-populations of dermal DCs expanded significantly in leading edge and lesional vitiligo skin. We also detected elevated tissue mRNA levels of IL-17A in leading edge skin biopsies of vitiligo patients, as well as IL-17A positive T cells by immunohistochemistry and immunofluorescence. Langerhans cells with activated inflammasomes were also noted in lesional vitiligo skin, along with increased IL-1ß mRNA, which suggest the potential of Langerhans cells to drive Th17 activation in vitiligo.

Conclusions/Significance

These studies provided direct tissue evidence that implicates active Th17 cells in vitiligo skin lesions. We characterized new cellular immune elements, in the active margins of vitiligo lesions (e.g. populations of epidermal and dermal dendritic cells subsets), which could potentially drive the inflammatory responses.     

Interferon-induced protein IFIT4 is associated with systemic lupus erythematosus and promotes differentiation of monocytes into dendritic cell-like cells

Introduction

Using oligonucleotide microarray, many IFN-inducible genes have been found to be highly expressed in peripheral blood mononuclear cells (PBMCs) from most patients with systemic lupus erythematosus (SLE). Among these IFN-inducible genes, IFN-induced protein with tetratricopeptide repeats 4 (IFIT4) is a novel gene whose function is unknown.

Methods

In this study we examined the role played by IFIT4 in monocyte differentiation and the correlation between IFIT4 expression and the clinical manifestation of SLE. To this end, we used plasmid transfection, flow cytometry, mixed leucocyte responses, ELISA, quantitative RT-PCR and Western blotting.

Results

We found that both IFIT4 mRNA and protein expression levels were significantly higher in PBMCs and monocytes from SLE patients than in those from healthy control individuals. IFIT4 expression was positively correlated with antinuclear antibodies, anti-double-stranded DNA, and anti-Sm auto-immune antibodies in SLE. Patients with SLE exhibiting higher expression of IFIT4 had a higher prevalence of leucopenia, thrombocytopenia and C3/C4 decrease. IFIT4 protein was localized exclusively to the cytoplasm, and it was significantly upregulated by IFN-α in normal PBMCs. To determine the role played by IFIT4 in monocyte differentiation, the monocytic cell line THP-1 was transfected with pEGFP-IFIT4 expression plasmid and stimulated with granulocyte-macrophage colony-stimulating factor/IL-4 to generate IFIT4-primed dendritic cell-like cells (DCLCs). IFIT4-primed DCLCs acquired morphological characteristics of dendritic cells more quickly, with greater resemblance to dendritic cells, as compared with DCLCs primed with pEGFP-C1 control plasmid trasfection. Furthermore, they exhibited higher expressions of CD40, CD86, CD80, HLA-DR and CD83, along with lower expression of CD14; increased IL-12 secretion; and an increased ability to stimulate T-cell proliferation. In addition, IFIT4-primed DCLCs enhanced IFN-γ secretion (about 2.4-fold) by T cells compared with controls.

Conclusion

Our findings suggest that IFIT4 might play roles in promoting monocyte differentiation into DCLCs and in directing DCLCs to modulate T-helper-1 cell differentiation; these actions might contribute to the autoimmunity and pathogenesis of SLE.     

The function and magnetic resonance imaging of immature dendritic cells under ultrasmall superparamagnetic iron oxide (USPIO)-labeling

Objective

To investigate the effects of ultrasmall superparamagnetic iron oxide (USPIO) labeling on the maturity or immune tolerance of immature dendritic cells (imDCs) as the success of immunotherapy with immature dendritic cells is highly dependent on immune tolerance.

Results

The feasibility of tracking implanted USPIO-labeled imDCs in vivo by magnetic resonance imaging (MRI) was explored. The effects of USPIO labeling on the immune tolerance of imDCs was examined. USPIO when higher than 200 μg/ml caused considerable damage to imDCs, induced imDC maturation, and impacted the immune tolerance of imDCs. USPIO labeling caused a dose-dependent increase in autophagosome formation in imDCs, and autophagy inhibitors prevented the maturation of imDCs while stimulating their immune tolerance.

Conclusions

We speculate that high concentrations of USPIO can be used to induce imDC maturation, and that this process is likely mediated through an autophagy-related pathway.

     

Increased expression of Mer tyrosine kinase in circulating dendritic cells and monocytes of lupus patients: correlations with plasma interferon activity and steroid therapy

Introduction

The requirement for the immunoregulatory Mer tyrosine kinase (Mer) for optimal removal of apoptotic cells prompted us to look at its expression in systemic lupus erythematosus (SLE), in which apoptotic cell clearance is abnormal. We compared the levels of expression of Mer in normal human subjects and in patients with SLE.

Methods

We used flow cytometry of isolated peripheral blood mononuclear cells to compare the levels of Mer on leukocyte subsets. We used a Mer-specific enzyme-linked immunosorbent assay (ELISA) to quantify soluble Mer (sMer) in plasmas.

Results

Monocytes, CD1c⁺ myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) from both normal individuals and from SLE patients expressed Mer. In both normal and SLE patients, the CD14⁺⁺CD16⁺ subpopulation of monocytes expressed the highest levels of Mer, with somewhat lower levels on the CD14^intCD16⁺ population. Mer levels on CD1c⁺ mDCs and pDCs, and sMer levels in blood were increased in SLE patients compared with controls. In patients, Mer levels on CD14^intCD16⁺, CD14⁺⁺CD16^- monocytes, and CD1c⁺ dendritic cells correlated positively with type I interferon (IFN-I) activity detected in blood. In SLE patients treated with corticosteroids, Mer expression on monocytes correlated with prednisone dose, CD1c⁺ myeloid dendritic cells in patients treated with prednisone had higher levels of Mer expression than those in patients not receiving prednisone.

Conclusions

We found no global defect in Mer expression in lupus blood. In contrast, we observed increased levels of Mer expression in DC populations, which could represent a response to increased IFN-I in SLE patients. Enhanced Mer expression induced by corticosteroids may contribute to its beneficial effects in SLE.     

ApoE receptor 2 regulates synapse and dendritic spine formation

Background

Apolipoprotein E receptor 2 (ApoEr2) is a postsynaptic protein involved in long-term potentiation (LTP), learning, and memory through unknown mechanisms. We examined the biological effects of ApoEr2 on synapse and dendritic spine formation—processes critical for learning and memory.

Methodology/Principal Findings

In a heterologous co-culture synapse assay, overexpression of ApoEr2 in COS7 cells significantly increased colocalization with synaptophysin in primary hippocampal neurons, suggesting that ApoEr2 promotes interaction with presynaptic structures. In primary neuronal cultures, overexpression of ApoEr2 increased dendritic spine density. Consistent with our in vitro findings, ApoEr2 knockout mice had decreased dendritic spine density in cortical layers II/III at 1 month of age. We also tested whether the interaction between ApoEr2 and its cytoplasmic adaptor proteins, specifically X11α and PSD-95, affected synapse and dendritic spine formation. X11α decreased cell surface levels of ApoEr2 along with synapse and dendritic spine density. In contrast, PSD-95 increased cell surface levels of ApoEr2 as well as synapse and dendritic spine density.

Conclusions/Significance

These results suggest that ApoEr2 plays important roles in structure and function of CNS synapses and dendritic spines, and that these roles are modulated by cytoplasmic adaptor proteins X11α and PSD-95.     

Cigarette smoke promotes dendritic cell accumulation in COPD; a Lung Tissue Research Consortium study

Background

Abnormal immune responses are believed to be highly relevant in the pathogenesis of chronic obstructive pulmonary disease (COPD). Dendritic cells provide a critical checkpoint for immunity by their capacity to both induce and suppress immunity. Although evident that cigarette smoke, the primary cause of COPD, significantly influences dendritic cell functions, little is known about the roles of dendritic cells in the pathogenesis of COPD.

Methods

The extent of dendritic cell infiltration in COPD tissue specimens was determined using immunohistochemical localization of CD83+ cells (marker of matured myeloid dendritic cells), and CD1a+ cells (Langerhans cells). The extent of tissue infiltration with Langerhans cells was also determined by the relative expression of the CD207 gene in COPD versus control tissues. To determine mechanisms by which dendritic cells accumulate in COPD, complimentary studies were conducted using monocyte-derived human dendritic cells exposed to cigarette smoke extract (CSE), and dendritic cells extracted from mice chronically exposed to cigarette smoke.

Results

In human COPD lung tissue, we detected a significant increase in the total number of CD83+ cells, and significantly higher amounts of CD207 mRNA when compared with control tissue. Human monocyte-derived dendritic cells exposed to CSE (0.1-2%) exhibited enhanced survival in vitro when compared with control dendritic cells. Murine dendritic cells extracted from mice exposed to cigarette smoke for 4 weeks, also demonstrated enhanced survival compared to dendritic cells extracted from control mice. Acute exposure of human dendritic cells to CSE induced the cellular pro-survival proteins heme-oxygenase-1 (HO-1), and B cell lymphoma leukemia-x(L) (Bcl-xL), predominantly through oxidative stress. Although activated human dendritic cells conditioned with CSE expressed diminished migratory CCR7 expression, their migration towards the CCR7 ligand CCL21 was not impaired.

Conclusions

These data indicate that COPD is associated with increased numbers of cells bearing markers associated with Langerhans cells and mature dendritic cells, and that cigarette smoke promotes survival signals and augments survival of dendritic cells. Although CSE suppressed dendritic cell CCR7 expression, migration towards a CCR7 ligand was not diminished, suggesting that reduced CCR7-dependent migration is unlikely to be an important mechanism for dendritic cell retention in the lungs of smokers with COPD.     

PMA withdrawal in PMA‐treated monocytic THP‐1 cells and subsequent retinoic acid stimulation,modulate induction of apoptosis and appearance of dendritic cells

Objectives

To analyse proliferation, differentiation and apoptosis in THP‐1 cells after stimulation with phorbol 12‐myristate 13‐acetate (PMA) and retinoic acid (RA).

Materials and methods

PMA and RA were used in a three‐step‐procedure: (i) treatment with 6, 30, 60 nm PMA, that induced initial, intermediate and advanced levels of monocyte‐macrophage transition, respectively; (ii) recovery in PMA‐free medium; (iii) incubation with 4 μm RA. Cultures were characterized cytokinetically (flow cytometry/bromodeoxyuridine uptake) and immunocytochemically (static cytometry) for expression of CD14, CD11b (monocyte‐macrophage) and DC‐SIGN (dendritic cell: DCs) markers.

Results

Some treatments determined appearance of monocyte/macrophage, dendritic and apoptotic phenotypes, percentages of which were related to PMA dose used in step 1, and dependent on presence/absence of PMA and RA. PMA withdrawal induced dedifferentiation and partial restoration of proliferative activity, specially in 6 and 30 nm PMA‐derived cells. Recovery in the presence of serum (fundamental to DC appearance) indicated that depending on differentiation level, cell proliferation and apoptosis were inversely correlated. Treatment with 30 nm PMA induced intermediate levels of monocytic‐macrophagic differentiation, with expression of alternative means of differentiation and acquisition of DCs without using cytokines, after PMA withdrawal and RA stimulation.

Conclusions

Our experimental conditions favoured differentiation, dedifferentiation and transdifferentiational pathways, in monocytic THP‐1 cells, the balance of which could be related to both cell proliferation and cell death.

     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

1,25-Dihydroxyvitamin D3 modulates the phenotype and function of Monocyte derived dendritic cells in cattle

Background

The active form of the vitamin D₃, 1,25-Dihydroxyvitamin D₃ (1,25-(OH)₂D₃) has been shown to have major effects not only on physiological processes but also on the regulation of the immune system of vertebrates. Dendritic cells are specialised antigen presenting cells which are in charge of the initiation of T-cell dependant immune responses and as such are key regulators of responses towards pathogens. In this study we set out to evaluate the effects of 1,25-(OH)₂D₃ on the phenotype of cattle monocyte-derived dendritic cells (MoDCs) and how the conditioning with this vitamin affects the function of these myeloid cells.

Results

MoDCs were generated from CD14⁺ monocytes with bovine IL-4 and GM-CSF with or without 1,25-(OH)₂D₃ supplementation for 10 days. Vitamin D conditioned MoDCs showed a reduced expression of co-stimulatory and antigen presenting molecules, as well as a reduced capability of endocytose ovalbumin. Furthermore, the capacity of MoDCs to induce proliferation in an allogeneic mixed leukocyte reaction was abolished when MoDCs were generated in presence of 1,25-(OH)₂D₃. LPS induced maturation of 1,25-(OH)₂D₃conditioned MoDCs resulted in lower secretion of IL-12 and higher IL-10 than that observed in MoDCs.

Conclusions

The typical immunotolerant phenotype observed in cattle DCs after exposure to 1,25-(OH)₂D₃ has a significant effect on the functionality of these immune cells, inhibiting the T-cell stimulatory capacity of MoDCs. This could have profound implications on how the bovine immune system deals with pathogens, particularly in diseases such as tuberculosis or paratuberculosis.

     

Myeloid dendritic cells display downregulation of C-type lectin receptors and aberrant lectin uptake in systemic lupus erythematosus

Introduction  

There is a growing body of evidence implicating aberrant dendritic cell function as a crucial component in the immunopathogenesis of systemic lupus erythematosus. The purpose of the present study was to characterize the phagocytic capacity and expression of receptors involved in pathogen recognition and self-nonself discrimination on myeloid dendritic cells from patients with lupus.